CLY series compounds, their preparation methods and uses of the prepared drugs

Abstract

The present invention discloses CLY series compounds and the preparation method and preparation drug use thereof, said CLY series compounds are compounds having the structure of formula I, and its tautomers, solvates or pharma- ceutical acceptable salts thereof. According to animal model experiments, all of the CLY series compounds can significantly reduce the tyrosinase level in the skin and blood of melasma mouse models, reduce the expression of hepatocyte factor (SCF) and C-kit protein in the skin, inhibit the pigmentation of melasma, significantly promote the wound healing of the skin and reduce the formation of scars; significantly promote the hair growth and reduce the damage of hair follicles in hair loss mouse models, and significantly promote the hair growth and hair follicle formation in rabbit ears. It can obviously alleviate the symptoms of acne models, reduce pore clogging and acne formation;CLY series compounds can obviously increase the survival time of acute GVHD mice, alleviate clinical symptoms, showing therapeutic effect against acute GVHD;CLY series compounds can obviously reduce MMP-2, MMP-9 levels in lung tissue of pulmonary fibrosis mouse models, increase TIMP-1 and VEGF levels, and at the same time increase SOD, CAT enzyme levels in peripheral blood;CLY series compounds can improve arthritis symptoms in rheumatoid arthritis mice by reducing IL-17 levels in peripheral blood and increasing inflammatory indicators such as IL-10. This compound can be used alone or in combination with other drugs, providing a new drug choice for the treatment of the above diseases. [Formula 1] TIFF2024532854000096.tif42170

Description

[Technical field]

[0001] The present invention relates to the field of pharmaceutical chemistry, specifically to CLY series compounds and their preparation methods and the use of the prepared drugs. [Background technology]

[0002] Melasma is an acquired pigmentation disorder that is likely to occur in middle-aged and adolescent women. Its pathogenesis is extremely complex and has many influencing factors, but the direct cause of melanoma is the deposition of melanin in the skin due to various causes. Tyrosinase produces melanin through a series of oxidation reactions. Tyrosine is oxidized to dopa under the action of tyrosinase in the melanin corpuscles, dopa is further oxidized to dopaquinone by dopa oxidase, and dopaquinone is finally oxidized under the action of tyrosinase to form melanin. Disturbances in the series of oxidation and antioxidant reactions in this process may cause and promote the occurrence and development of melasma, and an increase in tyrosine is the main material basis for the development of melasma. When the balance of the series of oxidation reactions is disturbed, oxygen free radicals are produced in excess in the body, while the activity of antioxidant enzymes such as superoxide dismutase (SOD) decreases, causing peroxidation of membrane lipids and producing lipid peroxides. Lipid peroxides are unstable and rapidly decompose to produce aldehydes, which in turn increases the final product malondialdehyde (MDA), which rapidly attacks phospholipids and proteins, causing oxidative damage to pigment cells, promoting the oxidation reaction of tyrosinase, increasing melanin, and depositing it in the basal layer of the skin. Therefore, reducing the amount of MDA in skin tissues and enhancing the activity of the antioxidant enzyme SOD is of great significance for the prevention and treatment of melasma. Melasma is prone to recurrence and difficult to treat. There are few products on the market that can fundamentally solve melasma, and pigment spots are prone to recurrence. Hydroquinone is the earliest and most widely used whitening agent, but its application in whitening and melasma treatment is regulated due to its uneven distribution of skin pigmentation, high irritation, and even possible carcinogenicity. Arbutin is one of the most widely used whitening agents in clinical practice, but its effect is limited.

[0003] Scarring is damage to human skin and soft tissues caused by physical, biological, chemical and other factors, which results in serious damage to the skin and soft tissues and cannot completely repair itself. Scarring causes great physical and mental suffering to patients, especially the scarring left after burns, scalding and severe trauma. Scarring is difficult to deal with, and at present, it is only possible to soften and lighten reddish and hard scars, narrow wide scars and thin thick scars, but it is impossible to completely eliminate scarring. Therefore, it is important to intervene in the early stage of wound healing, which can effectively inhibit scar formation, improve the appearance, correct deformity and restore function. At present, commonly used methods for the treatment of scarring include surgery, laser therapy, cryotherapy and drug therapy. Commonly used drugs include glucocorticoids and retinoids. Glucocorticosteroids have obvious anti-fibrotic effects, but have many toxic side effects. Retinoic acid is an intermediate product of vitamin A metabolism in the body. It reduces local inflammation, promotes epithelial cell proliferation, inhibits collagen synthesis, inhibits fibroblast DNA synthesis, and inhibits cell proliferation. The higher the concentration of retinoids, the more pronounced the antiproliferative effect. However, retinoic acid has poor efficacy and many toxic side effects when used in the system. External application of retinoic acid causes obvious skin irritation, which becomes more severe as the concentration increases.

[0004] Alopecia areata (AA) is a non-scarring type of alopecia in which the local skin is almost normal. It is usually a sudden patchy alopecia, and in severe cases, it can affect the entire scalp, in which case it is called alopecia totalis (AT), and when it affects the hair of the whole body, including the armpits and pubic hair, it is called alopecia universalis (AU), which can have a serious impact on the appearance and psychology of the patient. The etiology of the disease has not yet been fully elucidated, and abnormalities and instability of autoimmune function and neuropsychological factors are thought to be important related factors. Alopecia areata has a high chance of being cured, but the probability of recovery varies greatly depending on the etiology. Some patients recover naturally, while others may continue to have alopecia for several years. Minoxidil is a topical medication commonly used to treat alopecia areata, which promotes vasodilation of the skin, improves local blood circulation, and promotes hair growth. Glucocorticosteroids are commonly used in severe alopecia areata, mainly including prednisolone and conjugated betamethasone, and can be administered orally, topically, or by intradermal injection. Patients for whom glucocorticoids are not suitable can be treated with immunosuppressants, the most common being cyclosporine, methotrexate, glucocorticosteroids, and immunosuppressants, but these drugs have many side effects.

[0005] Androgen-dependent hereditary alopecia (AGA), also known as seborrheic alopecia, is a common and easily occurring disease. It is most common in men aged around 20 to 30 years. Hair loss occurs mainly on the top of the head, usually starting from the hairline on both sides of the forehead, but some people may also experience hair loss from the crown of the head. The hair loss area gradually spreads upwards, and the hair gradually becomes thinner and thinner, until the hair on the crown of the head is almost completely or completely lost, but the hair on the back of the head and the upper part of both sides of the head remains, giving the appearance of a horseshoe, and the hair in this band remains normal. The hair loss area has lighter skin, smaller pores, or a few fine, soft, fine hairs. The speed, extent, and severity of hair loss are influenced by genetics and individual differences. In general, the progression is fastest around the age of 30, and severe total hair loss is rare. Women often suffer from diffuse alopecia on the crown of the head, and the hair on the crown of the head thins. According to an epidemiological survey in China, the prevalence of male pattern baldness is 21.3% in men and 6.0% in women. The etiology and pathology of androgenetic alopecia have not yet been fully elucidated, and it is generally believed that androgens and their receptors play an important role in the development of the disease, and type II 5a-reductase is an important factor in its development. Under normal physiological conditions, androgens in the body play a certain role in stimulating hair growth and development, but they can induce hair loss in certain areas. Testosterone is the main androgen in the body, which is converted by 5a-reductase into dihydrotestosterone, the latter of which can transform terminal hair into vellus hair and ultimately cause hair loss. At present, the lack of an ideal treatment is a challenge facing the treatment of alopecia-related diseases. Minoxidil is a topical drug approved by the FDA as a nonspecific treatment for alopecia and the first choice for the treatment of alopecia, but its use may cause hirsutism on the face and limbs, and the therapeutic effect will gradually disappear if its use is discontinued. Since androgens play a major role in the onset of AGA, recent new treatments have attempted to terminate hair follicle miniaturization through antiandrogen effects. Finasteride is a selective inhibitor of type II 5a reductase, which has been shown to be effective in treating AGA in recent years and has been shown to improve hair growth sustainably.However, finasteride has side effects such as sexual dysfunction, a temporary decrease in sperm count, and abnormal breast development in men, and animal experiments have also shown that it is teratogenic, so it is not used in pediatrics or women of childbearing age. Cimetidine must be taken for more than five months, and side effects include breast development in men, impotence, and decreased libido. Oral contraceptives: Mainly sogonolone, levonorgestrel (levomethylnorethindrone), progesterone, norethindrone (oximenorethindrone), bisesternorethindrone, vinpocetine, etc. These are often used to treat AGA in women, and hair can improve to a certain extent with 6 to 12 months of treatment.

[0006] Acne (commonly known as acne) is a chronic inflammatory disease that is prone to develop in the sebaceous gland of the hair follicle, with a prevalence of about 9.4%. The occurrence of acne is closely related to the physiological and pathological changes of the skin during puberty. Clinical symptoms mainly include acne, papules, pustules, nodules, cysts, scars, etc., which have a serious impact on the appearance and psychology of patients. Acne is associated with several pathologies, and the formation of acne due to abnormal keratinization of the hair follicle opening is an important basis for the development of this disease, and inflammation and infection are factors in the development of acne. Acne patients have large sebaceous glands, increased sebaceous gland secretion, and a relatively low level of linoleic acid in sebum, which affects the synthesis of fat, resulting in a shortage of fatty acids in the hair follicle epithelium, which induces hyperkeratinization of the hair follicle, which prevents the epithelium from peeling off normally, and the excessively small opening of the hair follicle sebaceous gland causes sebum to be discharged smoothly, resulting in the formation of acne. The sebaceous gland of the hair follicle is blocked, creating a low-oxygen environment in the sebaceous gland, which allows the anaerobic acne bacteria to proliferate in large quantities, decompose sebum, produce chemokines, and accumulate white blood cells to form papules. A large number of neutrophils gather in the sebaceous gland of the hair follicle, engulf Propionibacterium acnes, and cause an inflammatory reaction, resulting in the accumulation of a large number of pus cells, the formation of pustules and cysts, and the formation of depressed scars after healing. Elevated androgen levels are an important part of promoting the development of acne, causing abnormal keratinization of the skin, blocking the duct of the sebaceous gland of the hair follicle, leading to the retention and proliferation of bacteria, and causing inflammation. Diseases caused by abnormal keratinization that are similar to acne include ichthyosis, keratosis pilaris (also known as lichen pilaris), Darier's disease, and porokeratosis. Keratosis pilaris is seen as an enlargement of the hair follicle opening with a keratin plug, while ichthyosis presents with a decrease in sweat glands and sebaceous glands and keratin plugs in the hair follicle. The above diseases are prone to recurrence and are difficult to treat. The main drug used to treat keratinization abnormalities and remove keratin plugs and acne is retinoids. Retinoids can inhibit keratinization, inhibit sebum secretion, promote normal keratinization of keratinocytes, and have immunomodulatory and anti-inflammatory effects, so they can inhibit the formation of acne, papules, and pustules, and are widely used in clinical practice to treat keratinization abnormalities such as acne, ichthyosis, keratosis pilaris, Darier's disease, and porokeratosis.However, topical retinoids tend to irritate the skin, causing redness, swelling, pain, and aggravation of existing lesions, and long-term application of retinoids can lead to thinning of the skin, photosensitivity, and damage to the skin barrier, while oral retinoids have side effects such as liver damage and elevated blood lipids. Therefore, there is a clinical need for more drugs to treat such diseases.

[0007] Psoriasis is a common chronic recurrent inflammatory skin disease with a global prevalence of 0.1-3% in the natural population. Patients with moderate to severe disease are at high risk of developing metabolic syndrome and cardiovascular disorders. Therefore, early diagnosis and treatment of psoriatic disease are important for controlling and improving symptoms and preventing complications. Topical treatment is the first choice for mild to moderate psoriasis. Topical glucocorticoids are more effective, but are not suitable for long-term, continuous, widespread use; topical vitamin A acid treatment is more effective for plaque-type psoriasis, but care must be taken to prevent skin irritation; vitamin D3 derivatives such as calcipotriol also have better efficacy, but are not suitable for use on the face or in skin folds; calcium-modulating neurophosphatase inhibitors (e.g., tacrolimus, pimecrolimus) can be used on the scalp, skin folds, genitals, etc., but long-term widespread use may increase the risk of lymphoma and skin cancer; various keratinogenesis promoters (e.g., tar preparations, anthralin ointment, 10-15% camptothecin ointment, salicylic acid ointment) can also be used topically, but their effectiveness is limited. Immunosuppressants are mainly used for erythroderma, pustular psoriasis, and psoriatic arthritis. Antibiotics should be used in patients with obvious infections or generalized pustular psoriasis. Immunosuppressants can be used to treat moderate to severe patients, but long-term use can lead to many side effects, including bone marrow suppression, liver dysfunction, kidney dysfunction, and increased risk of infection.

[0008] Eczema (also known as atopic dermatitis or atopic dermatitis) is an inflammatory reaction of the skin caused by various internal and external factors, accompanied by severe itching and prone to recurrence. The causes of eczema are complex. For mild to moderate eczema, topical therapy is mainly used. Depending on the skin lesion, appropriate dosage forms and drugs are used. For subacute and chronic eczema, appropriate glucocorticoid creams, tar-based preparations, or immunomodulators such as tacrolimus ointment and pimecrolimus ointment are used. Glucocorticoids and immunosuppressants can be used systemically in severe patients, but they have many side effects and are not suitable for long-term use.

[0009] Graft versus host disease (GVHD) is caused by T lymphocytes in the allograft after transplantation, stimulated by a series of "cytokine storms" initiated by the recipient, which significantly enhance the immune response to the recipient's antigens and launch cytotoxic attacks against the recipient's target cells, among which the skin, liver and intestinal tract are the main targets. The incidence rate of acute GVHD is 30-45%, and the incidence rate in chronic cases is lower than that of acute cases. Allogeneic hematopoietic stem cell transplantation is an effective treatment for curable hematologic tumors and hematopoietic dysfunction disorders. Acute GVHD is its major serious complication, with high morbidity, mortality and disability, and is an important cause of non-recurrent death in malignant hematologic diseases. Steroids are the first-line drug for the treatment of acute GVHD, but about 50% of patients have hormone resistance and cannot control the GVHD reaction. Although other second-line treatments for GVHD reactions, such as monoclonal antibodies against T cell surface antigens (e.g., CD3, CD25) and chemotherapy drugs (e.g., mortimethimechloride, tacrolimus), have definite therapeutic effects against GVHD, the subsequent immunodeficiency and opportunistic infections significantly reduce the benefits of these drugs and ultimately do not extend the survival of patients. Therefore, there is an urgent need to find new therapeutic approaches to effectively control hormone-resistant acute GVHD.

[0010] Pulmonary fibrosis is a diffuse lung disease with unknown causes and complex pathology, which is currently recognized as the result of excessive deposition of extracellular matrix due to excessive repair after lung injury. Although the pathogenesis of pulmonary fibrosis is unknown, it is now recognized that repeated injury and excessive repair of the alveolar epithelium are the key to its onset. This pathology is characterized by the aggregation of extracellular matrix metalloproteinases (MMPs) caused by long-term chronic pneumonia and persistent alveolar injury, which leads to an abnormal increase in MMP-2 and MMP-9, in particular, and a decrease in tissue inhibitor of metalloproteinase-1 (TIMP-1), which causes an imbalance, resulting in a large amount of aggregation of the extracellular matrix in the lung, remodeling of histiocytes, and excessive deposition of collagen. At the same time, it inhibits the expression of vascular endothelial growth factor (VEGF) in tissues, reduces the permeability of pulmonary microvessels, inhibits the division and proliferation of vascular endothelial cells and vascular regeneration, which may aggravate lung tissue damage and ultimately cause pulmonary fibrosis, a diffuse interstitial lung disease. At present, there is no effective anti-fibrotic drug, and glucocorticoid anti-inflammatory drugs are commonly used to suppress the anti-fibrotic process, but they have limited efficacy and many side effects. Current treatments are far from meeting clinical needs, and it is necessary to find more new drugs with high efficacy and few side effects to inhibit disease progression, reduce recurrence and complications, and lower mortality.

[0011] Autoimmune diseases have a high incidence rate. There are at least hundreds of millions of patients worldwide, including rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, lupus erythematosus, dermatomyositis, scleroderma, and sicca syndrome. When these diseases become severe, they affect multiple organs, causing damage to the heart, liver, kidneys, blood vessels, lungs, joints, and brain, and have a high mortality rate second only to malignant tumors. Rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, and Crohn's disease share a common path of onset. The etiology and pathology of these diseases are very complex, and currently cannot be cured completely, requiring long-term drug therapy to suppress the progression of the disease. The therapeutic drugs commonly used in clinical practice are mainly glucocorticoids and immunosuppressants, but the efficacy rate of these drugs is only about 50%, and they have side effects such as bone marrow suppression, liver and kidney dysfunction, osteoporosis, and the induction of infections and tumors, so their long-term use is limited. Current new biologics also have immunosuppressive effects, carry the risk of inducing infections and tumors, and are expensive, limiting their long-term general use.

[0012] For the above diseases, current treatments are far from meeting clinical needs, and there is a need to find new drugs with high efficacy, fewer side effects and lower cost to inhibit disease progression and reduce the occurrence of recurrence and complications. Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a CLY series compound or a pharma- ceutical acceptable salt thereof having medicinal properties. A further object of the present invention is to provide a method for preparing said compound.A further object of the present invention is to provide uses of said compound. The object of the present invention can be achieved by the following means: The present invention provides compounds having the structure shown in Formula I, a tautomer thereof, a solvate thereof, or a pharma- ceutically acceptable salt thereof,

[0014] [ka]

[0015] however: R 1 is a substituted or unsubstituted 5-6 membered heterocycle containing at least one of N, O, S, benzoheterocycle (e.g., isoquinolinyl), or a bicycle having at least two of the above heterocycles, where parallel rings refer to a bicycle in which the two ring structures are formed by two adjacent atoms, such as pyrrolopyridine, and the substituents are hydrogen, halogen, or (C1-C4) alkyl; R 2 is hydrogen, halogen, hydroxyl, methoxy, ethoxy, amino, methyl or ethyl; R 3 is hydrogen, halogen, hydroxyl, methoxy, ethoxy, amino, (C1-C3) alkyl or the following groups:

[0016] [ka]

[0017] and; R 4 is a substituted or unsubstituted 5- to 6-membered cycloalkyl group, or a 4- to 7-membered heterocycle having 1 to 3 heteroatoms selected from N and O, and the substituents are hydrogen, -NH 2 , -OH, (C1-C4) alkyl, (C1-C4) alkoxy, amino, and (C1-C4) alkylamino; Preferably, R 4 teeth

[0018] [ka]

[0019] It is.

[0020] Preferably, R 3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl or the following groups:

[0021] [ka]

[0022] If R 4 teeth

[0023] [ka]

[0024] and R 3 teeth

[0025] [ka]

[0026] If R 4 teeth

[0027] [ka]

[0028] and; however: R 6 and R 8 are each independently hydrogen, methyl, halogen, or (C-C) alkyl, with the proviso that R 6 and R 8 are not both halogens at the same time. 6 is hydrogen or methyl, and R 8 is hydrogen; R 7 is hydroxy, (C1-C4)alkoxy, (C1-C4)alkoxycarbonyloxy(C1-C4)alkyl, or (C1-C4)alkylcarbonyloxy(Cl-C4)alkyl; R l0 and R 11 are each independently hydrogen, (C1-C4) alkyl, or (C3-C6) cycloalkyl; R 12 is selected from hydrogen, halogen, -OH, -NH2 or (C1-C3) alkyl; R 13 is hydrogen, (C1-C4)alkyl, (C1-C4)alkylcarbonyloxy(C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl; R 14 is hydrogen, (C1-C4)alkyl, (C1-C4)alkylcarbonyloxy(C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl; R 15 is hydroxy, tetrazolyl, (C1-C2) alkylsulfonyl or trifluoromethylsulfonyl; R 16 is hydrogen, (C1-C4)alkyl, (C1-C4)alkylcarbonyloxy(C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl).

[0029] In some embodiments, R l is isoquinolin-1-yl, R l is optionally monosubstituted with chlorine or methyl.

[0030] In some embodiments, R 2 is hydrogen, hydroxy, or methyl.

[0031] In some embodiments, R l is a substituted or unsubstituted pyridinyl or pyrrolopyridinyl, said substituents being hydrogen, chloro, or methyl.

[0032] In some embodiments, R l2 is selected from hydrogen, halogen, -OH, -NH2, or methyl; in some embodiments, R 12 , H.

[0033] The present invention provides a compound having the structure shown in formula Ia, a tautomer thereof, a solvate thereof, or a pharma- ceutically acceptable salt thereof:

[0034] [ka]

[0035] However, R 3 is H, halogen, hydroxyl, methoxy, amino, methyl, or any of the following substituted or unsubstituted groups:

[0036] [ka]

[0037] Selected from R 4 teeth

[0038] [ka]

[0039] and; However, R 6 , R 7 , R 8 , R l0 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 The limitation is consistent with any one of the limitations above. Preferably, R 3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl or any of the following substituted or unsubstituted groups:

[0040] [ka]

[0041] If R 4 teeth

[0042] [ka]

[0043] and; R 3 teeth

[0044] [ka]

[0045] In the case of R 4 teeth

[0046] [ka]

[0047] and Here, R 6 and R 8 are each independently hydrogen, methyl, halogen, or (C-C) alkyl, with the proviso that R 6 and R 8 are not both halogens at the same time. 6 is hydrogen or methyl, and R 8 is hydrogen; R 7 is hydroxyl, (C1-C4)alkoxy, (C1-C4)alkoxycarbonyloxy(C1-C4)alkoxy, or (C1-C4)alkylcarbonyloxy(Cl-C4)alkoxy; R l0 and R 11 are each independently hydrogen, (C1-C4) alkyl, or (C3-C6) cycloalkyl; R 12 are hydrogen, halogen, -OH, -NH 2 or (C1-C3) alkyl; R 13is hydrogen, (C1-C4)alkyl, (C1-C4)alkylcarbonyloxy(C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl; R 14 is hydrogen, (C1-C4)alkyl, (C1-C4)alkylcarbonyloxy(C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl; R 15 is hydroxy, tetrazolyl, (C1-C2)alkylsulfonyl or trifluoromethylsulfonyl, and R16 is hydrogen, (C1-C4)alkyl, (C1-C4)alkylcarbonyloxy(C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl.

[0048] In some embodiments, the compound having the structure shown in formula Ia, its tautomer, solvate, or pharma- ceutically acceptable salt thereof, has R 6 is H and R 8 is H; In some embodiments, the compound having the structure shown in formula Ia, its tautomer, solvate, or pharma- ceutically acceptable salt thereof, has R 12 are hydrogen, halogens, -OH, -NH 2 or methyl; in some embodiments, R 12 is selected from hydrogen.

[0049] In some embodiments, the compound having the structure shown in formula Ia, its tautomer, solvate, or pharma- ceutically acceptable salt is R 11 is H and R 10 is H or methyl, R 13 teeth

[0050] [ka]

[0051] It is.

[0052] The present invention provides a compound having the structure shown in formula Ib, a tautomer thereof, a solvate thereof, or a pharma- ceutically acceptable salt thereof:

[0053] [ka]

[0054] However, R 3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl, or any of the following substituted or unsubstituted groups:

[0055] [ka]

[0056] and R 4 teeth

[0057] [ka]

[0058] and R 6 , R 8 , R l0 , R 11 , R 12 , R 13 The limitation is consistent with any one of the limitations above.

[0059] Preferably, R 3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl or any of the following substituted or unsubstituted groups:

[0060] [ka]

[0061] If R 4 teeth

[0062] [ka]

[0063] and; R 3 teeth

[0064] [ka]

[0065] In the case of R 4 teeth

[0066] [ka]

[0067] It is.

[0068] Here, R 6 , R 8 , R l0 , R 11 , R 12 , R 13 The limitation is consistent with any one of the limitations above.

[0069] In some embodiments, the compound having the structure shown in formula Ib, its tautomer, solvate, or pharma- ceutically acceptable salt thereof, is R 12 are hydrogen, halogens, -OH, -NH 2 or methyl; in some embodiments, R 12 is selected from hydrogen.

[0070] In some illustrative embodiments, the present invention provides the following compounds, tautomers thereof, solvates thereof, or pharma- ceutically acceptable salts thereof, but is not limited to the following compounds:

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] [ka]

[0075] The present invention also provides a process for the preparation of a compound of formula (I):

[0076] [ka]

[0077] raw material

[0078] [ka]

[0079] By preparation of

[0080] [ka]

[0081] is generated, and

[0082] [ka]

[0083] and

[0084] [ka]

[0085] produces the final product I, Here, R 1 , R2 , R 3 , R 4 is as defined above.

[0086] The present invention also provides a pharmaceutical composition comprising a compound described in the present invention or a pharma- ceutically acceptable salt thereof as an active ingredient or main active ingredient, and a pharma- ceutically acceptable carrier.

[0087] The present invention also provides the use of a compound according to the invention in the preparation of a medicament for treatment and / or prophylaxis.

[0088] In some embodiments, the present invention also provides the use of the compounds described in the present invention in medicine for the treatment and / or prevention of diseases such as melasma, scarring, androgenic alopecia, seborrheic alopecia, alopecia areata, acne, pulmonary fibrosis, psoriasis, eczema, atopic dermatitis, etc.

[0089] The compounds or compositions according to the invention may be in any pharma- ceutically acceptable dosage form, for example, any suitable dosage form for oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, nasal, inhalation, vaginal, intraocular, topical, parenteral dermal, subcutaneous, intraadipose, intraarticular, intraperitoneal or intrathecal administration.

[0090] In a preferred embodiment, the dosage form described in the present invention is a gel, emulsion, cream, liniment, lotion, spray, solution, tablet, punch, oral liquid, capsule, drops, enema, film or injection.

[0091] Definitions in the invention:

[0092] "C 5 ~C 6 "Monohydric alcohol" means a saturated aliphatic hydrocarbon group containing 5 or 6 carbon atoms substituted with one hydroxyl group, including straight-chain and branched-chain groups.

[0093] "Heterocyclic" refers to a saturated cyclic group of 4 to 7 ring atoms, in which one or two or three of the ring atoms are heteroatoms selected from N, O or S(O)m, where m is an integer from 0 to 2, and the remaining ring atoms are C, in which one or two of the C atoms are optionally substituted with a carbonyl group. The rings of the heterocyclic group may be optionally and independently substituted with one, two or three substituents.

[0094] "Alkyl" refers to saturated aliphatic hydrocarbon groups having 1 to 20 carbon atoms, including both straight-chain and branched-chain groups. (Numerical ranges referred to in this application, such as "1-4," refer to alkyl groups that may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Alkyl groups may be substituted or unsubstituted.)

[0095] "Cycloalkyl" refers to all-carbon monocyclic or closely packed ring groups in which one or more of the rings does not have a completely connected pi-electron system ("packed" rings means that each ring in the system shares an adjacent pair of carbon atoms with the other ring in the system); examples of cycloalkyl groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadienyl, cycloheptane, and cycloheptatriene.

[0096] "Alkoxy" refers to -O-(unsubstituted alkyl) or -O-(unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.

[0097] "Alkylamino" refers to -NH- (unsubstituted alkyl), -NH- (unsubstituted cycloalkyl), -N- (unsubstituted alkyl) 2 or -N-(unsubstituted cycloalkyl) 2Representative examples include, but are not limited to, methylamino, ethylamino, propylamino, butylamino, cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, and the like.

[0098] "(C1-C4)alkoxycarbonyloxy(C1-C4)alkyl" refers to (C1-C4)alkyl-OC(O)-O-(C1-C4)alkyl-.

[0099] "(C1-C4)alkoxycarbonyloxy(C1-C4)alkyl" refers to (C1-C4)alkyl-OC(O)-O-(C1-C4)alkyl-. Effect of the Invention

[0100] The compound CLY series of the present invention or its pharmacologic acceptable salt can be applied to the pharmaceutical field. According to the animal experimental model, all of the compounds CLY series can significantly reduce the level of tyrosinase in the skin and blood of melasma model mice, reduce the expression of hepatic cytokines (SCF) and C-kit protein in the skin, inhibit the formation of melasma pigmentation, significantly promote the healing of skin wounds, and reduce the formation of scars. It can significantly promote hair growth in male pattern baldness mouse model and reduce the destruction of hair follicles by androgen. The CLY series can significantly inhibit the inflammatory response in mouse models of psoriasis and eczema. The CLY series compounds can significantly improve the survival time of acute GVHD mice and reduce clinical symptoms, showing a therapeutic effect on acute GVHD. The CLY series compounds can significantly reduce the levels of MMP-2 and MMP-9, increase the levels of TIMP-1 and VEGF in the lung tissue of a mouse model of pulmonary fibrosis, and simultaneously increase the levels of SOD and CAT enzymes in the peripheral blood, showing an inhibitory effect on pulmonary fibrosis. The CLY series compounds can improve the arthritis symptoms of rheumatoid arthritis mice by reducing the IL-17 level in the peripheral blood and increasing inflammatory indicators such as IL-10. Since many diseases have common development pathways, the efficacy of the compounds includes, but is not limited to, the above diseases. The compounds can be used alone or in combination with other drugs, providing a new option of drugs for treating the above diseases. [Brief description of the drawings]

[0101] [Figure 1] The CLY series compounds can significantly promote hair growth in alopecia model mice. [Diagram 2] The CLY series compounds can significantly suppress psoriasis-like inflammatory responses in mice. [Diagram 3] CLY-2 nuclear magnetic detection results. [Figure 4] CLY-8 nuclear magnetic detection results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] The present invention will be further described below with reference to examples. The specific examples described herein are for the purpose of illustrating the present invention, and are not intended to limit the present invention. It should be understood that simple improvements to the preparation method of the present invention, based on the concept of the present invention, are also within the scope of protection of the present invention. In the following examples, the experimental methods for which no specific conditions are shown are generally in accordance with known means in the art. The test materials used in the following examples can be purchased from ordinary biochemical reagent stores unless otherwise specified.

[0103] Example 1 Preparation of (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)-N-(1-methylpiperidin-3-yl)benzamide (abbreviation: CLY-1) and (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)-N-(1-ethylpiperidin-3-yl)benzamide (abbreviation: CLY-2): 1. Synthesis route

[0104] [ka]

[0105] 2. Specific implementation methods (1) Synthesis of (R)-tert-butyl 3-((3-chloropyridin-2-yl)amino)piperidine-1-formate Add (R)-1-Boc-3-aminopiperidine, 2-bromo-3-chloropyridine, sodium tert-butoxide, and dioxane to a 250 ml container bottle, and under nitrogen gas protection, start stirring. Add RuPhosPd G3 and ligand RuPhos, heat to 100 degrees, and react for 7 hours. Then stop the reaction, add to water, and extract with ethyl acetate in several portions. After combining the ethyl acetate layers, wash with water three times. After collecting the ethyl acetate layers, obtain the remaining paste, mix with silica gel, and obtain (R)-3-((3-chloropyridin-2-yl)amino)piperidine-1-tert-butyl formate by silica gel column chromatography.

[0106] (2) Synthesis of piperidine amide Add 4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)benzoic acid to a container bottle, heat and stir the pre-dried toluene, N,N-dimethylformamide, and thionyl chloride at 30°C until clarified, and recover the solvent under reduced pressure to obtain a residual solid. Add 50 ml of pre-dried tetrahydrofuran, (R)-tert-butyl 3-((3-chloropyridin-2-yl)amino)piperidine-1-carboxylate, stir until clarified, place in an ice bath, add lithium bis(trimethylsilyl)amine group, stir for 1 hour, remove the ice bath, stir for 2 hours, pour into water, extract with ethyl acetate, wash the ethyl acetate layer, recover the solvent, and obtain a paste under reduced pressure.

[0107] (3) Synthesis of piperidine amide Piperidine amide, dichloromethane, and trifluoroacetic acid are stirred at room temperature overnight, poured into water, and sodium bicarbonate is added to adjust the pH to 10-11. The mixture is extracted with dichloromethane and washed. The solvent is removed under reduced pressure to obtain a paste-like residue, which is then purified by silica gel column chromatography to obtain piperidine amine.

[0108] (4) Synthesis of CLY-1 Piperonylamine is dissolved in dichloromethane, iodomethane and silver carbonate are added, and the mixture is stirred at room temperature for 48 hours away from light. The reaction solution is then directly subjected to silica gel column chromatography to obtain CLY-1.

[0109] The chemical formula of CLY-1 is C 23 H 22 ClN 7 It is O. Hydrogen spectrum d4-MeOH: 8.83 (1H), 8.60 (1H), 8.54 (1H), 8.29 (2H), 7.82(1H), 7.61(3H), 7.42 (1H), 5.06(1H), 3.82(1H), 3.63(1H), 3.39 (1H), 2.95 (1H), 2.40-1.87(6H), 1.55-1.41 (1H).

[0110] (5) Synthesis of CLY-2 Piperonylamine is dissolved in dichloromethane, and iodoethane and silver carbonate are added. The mixture is stirred at room temperature for 48 hours away from light, and the reaction solution is directly subjected to silica gel column chromatography to obtain CLY-2. The mass spectrum of CLY-2 is shown in Figure 3.

[0111] Example 2 Preparation of (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)-N-(pyrrolidin-3-yl)benzamide (abbreviation: CLY-8): 2. Synthesis route

[0112] [ka]

[0113] 2. Specific synthesis steps (1) Synthesis of ETH-1 Add 11.8g of (R)-1-Boc-3-aminopyrrolidine, 11.0g of 2-bromo-3-chloropyridine, 10.0g of sodium tert-butoxide, and 70ml of toluene to a 250ml three-neck flask, start stirring under the protection of nitrogen gas, add 0.1g of palladium acetate, 1,1'-bian-2-naphthol, and tris(dimethylamino)phosphine, and heat to 100°C. After 7 hours of reaction, stop the reaction, pour into 500ml of water, and extract with 1000ml of ethyl acetate in several portions, combine the ethyl acetate layer, wash with 200*3 times of water, and collect the ethyl acetate layer to obtain the residual paste-like residue, mix with silica gel, and obtain about 7g of ETH-1 by silica gel column chromatography.

[0114] (2) Synthesis of pyrrolidine amide Dissolve 4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)benzoic acid in a solvent, add oxalyl chloride and catalyst N,N-dimethylformamide, react at 25-55 ° C until clarified, recover the solvent under reduced pressure, add the residue to the solvent, add tert-butyl (R)-3-((3-chloropyridin-2-yl)amino)pyrrolidine-1-carboxylate, cool to 5 ° C in an ice bath, add lithium bis(trimethylsilyl)amide to obtain pyrrolidine amide, i.e., (R)-3-(4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)benzamide)pyrrolidine-1-carboxylate tert-butyl.

[0115] (3) Synthesis of compound CLY-8 10g of pyrrolidine amide, 50ml of dichloromethane, and 10ml of 4mol / L hydrochloric acid in methanol are stirred at room temperature overnight. Pour into 100ml of water, add sodium bicarbonate to adjust the pH to 10-11, extract with 100ml of dichloromethane, wash, and recover the solvent under reduced pressure to obtain a paste-like residue. By silica gel column chromatography, 3.2g of compound (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)-N-(pyrrolidine-3-substrate)benzamide (abbreviation: CLY-8) is obtained. M+H=420.1 (see Figure 4 for mass spectrum analysis results). The compounds in Table 1 below can also be synthesized by using the same method as above.

[0116] [Table 1-1]

[0117] [Table 1-2]

[0118] [Table 1-3]

[0119] Example 3: Synthesis of (R)-N-(3-chloropyridin-2-yl)-N-(pyrrolidin-3-yl)-3-(1H-tetrazol-5-yl)benzenemethanamine (abbreviation: CLY-14)

[0120] [ka]

[0121] The (R)-tert-butyl 3-(N-(3-chloropyridin-2-yl)-3-cyanobenzamido)pyrrolidine-1-carboxylate can be obtained in a similar manner to Step 1, Example 3.

[0122] [ka]

[0123] Step 2: 4.2g of tert-butyl (R)-3-(N-(3-chloropyridin-2-yl)-3-cyanobenzamide)pyrrolidine-1-carboxylate, 1g of sodium azide, and 2g of triethylamine hydrochloride are added to 50ml of N,N-dimethylformamide, stirred at 100°C for 20 hours, cooled, poured into 200ml of water, and added with concentrated hydrochloric acid until the pH value reaches 2-3. The solid is filtered, washed with water, and dried. 50ml of dichloromethane and 10ml of methanol are dissolved, and 10ml of 4mol / L hydrogen chloride dioxane solution is added, stirred at room temperature overnight, and concentrated under reduced pressure. The solid obtained is subjected to silica gel chromatography to obtain 2.7g of (R)-N-(3-chloropyridin-2-yl)-N-(pyrrolidin-3-yl)-3-(1H-tetrazol-5-yl)benzamide. MS(ES+):370(M+H). Chemical formula: C17H16ClN7O Molecular weight: 369.81 Hydrogen Spectrum CDCl 3 Data at: 0.77(1H), 2.29(2H), 2.59(2H), 3.29(2H), 4.53(1H), 6.92(1H), 7.24(1H), 7.43(1H), 7.63(1H), 7.83(1H), 8.21(1H), 8.64(1H), 8.72(1H).

[0124] The compounds in Table 2 below can also be synthesized by referring to a similar method.

[0125] [Table 2-1]

[0126] [Table 2-2]

[0127] [Table 2-3]

[0128] [Table 2-4]

[0129] [Table 2-5]

[0130] [Table 2-6]

[0131] [Table 2-7]

[0132] Example 4: Synthesis of (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)-N-(tetrahydro-2H-pyran-3-yl)benzamide (abbreviation: CLY-42).

[0133] Step 1: Synthesis of (R)-3-chloro-N-(tetrahydro-2H-pyran-3-yl)pyridin-2-amine.

[0134] [ka]

[0135] Add 10.1g of (R)-tetrahydro-2H-pyran-3-amine, 14.0g of 2-bromo-3-chloropyridine, 12.0g of sodium tert-butoxide, and 100ml of tetrahydrofuran to a 250ml three-neck flask, under the protection of nitrogen gas, start stirring, add 0.1g of palladium acetate, 1,1'-bian-2-naphthol, and tris(dimethylamino)phosphine, heat to 65℃, react for 12 hours, then stop the reaction, pour into 500ml of water, extract with 1000ml of ethyl acetate in several portions, combine with the ethyl acetate layer, wash with 200 * 3 times of water, collect the ethyl acetate layer to obtain a paste-like residue, mix with silica gel, and obtain about 8g of (R)-3-chloro-N-(tetrahydro-2H-pyran-3-yl)pyridin-2-amine by silica gel column chromatography.

[0136] Step 2:

[0137] [ka]

[0138] 5g of 4-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)benzoic acid was dissolved in 50ml of dichloromethane, 2.5ml of oxalyl chloride and 0.2ml of catalyst N,N-dimethylformamide were added, and the mixture was reacted at room temperature until clarification. The solvent was collected under reduced pressure, and the residue was added to 50ml of tetrahydrofuran solvent, and the product (R)-3-chloro-N-(tetrahydro 2H-pyran-3-yl)pyridin-2-amine from the previous step was added to tetrahydrofuran solution of 4g. The mixture was cooled to below 5℃ in an ice bath, and 20ml of bis(trimethylsilyl)aminolithium 1M solution was added. The mixture was stirred at room temperature overnight and reacted, then poured into water, extracted with 100ml*3 of ethyl acetate, washed with water, and the residue was collected under reduced pressure. The (R)-4-(3H-[1,2,3 About 6 g of triazolo[4,5-b]pyridin-3-yl)-N-(3-chloropyridin-2-yl)-N-(tetrahydro-2H-pyran-3-yl)benzamide is obtained. MS (ES+): 435 (M+H).

[0139] Using a similar method

[0140] [ka]

[0141] (CLY-43) can also be synthesized.

[0142] Example 5: Synthesis of ethyl 1-(5-(4-(4-(3-chloropyridin-2-yl)((R)-pyrrolidin-3-yl)carbamoyl)phenyl)-1-methyl-1H-pyrazol-5-yl)-2H-tetrazol-2-yl)isobutyrate (abbreviation: CLY-44).

[0143] Step 1:

[0144] [ka]

[0145] (1) Synthesis of (R)-3-(((3-chloropyridin-2-yl)amino)pyrrolidine-1-carboxylate tert-butyl ester. Add 11.8g of (R)-1-tert-butoxycarbonyl-3-aminopyrrolidine, 11.0g of 2-bromo-3-chloropyridine, 10.0g of sodium tert-butoxide, and 70ml of toluene to a 250ml three-neck flask, under the protection of nitrogen gas, start stirring, add 0.1g of palladium acetate, 1,1'-bi-naphthalene-2-phenol, and tris(dimethylamino)phosphine, heat to 100℃, react for 7 hours, then stop the reaction, pour into 500ml of water, extract with 1000ml of ethyl acetate in several portions, combine with the ethyl acetate layer, wash with 200*3 times of water, collect the ethyl acetate layer to obtain a paste-like residue, mix with silica gel, and obtain about 7g of tert-butyl (R)-3-(((3-chloropyridin-2-yl)amino)pyrrolidine-1-carboxylate by silica gel column chromatography.

[0146] Step 2:

[0147] [ka]

[0148] At 0°C, 5g of tert-butyl (R)-3-((3-chloropyridin-2-yl)amino)pyrrolidine-1-carboxylate and 4g of 4-bromobenzoyl chloride in 100ml of tetrahydrofuran solution are added dropwise to 20ml of 1M lithium bis(trimethylsilyl)amidoyl solution, stirred overnight at room temperature, diluted with 300ml of ethyl acetate, washed with 200ml of water*3 times, and the ethyl acetate layer is concentrated under reduced pressure. The residue is subjected to column chromatography to obtain about 4g of tert-butyl (R)-3-(4-bromo-N-(3-chloropyridin-2-yl)benzamide)pyrrolidine-1-carboxylate.

[0149] Step 3: Preparation of (R)-tert-butyl 3-(N-(3-chloropyridin-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)pyrrolidine-1-carboxylate

[0150] [ka]

[0151] Add 3g of tert-butyl (R)-3-(4-bromo-N-(3-chloropyridin-2-yl)benzamide)pyrrolidine-1-carboxylate, 3g of bis(pinacolato)diboron, and 2g of potassium acetate to 30ml of 1,4-dioxane and stir. Under the protection of nitrogen gas, add 0.6g of palladium chloride, heat to 100°C, and stir for 5 hours. Concentrate under reduced pressure. Purify the residue by silica gel column chromatography to obtain 3.1g of tert-butyl (R)-3-(N-(3-chloropyridin-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)pyrrolidine-1-carboxylate.

[0152] Step 4: Synthesis of ethyl 1-(5-(4-(4-((3-chloropyridin-2-yl)((R)-pyrrolidin-3-yl)carbamoyl)phenyl)-1-methyl-1H-pyrazol-5-yl)-2H-tetrazol-2-yl)isobutyrate

[0153] [ka]

[0154] 3g of tert-butyl (R)-3-(N-(3-chloropyridin-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)pyrrolidine-1-carboxylate, 2.5g of 1-(5-(4-iodo-1-methyl-1H-pyrazol-5-yl)-2H-tetrazol-2-yl)ethyl isobutanoate, 5g of tris-potassium phosphate, and 0.1g of methanesulfonate (2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) were added to a 50% aqueous solution of 1,4-dioxane, and the mixture was reacted at 100°C for 5 hours under the protection of nitrogen gas. After the reaction mixture returns to room temperature, it is diluted with 200ml of ethyl acetate, the ethyl acetate layer is separated, washed with 100ml*3 of water, dried over anhydrous sodium sulfate, and the ethyl acetate is collected under reduced pressure to obtain the residue. The residue is dissolved in 30ml of dichloromethane, and 4ml of 4mol / L·1,4-dioxane hydrogen chloride aqueous solution is added, stirred for 2 hours, and the solvent is collected under reduced pressure until it becomes dry, and separated by silica gel chromatography to obtain about 0.5g of 1-(5-(4-(3-chloro-pyridin-2-yl)(((R)-pyrrolidin-3-yl)carbamoyl)phenyl)-1-methyl-1H-pyrazol-5-yl)-2H -tetrazol-2-yl)isobutyric acid ethyl ester. MS (ES+): 565 (M+H).

[0155] Using a similar method

[0156] [ka]

[0157] (CLY-45) can also be synthesized.

[0158] Example 6 Study of drug properties using rats 1. Experimental Method: Male SD rats were purchased and randomly assigned. The 12 rats used for each test compound were further randomly divided into two groups, six of which were intravenously administered (1 mg / kg) and blood samples were taken at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration, and plasma samples were separated. The other six were orally administered (5 mg / kg) and blood samples were taken at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration, and plasma samples were separated. The plasma concentrations of each compound were measured by LC / MS / MS, and the relevant pharmacokinetic parameters were calculated by Phocnix WinNonlin 6.2.1 software, and the bioavailability in rats was calculated to evaluate the pharmacokinetic properties of each compound.

[0159] 2. Experimental Results In all the above compounds, compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-5, CLY-8, CLY-11, CLY-14, CLY-17, CLY-18, CLY-19, CLY-21, CLY-26, CLY-28, CLY-30, CLY-32, CLY-35, CLY-36, CLY-37, CLY-44 were males. The bioavailability of the compounds was 69.8%, 72.6%, 69.5%, 59.5%, 53.8%, 78.2%, 63.5%, 52.3%, 52.5%, 46.9%, 66.3%, 49.6%, 48.5%, 42.6%, 46.2%, 58.6%, 62.2%, 68.6%, 56.5%, and 50.6% in male and female SD rats, respectively, indicating that the compounds have good pharmacokinetic properties.

[0160] Example 7 Effect of compound CLY on melasma model in guinea pigs 1. Experimental Method 1.1 Experimental materials 1.1.1 Reagents: Progesterone (20 mg / ml) was purchased from Shanghai General Pharmaceutical Co. Ltd., arbutin ointment was purchased from Shanghai Asia Pioneer Pharmaceutical Co. Ltd., tyrosine, malondialdehyde (MDA), and superoxide dismutase (SOD) kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0161] 1.1.2 Equipment: DY89-II electric glass homogenizer was purchased from Ningbo Xinlan Biotechnology Co., Ltd., and lineage biological microscope (Image-Pro Plus 6.0) was purchased from Media Cybernetics, USA.

[0162] 1.1.3 Experimental animals: SPF grade healthy purebred female guinea pigs, weighing (230±30) g, were obtained from Shanghai Slac Laboratory Animal Co. Ltd.

[0163] 1.1.4 Preparation method of therapeutic cream: The excipient matrix composition is methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), octadecanol (5%), glycerol (20%), alkylaryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl p-hydroxybenzoate (0.1%), distilled water (about 31-55%), mixed with an appropriate amount of CLY series compound to form a 0.25% mixed emulsion. The cream matrix used in this embodiment is the matrix composition of the cream from which the active ingredient has been removed.

[0164] 1.2 Animal grouping and modeling They were numbered according to body weight and randomly assigned to the following groups: model control group (cream matrix application), blank control group (cream matrix application), CLY-1 treatment group (0.25% CLY-1 cream applied to the skin), CLY-2 treatment group (0.25% CLY-2 cream applied to the skin), CLY-3 treatment group (0.25% CLY-3 cream applied to the skin), CLY-4 treatment group (0.25% CLY-4 cream applied to the skin), and The guinea pigs were divided into groups of six each: CLY-5 treatment group (0.25% CLY-5 cream applied to the skin), CLY-14 treatment group (0.25% CLY-14 cream applied to the skin), CLY-19 treatment group (0.25% CLY-19 cream applied to the skin), CLY-36 treatment group (0.25% CLY-36 cream applied to the skin), and positive treatment group (0.25% arbutin cream applied to the skin). Except for the blank control group, all guinea pigs were injected with 20mg / ml progesterone injection (7.5mg / kg) once a day at the base of the hind legs for 30 consecutive days to establish the melasma model. The model was successfully reproduced when the skin in the model area on the back of the guinea pig showed stable dark brown spots with clear and uniform junctions. After modeling, the guinea pigs in the model control group, blank control group, CLY series treatment group, and positive treatment group were applied with the corresponding cream on their backs once a day for 30 consecutive days.

[0165] 1.3 Observation indicators (1) Measurement of tyrosine, MDA content and SOD activity A piece of spare skin tissue was taken from each guinea pig, washed with pre-cooled physiological saline, and wiped dry to remove subcutaneous fat and other connective tissue. Each piece of skin tissue was then cut into 0.5g pieces and placed in five small test tubes containing 2.0ml of pre-cooled physiological saline, homogenized with a high-speed disperser at a speed of 10r / min for 10 seconds, and then centrifuged at a speed of 3500r / m for 15 minutes, and the supernatant was collected. Tyrosine was measured by high-performance liquid chromatography, MDA by the thiobarbituric acid method, and SOD by the xanthine oxidase method, and tyrosine, MDA content, and SOD activity were measured according to the kit instructions.

[0166] (2) Pathological and morphological observations of skin melanophores A spare skin tissue piece of approximately 2cm x 1cm was taken from each guinea pig, fixed with 10% paraformaldehyde, histopathological histology was measured, immunohistochemistry was performed, melanophores were stained and the number of cells was observed, and the positive cells were judged according to the literature: none: 0 points, less than 15%: 0.5 points, less than 30%: 1 point, more than 30%: 2 points. Each section was observed in five fields to find positive targets in the cytoplasm of epidermal cells and accessory epithelial cells of the skin that showed brown reaction, and then quantitatively analyzed using the BX50F4 Beihang Pathological Image Analysis System to calculate the average area of ​​melanin-positive targets in the five fields of view from each guinea pig, the ratio of targets to the area of ​​the statistical field (surface density), the ratio of the number of targets to the area of ​​the statistical field (total count density), the average gray scale, the average optical density, and the integrated optical density.

[0167] 1.4 Statistical methods SPSS16.0 software was used for statistics, and the measured values ​​were expressed as the mean ± standard deviation (x ± s). One-way analysis of variance was performed for comparisons between multiple groups, and t-tests were used for comparisons between groups. If P<0.05, it was considered that there was a significant difference.

[0168] 2. Experimental Results (1) Tyrosine, MDA content and SOD activity in guinea pigs of each group The results of tyrosine, MDA content and SOD activity in guinea pigs of each group are shown in Table 3. The tyrosine and MDA content of the skin of guinea pigs in the model group was higher than that of the blank group, and the SOD activity was lower than that of the blank group, indicating that the melasma model was successfully established. The tyrosine and MDA content of the skin of the CLY series treatment group and the positive treatment group was lower than that of the model group, and the SOD activity was increased, with statistically significant differences (P<0.05).

[0169] [Table 3]

[0170] (2) The area, number and color shade of melanophores in each group of guinea pigs The area, number and color of melanophores in each group of guinea pigs are shown in Tables 4 and 5. Compared with the blank group, the guinea pigs in the model group showed an increase in melanin deposition area, an increase in melanocyte number, an increase in optical density and an increase in color, and compared with the model group, the guinea pigs in the CLY series treatment group and the positive treatment group showed a decrease in melanin deposition area, a decrease in melanophore quantity, a decrease in optical density and a decrease in color.

[0171] [Table 4]

[0172] [Table 5]

[0173] 3. Experimental Conclusion CLY-1, CLY-2, CLY-3, CLY-4, CLY-5, CLY-14, CLY-19, and CLY-36 can treat melasma by increasing the activity of SOD enzyme in skin tissue, decreasing the content of tyrosine and MDA, inhibiting the tyrosinase activity of melanophores and melanoma cells, enhancing the redox reaction of skin cells, decreasing the production of free radicals, and suppressing the production of melanin.

[0174] Example 8: Effect of the CLY series of compounds on a rat scar model 1. Experimental Method 1.1 Preparation method of therapeutic cream: The excipient matrix composition is methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), octadecanol (5%), glycerol (20%), alkylaryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl p-hydroxybenzoate (0.1%), distilled water (about 31-55%), mixed with an appropriate amount of CLY series compound to form a mixed emulsion. The emulsion matrix used in this embodiment is the matrix composition of the emulsion from which the active ingredient has been removed.

[0175] 1.2 Experimental animal grouping and modeling: SPF grade male rats, weighing (210±28) g, were obtained from the Animal Center of Nanjing Medical University. They were numbered according to their body weight and randomly assigned to the following groups: model control group (cream matrix application), CLY-1 treatment group (0.5% CLY-1 cream applied to the skin), CLY-2 treatment group (0.5% CLY-2 cream applied to the skin), CLY-3 treatment group (0.5% CLY-3 cream applied to the skin), CLY-4 treatment group (0.5% CLY-4 cream applied to the skin), CLY-8 treatment group (0.5% CLY-8 cream applied to the skin), CLY-14 treatment group (0.5% CLY-14 cream applied to the skin), CLY-19 treatment group (0.5% CLY-19 cream applied to the skin), and CLY-36 treatment group (0.5% CLY-36 cream applied to the skin), with six mice in each group. Each group of rats was anesthetized with 2% pentobarbital sodium (120 mg / kg) by intraperitoneal injection and fixed to the operating table. After that, a 4 × 5 cm piece of intact skin was selected from the left side of the central back and depilated with 8% sodium sulfide. After that, a circular incision with a diameter of 2.4 cm was made at each depilated site with tissue scissors to a depth of fascia, and part of the superficial fascia was destroyed. To prevent the rats from biting or licking, the animals were kept in separate cages. The wounds were disinfected with 2% iodine tincture every day, and the rats were observed for wound healing.

[0176] 2. Experimental Results 2.1 Observation of rat wounds The wounds were disinfected regularly every day, and the wounds of the rats were observed on days 1, 3, 5, 7, 12, and 20. From day 5 onwards, the wound healing speed of each CLY series treatment group was found to be significantly faster than that of the model group, and the wound area gradually decreased. By day 12, the wounds of each treatment group had mostly healed, while the wounds of the model group were still about 0.5 cm2. 2 By day 20, all wounds had healed, with obvious scarring remaining in the model group and only variable amounts of pigmentation remaining in the treatment groups.

[0177] 3. Experimental Conclusion CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-14, CLY-19, and CLY-36 all significantly promote the healing of skin wounds and inhibit scar formation.

[0178] Example 9: Effect of CLY series on rat hair loss model 1. Experimental Method 1.1 Material (1) Method for preparing tinctures of compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-14, CLY-19, and CLY-36: Mix appropriate amounts of each of the above compounds with 75% ethanol to prepare tinctures of different concentrations. (2) Positive therapeutic drug: minoxidil 5% tincture (product name: Lundi, Zhejiang Wanma Pharmaceutical Co., Ltd.) (3) Experimental animals: SPF grade Wistar rats, male, obtained from Shanghai Slac Laboratory Animal Co. Ltd.

[0179] 1.2 Animal grouping and modeling Wistar rats were divided into four groups based on body weight: negative control group (75% ethanol application), model group (75% ethanol application), positive control group (5% minoxidil tincture application), CLY-1 application group (5% CLY-1 tincture application), CLY-2 application group (5% CLY-2 tincture application), CLY-3 application group (5% CLY-3 tincture application), CLY-4 topical group (5% CLY-4 tincture topical application), CLY-5 topical group (5% CLY-5 tincture topical application), CLY-14 topical group (5% CLY-14 tincture topical application), CLY-19 topical group (5% CLY-19 tincture topical application), CLY-36 topical group (5% CLY-36 tincture topical application), CLY-1 intravenous injection group (2 mg / kg.d), CLY-2 intravenous injection group (2 mg / kg.d), CLY-3 intravenous injection group (2 mg / kg.d), CLY-4 intravenous injection group (2 mg / kg.d), CLY-5 intravenous injection group (2 mg / kg.d), CLY-14 intravenous injection group (2 mg / kg.d), CLY-19 intravenous injection group ( The rats were randomly divided into two groups: 1) 10-mg / kg.d (2mg / kg.d), 2) 10-mg / kg.d (2mg / kg.d), and 3) 10-mg / kg.d (2mg / kg.d) intravenous injection of CLY-36. Before the experiment, a 4cmx5cm area was selected on the back of each rat to be depilated and used as the observation site. Except for the negative control group, the rats were subcutaneously injected with testosterone propionate injection [5 ml / (kg-d)] on the back of the neck once a day for 60 consecutive days to establish the SA model. After 4 weeks of subcutaneous injection of testosterone propionate, the rats gradually showed hair loss. The remaining hair became thin and brittle, proving that the alopecia model was successfully established. Simultaneously, testosterone was applied to the observation site on the back of the rats in the corresponding drug group at a dose of 2mL / (rat·time) twice a day with an interval of 8 hours. The negative control group and the model group were administered with 75% ethanol solution at a dose of 2mL / (rat·time) twice a day for 60 days.

[0180] 1.3 Observation indices and test methods Every 15 days after drug administration, 10 hairs were removed from the observation site on the back of each rat, and the length of the hair was measured with a vernier caliper. After 60 days of drug administration, the skin from the experimental observation site was collected and subjected to routine tissue dehydration, paraffin embedding, HE staining and light microscopy to observe the histopathological changes of the hair follicles and sebaceous glands of the rat skin. A semi-quantitative analysis of the lesions in each group was performed. The grading criteria are as follows: normal skin dermal tissue cells and the structure of subcutaneous hair follicles and sebaceous glands are recorded as "one". If the skin dermis does not show hyperplasia, the lesions of the hair follicles and sebaceous glands are not limited, and no subcutaneous inflammation is observed, it is recorded as "±". The skin dermal tissue has obvious hyperplasia, and the hair follicles and sebaceous glands are obviously cystic degeneration. There is obvious cystic change in the hair follicles. If there is no obvious hyperplasia in the sebaceous glands or subcutaneous inflammation, it is recorded as "+". The skin dermis tissue has segmental hyperplasia, which is not obvious, a small part of the hair follicles has hair follicle lesions, and the sebaceous glands have mild hyperplasia and hypertrophy. There is no obvious inflammation in the subcutaneous tissue, which is recorded as "++": the skin tissue cells in the dermis have different degrees of segmental hyperplasia, some hair follicles have cystic changes, the performance of the hair follicles is not uniform in size, and the peripheral part is acellular. There is hyperplasia in the sebaceous glands, the hyperplastic glands have fewer nuclei, and the subcutaneous tissue of individual rats has mild inflammatory hyperplasia, which is recorded as "+++".

[0181] 2. Experimental Results 2.1 Effect of CLY on hair growth in rats On the 15th, 30th, 45th and 60th day after drug administration, the hair length of the rats in each treatment group was longer than that of the model group, and the difference was statistically significant (P<0.01), and the difference was statistically significant (P<0.05) compared with the positive treatment group. See Table 6.

[0182] [Table 6]

[0183] 2.2 Effect of CLY on hair follicle morphology in the superficial dermis of skin tissue at observation sites in rats The dermal tissue cells of some of the skin of the rats in the model group had different degrees of segmental thickening, and mild lymphocytosis was observed in the subcutaneous tissue of the rats. Some of the subcutaneous hair follicles of the rats had obvious cystic degeneration, the size of the hair follicles was different, and the lumen of the enlarged hair follicles showed shedding of keratinized material. Mild fibrosis was observed in the periphery, the cells around the hair follicles disappeared or the cell level was obviously reduced, some calcified material in the lumen appeared to be stained blue, the number of sebaceous glands increased, some sebaceous glands were enlarged, the nuclei of the enlarged sebaceous glands were obviously reduced, and the number of normal hair follicles was reduced. The lesions of the skin dermal tissue cells and subcutaneous hair follicles and sebaceous glands of the rats in all treatment groups were alleviated to different degrees compared with the model group. The number of damaged hair follicles in the skin of the rats in each treatment group was significantly reduced compared with the model control group, and the difference was statistically significant (P<0.01). Compared with the model control group, the skin dermis tissue cells, subcutaneous hair follicles, and sebaceous gland lesions of rats in all treatment groups were significantly reduced, and the difference was statistically significant (P<0.01). Compared with the positive treatment group, the difference was statistically significant (P<0.05). See Table 7.

[0184] [Table 7]

[0185] 3. Experimental Conclusion CLY-1, CLY-2, CLY-3, CLY-4, CLY-5, CLY-14, CLY-19, and CLY-36 significantly promoted hair growth, reduced damage to subcutaneous hair follicles and sebaceous glands, and showed no significant side effects in a rat alopecia model when applied topically and systemically.

[0186] Example 10: Effect of CLY series on mouse alopecia model 1. Experimental Method 1.1 Material (1) Method for preparing tinctures of compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-11, CLY-19, and CLY-36: 60% ethanol and the above compounds were mixed to prepare tinctures with a concentration of 2%. (2) Positive treatment drug: 2% minoxidil tincture (manufactured by the Institute of Dermatology, Chinese Academy of Medical Sciences) (3) Experimental animals: SPF-grade healthy C57BL / 6 male mice, half sex, 6-8 weeks old, weighing 20-25 g, were provided by GemPharmatech (Chengdu) Co., Ltd.

[0187] 1.2 Animal grouping and modeling The animal house had a 12-12 hour day-night shift system, the animals were allowed to eat and drink freely, and the temperature was 23-25°C. The experimental animals were acclimatized in the animal house for one week before entering the experiment. The experimental mice were divided into 11 groups: negative control group (60% ethanol application), model group (60% ethanol application), positive control group (2% minoxidil tincture application), CLY-1 group (2% CLY-1 tincture application), CLY-2 group (2% CLY-2 tincture application), CLY-3 group (2% CLY-3 tincture application), CLY-4 group (2% CLY-4 tincture), CLY-8 group (2% CLY-8 tincture), CLY-14 group (2% CLY-11 tincture), CLY-19 group (2% CLY-19 tincture), and CLY-36 group (2% CLY-36 tincture). Each group consisted of 6 mice, half male and half female. A 4cm x 5cm area was selected on the back of the experimental mice, and hair was removed. The hairless area was painted yellow with 3% picric acid solution as the observation area for the hair removal experiment. Testosterone propionate injection [8ml / (kg·d)] was subcutaneously injected once a day for 60 consecutive days into the back of the neck of the mice in the other groups, except for the negative control group, to establish the SA model. Drugs were administered simultaneously with the modeling. The drug was applied twice a day at 1ml / (mouse·time) to the observation site on the back of the rats in the corresponding drug group with an interval of 2 hours. The normal control group and the model subject group were applied with the vehicle (60% ethanol solution) twice a day at 1ml / (mouse·time) with an interval of 2 hours for 60 consecutive days.

[0188] 1.3 Observation Indicators and Test Methods Every 15 days after drug administration, 10 hairs were removed from the observation site on the back of the mouse, and the hair length was measured with a vernier caliper. 60 days after drug administration, the skin of the experimental observation site was collected and subjected to routine tissue dehydration, paraffin embedding, HE staining and light microscopy to observe the histopathological changes of the mouse skin hair follicles and sebaceous glands. The lesions of each group were analyzed semi-quantitatively. The grading criteria are as follows: Normal skin dermal histiocytes, subcutaneous hair follicles and sebaceous gland structures are recorded as "one". Those without dermal hyperplasia, limited lesions of hair follicles and sebaceous glands, and no subcutaneous inflammation are recorded as "±". Those without obvious dermal hyperplasia, obvious cystic degeneration of hair follicles, no obvious hyperplasia of sebaceous glands, and no subcutaneous inflammation are recorded as "+". Segmental hyperplasia of the dermal tissue is not evident, a few hair follicles have follicular changes, the sebaceous glands have mild hyperplasia and hypertrophy, and there is no obvious inflammation in the subcutaneous layer. Segmental hyperplasia of the dermal tissue cells of the skin is marked as "++", with some hair follicles showing cystic changes, follicles of uneven size, absence of cells around the follicular area, hyperplastic sebaceous glands, few nuclei in the hyperplastic glands, and some mild inflammatory hyperplasia in the subcutaneous layer.

[0189] 2. Experimental Results 2.1 Effect of CLY series on mouse hair growth On the 15th, 30th, 45th and 60th day of administration, the hair length of the mice in all groups was longer than that of the model control group, and the difference was statistically significant (P<0.01), and the difference was statistically significant (P<0.05) compared to the positive treatment group. See Table 8. The hair growth of the mice in each group on the 30th day of drug administration is shown in Figure 1.

[0190] [Table 8]

[0191] 2.2 Effect of CLY on the morphology of hair follicles in the epidermis layer of skin tissue at the observation sites in mice In the model group, some of the mice's skin showed segmental thickening of dermal tissue cells to different degrees, and mild lymphoid hyperplasia was observed in the subcutaneous skin. Some of the mice's subcutaneous hair follicles showed obvious cystic degeneration, the size of the hair follicles varied, and the lumen of the enlarged hair follicles showed shedding of keratinized material. Mild fibrosis was observed in the periphery, cells around the hair follicles disappeared or the cell level was obviously reduced, some calcified material in the lumen appeared to be stained blue, the number of sebaceous glands increased, some sebaceous glands were enlarged, the nuclei of the enlarged sebaceous glands were obviously reduced, and the number of normal hair follicles was reduced. The lesions of the skin dermal tissue cells and subcutaneous hair follicles and sebaceous glands of the mice in all treatment groups were alleviated to different degrees compared with the model group. The number of damaged hair follicles in the skin of the mice in each treatment group was significantly reduced compared with the model control group, and the difference was statistically significant (P<0.01). Compared with the model control group, the skin dermis tissue cells, subcutaneous hair follicles and sebaceous gland lesions of the mice in the treatment group were significantly reduced, and the difference was statistically significant (P<0.01). See Table 9.

[0192] [Table 9]

[0193] 3. Experimental Conclusion CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-11, CLY-19, and CLY-36 significantly promoted hair growth and suppressed damage to subcutaneous hair follicles and sebaceous glands in a mouse model of alopecia.

[0194] Example 11: Effect of CLY series on rabbit ear acne model 1. Experimental Method 1.1 Material (1) Preparation method of therapeutic cream: The excipient matrix composition is methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), octadecanol (5%), glycerol (20%), alkylaryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl p-hydroxybenzoate (0.1%), distilled water (about 31-55%), mixed with an appropriate amount of CLY series compound to form a mixed emulsion. The emulsion matrix used in this example is the matrix composition of the emulsion from which the active ingredient has been removed. (2) Positive treatment drug: 0.1% adapalene gel (trade name: Daphne, manufactured by Galderma, France) (3) Experimental animals: general grade New Zealand rabbits, 1.8-2.1 kg, male, obtained from Shanghai Slac Laboratory Animal Co. Ltd.

[0195] 1.2 Animal grouping and modeling The New Zealand rabbits were numbered according to body weight and randomized to the following groups: model matched group (cream applied), blank matched group (cream applied), positive treatment group (Daphne applied to skin), CLY-1 topical treatment group (0.25% CLY-1 cream applied to skin), CLY-2 topical treatment group (0.25% CLY-2 cream applied to skin), CLY-3 topical treatment group (0.25% CLY-3 cream applied to skin), CLY-4 topical treatment group (0.25% CLY-4 cream applied to skin), CLY-8 topical treatment group (0.25% CLY-4 cream applied to skin), CLY-8 topical treatment group (0.25% CLY-2 cream applied to skin), 0.25% CLY-3 cream applied to skin), CLY-4 topical treatment group (0.25% CLY-4 ... applied to the skin), CLY-8 topical therapy group (0.25% CLY-8 cream applied to the skin), CLY-9 topical therapy group (0.25% CLY-9 cream applied to the skin), CLY-14 topical therapy group (0.25% CLY-14 cream applied to the skin), CLY-19 topical group (0.25% CLY-19 cream applied to the skin), CLY-36 topical group (0.25% CLY-36 cream was applied to the skin); CLY-1 intravenous injection group (1mg / kg.d), CLY-2 intravenous injection group (1mg / kg.d), CLY-3 intravenous injection group (1mg / kg.d), CLY-4 intravenous injection group (1mg / kg.d), CLY-8 intravenous injection group (1mg / kg.d), CLY-9 intravenous injection group (1mg / kg.d), CLY-14 intravenous injection group (1mg / kg.d), CLY-19 intravenous injection group (1mg / kg.d), CLY-36 intravenous injection group (1mg / kg.d), each group consisted of 10 mice. The right ear of the depilated rabbit was used as the observation site, and 95% alcohol was applied to the left ear of all rabbits as a self-negative control. 2% coal tar was applied evenly to the right ear of both the model group and the treatment group (2% coal tar solution prepared with 95% alcohol, manufactured by Alfa Aesar China Co., Ltd.) and applied evenly to the opening of the external auditory canal on the inner ear side of the rabbit once a day, 0.5 mL at a time, over an area of ​​approximately 2 cm x 2 cm with a sterile cotton swab. The previous application site was wiped with warm water, and the application was continued for 14 consecutive days to establish the acne microacne model. Local skin changes, including the thickness, hardness, roughness, and the presence or absence of black keratin plugs at the hair follicle opening, were observed with the naked eye.Eighteen hours after the last application, the test subjects were killed, and a 5 mm hole was punched at the application site, and the skin tissue was collected, fixed in 10% formaldehyde, embedded in paraffin, sectioned, stained with HE, and then subjected to histopathological observation and analysis.

[0196] 1.3 Observation indicators There are three grades based on the histological criteria of the acne model. Grade 0 (one) is loose keratinocytes in the infundibulum but no acne is produced. Grade 1 (+) is reddened skin on the surface of the rabbit's ear or a small amount of dense keratinized material in the infundibulum of the hair follicle, with no infundibulum dilation; Grade 2 (2+) is a moderate amount of dense keratinized material in the infundibulum of the hair follicle, extending toward the sebaceous gland, with hyperplasia of the sebaceous duct, and infundibulum dilation; Grade 3 (3+) is extensive keratinized material in the hair follicle, severe dilation of the hair follicle with dense keratin embolism in the hair follicle, marked hyperplasia of the sebaceous duct epithelium, raised and scarred skin, and degenerative changes in the sebaceous gland.

[0197] Histopathological changes were observed under a microscope, and the epidermal thickness at five points in one section was measured using the image analysis system Biomias99 and the average value was calculated. The areas of two hair follicles and the diameters of four sebaceous glands in the four sections where the structure was best preserved at the same position were measured and the average values ​​for each were calculated. The data for the left and right external auditory canals of each group were subtracted to determine the differences in epidermal thickness, hair follicle area, and sebaceous gland diameter between the left and right ears of each group of rabbits.

[0198] 1.4 Statistical methods For statistics, SPSS16.0 software was used, and paired t-tests were performed for left-right contrast and t-tests for comparison between groups. If P<0.05, it was considered that there was a significant difference.

[0199] 2. Experimental Results Visual Observation: 14 days after application of coal tar, the skin of the left ear of all rabbits was soft, the hair follicle openings of the ear canal were in good order, and no acne, pimples, or pustules were observed. The right ear of the rabbits in the model control group was thickened after application of coal tar, the skin became hard, the surface was rough, most of the hair follicle openings had black spots and pimples, it was hard to the touch, and some had healed into one piece. The right ear of each topical treatment group showed mild roughness and thickening of the skin, and a small amount of black spots. In all intravenous injection groups, most of the hair follicle papules in the right ear of the rabbits had subsided, the skin became thin and soft, the acne was significantly reduced, the pores were significantly reduced, no desquamation was observed, and the condition was basically close to that of a normal rabbit ear. The right ear of the rabbits in the positive treatment group showed mild redness of the skin, some peeling, and a small amount of acne compared to the left ear.

[0200] Histological section observation: The left ear of the model group had a thin epidermis, hair follicles could be seen, and the junction between the dermis and epidermis was clear. The right ear of the model group showed thickening of the epidermis, hyperkeratosis, thickening of the granular layer and spinous layer after the model was established, keratin plugs blocked the mouths of the hair follicles, the hair follicles were enlarged and extended to the sebaceous glands, the infundibulum of the hair follicles was full of keratinized material, and the infundibulum of the hair follicles was enlarged like a vase; the superficial layer of the dermis had dilated capillaries, inflammatory cells were scattered around the hair follicles, and a small amount of neutrophils was seen; the number of sebaceous glands increased, and the volume of the sebaceous glands expanded.

[0201] Histological evaluation of experimental acne under a microscope in each group (see Table 10): the difference between the right ear of the rabbit in the model group (experimental control) and its left ear (blank control) was statistically significant (P<0.05), indicating that the modeling of the rabbit ear acne model was successful; the comparison between the right ear of the rabbit in each treatment group and the right ear of the rabbit in the model group showed that the difference was statistically significant (both P<0.05), indicating that the positive control group and each treatment group could improve acne acne skin damage.

[0202] Table 10. Histological grade of acne in each group As shown in Table 11, the epidermal thickness, hair follicle visualization area, and

[0203] [Table 10]

[0204] The epidermal thickness, hair follicle visualization area and sebaceous gland diameter in the right ear of rabbits in each treatment group were all decreased compared with those in the right ear of rabbits in the model group, with statistically significant differences (P<0.05), suggesting that the positive control group and each treatment group could improve the pathological damage of acne skin.

[0205] [Table 11]

[0206] 3. Experimental Conclusion Compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-9, CLY-14, CLY-19 and CLY-36 all significantly alleviated acne symptoms and inhibited clogged pores and pimple formation in rabbit ear acne models, demonstrating significant therapeutic effects against acne.

[0207] Example 12 Suppression of psoriasis-like inflammatory responses in mice by the CLY series of compounds

[0208] 1, Material: Positive medication (glucocorticoid drug): Mometasone furoate cream (Erozone), product of Schering-Plough Ltd (Shanghai). Animals: SPF grade healthy inbred mice (C57BL / 6); 8 weeks old. Preparation of CLY cream: The substrate composition was methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), octadecanol (5%), glycerol (20%), alkylaryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl p-hydroxybenzoate (0.1%), distilled water (about 31-55%), and an appropriate amount of CLY series compound liquid to form a mixed emulsion.), mixed with an appropriate amount of CLY series compound to form a mixed emulsion.

[0209] The cream base used in this example is a cream base composition from which the active ingredient has been removed.

[0210] 2. Experimental method: (1) Eight-week-old SPF grade female C57BL / 6 mice were purchased and randomly divided into blank control group, model group, positive control group (application of Erozon cream), CLY-1 treatment group (application of 0.5% CLY-1 cream), CLY-2 treatment group (application of 0.5% CLY-2 cream), CLY-8 treatment group (application of 0.5% CLY-8 cream), CLY-14 treatment group (application of 0.5% CLY-14 cream), CLY-19 treatment group (application of 0.5% CLY-19 cream), and CLY-36 treatment group (application of 0.5% CLY-36 cream), each group consisting of 5 mice. Pentobarbital sodium 80 mg / kg was intraperitoneally injected for anesthesia, and the hair on the back was shaved to an area of ​​approximately 2 cm x 3 cm, and the mice were kept in independent cages for 1 day. (2) Vaseline was applied to the blank control group, and 62.5 mg of 5% imiquimod cream was applied to the backs of the model group, positive control group, and CLY treatment group at the same time each day for six consecutive days. Photographs were taken every day, and PASI scoring was performed. (3) On the first day of modeling, the blank control group and model group were applied with cream matrix twice a day, and the treatment group was applied with 0.5% CLY series cream twice a day.

[0211] 3. Experimental results: (1) As shown in Figure 2, after 6 consecutive days of application of 5% imiquimod cream, all mice in the model group showed obvious erythema, scales, and infiltration at the application sites on the backs of mice in each CLY treatment group, whereas the erythema, scales, and infiltration at the application sites on the backs of mice in each CLY treatment group were significantly milder than those in the model group, and the erythema, scales, and infiltration in the treatment groups were close to those in the positive drug treatment group. This indicates that the CLY series compounds can significantly suppress inflammatory responses in psoriasis-like model mice.

[0212] Example 13: Inhibition of inflammatory responses in mouse eczema models by CLY series compounds 1. Experimental materials: Ovalbumin (OVA): Prepare 20 g / L in PBS and store at -20°C. Calcipotriol topical solution (product name: Darex topical solution): a product of Leo Pharmaceutical Products. Positive medication (glucocorticoid drug): Mometasone furoate cream (Erozone), product of Schering-Plough Ltd (Shanghai). Preparation of CLY cream: The matrix composition is methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), octadecanol (5%), glycerol (20%), alkylaryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl p-hydroxybenzoate (0.1%), distilled water (about 31-55%), and contains an appropriate amount of CLY series compound liquid to form a mixed emulsion. ), mixed with an appropriate amount of CLY series compound to form a mixed emulsion. The cream matrix used in this example is the matrix composition of the cream from which the active ingredient has been removed. Animals: SPF grade healthy inbred mice (C57BL / 6); 8 weeks old.

[0213] 2. Experimental method: Eight-week-old (0.02 kg) SPF grade female C57BL / 6 mice were purchased and randomly assigned to blank control group, model group, positive drug group, CLY-1 treatment group (0.1% CLY-1 cream topical application), CLY-2 treatment group (0.1% CLY-2 cream topical application), CLY-3 treatment group (0.1% CLY-3 cream topical application), CLY-4 treatment group (0.1% CLY-4 cream topical application), CLY-8 treatment group (0.1% CLY-8 cream topical application), CLY-14 treatment group (0.1% CLY-8 cream topical application), CLY-14 treatment group (0.1% CLY-3 cream topical application), CLY-19 treatment group (0.1% CLY-19 cream topical application) and CLY-36 treatment group (0.1% CLY-36 cream topical application), with 6 mice in each group.

[0214] Modeling: 14.3 ul of 75% ethanol was applied to both ears of normal control mice, and 14.3 ul of 1 nmol / L calcipotriol was applied to both ears of model group, positive drug group and each treatment group at a fixed time every day, then air-dried, and 25 ul of 20 g / L OVA was applied once a day, and modeling was performed for 12 consecutive days.

[0215] Starting from 4 days after the start of modeling, cream matrix was applied to the ear skin of mice in the blank control group and model group, Eloxon was applied to the ear skin of mice in the positive drug group, and treatment cream was applied to the ear skin of mice in each treatment group twice a day, morning and evening, for 10 consecutive days, and photographs were taken and scored every day.

[0216] The thickness of the mouse's ears was measured with an ear thickness gauge before modeling and on day 14, and the thickness of the mouse's outer ear was recorded. On day 14 after the measurement, the mouse was killed by the neck, and blood was collected and serum was separated.

[0217] The rabbit anti-mouse interleukin (IL)-4 antibody was sealed in an enzyme-labeled plate, left overnight at 4°C, and stained according to the instructions of the ELISA kit to terminate the reaction and detect serum IL-4 levels. All ELISA kits were purchased from Raybiotech, USA.

[0218] 3. Experimental results: (1) Comparison of mouse ear thickness: Before modeling, the difference in ear thickness between each group was not statistically significant (P>0.05). After modeling, the ear thickness of each group of mice is shown in Table 12. The model group was significantly higher than each treatment group, the positive drug group and the blank control group (all P<0.01), and the difference between the positive drug group and each treatment group was not statistically significant (all P>0.05).

[0219] [Table 12]

[0220] (2) Serum IL-4 concentration: Before modeling, the difference in serum IL-4 concentration between each group was not statistically significant (P>0.05). The serum IL-4 concentration in the peripheral blood of mice in each group after modeling is shown in Table 13. The model group was more significant than the other groups (all P<0.01), and the difference between the positive drug group and each treatment group was not statistically significant (P>0.05).

[0221] [Table 13]

[0222] 4. Experimental conclusions: Compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-14, CLY-19, and CLY-36 all suppressed inflammatory responses in a mouse model of eczema by reducing serum IL-4 levels.

[0223] Example 14 Effect of CLY series compounds on mouse graft-versus-host disease model 1. Experimental Method 1.1 Experimental animals: SPF grade male C57BL / 6 mice (H-2b) and female BALB / c mice (H-2d), aged 6-8 weeks, weighing 16-21 g. All experimental mice were housed in SPF grade isolation cages and provided with sterilized food and bedding. They were kept for 1 week before being used in the experiments.

[0224] 1.2 Animal grouping and modeling Preparation of bone marrow cells: Donor mice were killed by breaking their necks, and immersed in 75% ethanol by volume for 5 minutes. Two femurs and tibias of the mice were removed under aseptic conditions on an ultraclean table and placed in RPMI1640 medium containing 2% fetal bovine serum by volume. The bone marrow cavity was washed with RPMI1640 medium, and bone marrow cells were removed. The bone marrow suspension was blown into a sterile washer tube, filtered through a 200 mesh filter, and centrifuged at 1,500 r / min for 10 minutes to recover the cell precipitate, which was then resuspended by adding saline. In addition, a small amount of the cell suspension was taken, and red blood cells were removed with red blood cell lysis solution, after which the amount of nucleated cells was counted. The nucleated cell concentration in the bone marrow suspension was then adjusted to 5×1010 L-1.

[0225] Preparation of splenic mononuclear cells: The splenic foreskin was cut with sterile scissors, placed on a 200 mesh filter, and immersed in RPMI1640 medium containing 2% fetal bovine serum by volume. The spleen was lightly crushed with the handle of a sterile syringe, the filter was rinsed with medium several times, the filtered splenic cell suspension was collected, and centrifuged at 1 500 r / min for 10 minutes to collect the splenic cell precipitate. After resuspension in PBS, the single cell suspension was blown, lightly placed on the upper layer of mouse splenic lymphocyte separation fluid, and centrifuged at 1 800 r / min for 20 minutes to collect the cells from the white blood cell layer. The cells were washed twice with PBS, counted, and the cell density was adjusted to 5 × 1010 L-1, and stored in a refrigerator at 4 °C for storage.

[0226] C57BL / 6 mice were used as bone marrow transplant donor mice, and BABL / c mice were used as recipient mice. BABL / c mice were numbered according to body weight and randomly divided into a control group, a model group, and a CLY series treatment group (each group was intravenously injected with the corresponding CLY compound at 5 mg / kg.d), with six mice in each group. BALB / c mice were given sterile acidic water containing 200 mg / L gentamicin one week before transplantation, and 8.5 Gy total body irradiation (cesium source) was given within 24 hours before transplantation. To construct acute GVHD model mice, 0.5 mL of a mixture of C57BL / 6 mouse bone marrow nucleated cells (containing 1 × 107 cells) and splenic mononuclear cells (containing 5 × 106 cells) was injected into the tail vein of the model group and each CLY series treatment group, and 0.5 mL of saline was injected into the tail vein of the control group BABL / c mice. At the same time, the CLY series treatment group was intravenously injected with the CLY series compound solution at 5 mg / kg / d, and the other groups were intravenously injected with saline at 5 mg / kg / d.

[0227] 1.3 Observation indices and test methods 1.3.1 Mouse GVHD symptom score: To determine whether the mice had acute GVHD, the general condition of the mice, including body size, fur, posture, mobility, diarrhea, and mental state, was observed every day after transplantation. According to the clinical GVHD symptom scoring criteria, the clinical GVHD symptoms of the mice in each group were scored on the 11th day after transplantation.

[0228] [Table 14]

[0229] 1.3.2 Mouse survival time analysis: Survival data of mice in each group was collected 3 weeks after transplantation, and the survival curve was plotted using the Kaplan-Meier method. The difference in survival rate of mice in each group was compared using log-rank analysis.

[0230] 1.3.3 Histopathological examination: Two weeks after transplantation, three mice with GVHD were selected from each group, and their dorsal skin tissues, liver tissues, and small intestine tissues were collected and fixed with 40 g / L paraformaldehyde, embedded in paraffin, and stained with hematoxylin-eosin. The pathological changes in the tissues were observed under an optical microscope.

[0231] 2. Experimental Results 2.1 Effects of CLY series compounds on clinical symptoms and survival time of acute GVHD mice

[0232] 2.1.1 Clinical symptoms: Mice in the irradiated control group showed weight loss and decreased activity, but did not develop GVHD symptoms. In the acute GVHD group, mice began to show symptoms such as arched back, significant weight loss, decreased activity, tremors, and diarrhea from the sixth day after transplantation. Although GVHD symptoms also developed in the CLY series treatment group, the symptoms were significantly alleviated. The clinical symptom scoring results on the 11th day after transplantation were 7.48±0.77 in the acute GVHD group, and 5.28±0.53, 4.68±0.42, 4.91±0.46, 4.56±0.39, 5.06±0.41, and 4.93±0.39 in the CLY-1, CLY-2, CLY-5, CLY-8, CLY-11, and CLY-36 groups, respectively. The clinical symptom scores in the CLY series treatment groups were lower than those in the acute GVHD group (P<0.01).

[0233] 2.1.2 Survival of mice: The survival curves were plotted by Kaplan-Meier method, and the survival rates of mice in each group were compared by Log-rank analysis. The survival rates of mice in the acute GVHD group, CLY-1 group, CLY-2 group, CLY-5 group, CLY-8 group, CLY-11 group, and CLY-36 group were 12.13%, 61.26%, 68.36%, 58.79%, 63.13%, 62.60%, and 67.75%, respectively. Compared with the acute GVHD group, all CLY series groups showed a prolonged survival time of mice (P < 0.01).

[0234] 2.1.3 Pathological changes in skin, liver and small intestine tissues: Two weeks after transplantation, the skin, liver and small intestine tissues of mice that developed GVHD symptoms were collected and stained with hematoxylin-eosin to observe the histopathological changes. The skin tissues of the mice in each group were thinned, and no obvious inflammatory cell infiltration was observed; the pathological sections of the livers of the mice in the acute GVHD group showed multiple necrotic foci and a large number of inflammatory cells infiltrated, while the livers of the mice in the CLY series compound group only showed small necrotic foci, and a small number of inflammatory cell infiltrations or inflammatory factor infiltrations into the hepatic duct area were observed, but these were significantly reduced compared with the mice in the acute GVHD group.

[0235] 3. Experimental Conclusion The CLY series compounds CLY-1, CLY-2, CLY-5, CLY-8, CLY-11, and CLY-36 all significantly extended the survival time and alleviated clinical symptoms in mice with acute GVHD, demonstrating therapeutic efficacy against acute GVHD.

[0236] Example 15: Effect of CLY series compounds on a rat pulmonary fibrosis model 1. Experimental Method 1.1 Materials: (1) Reagents: bleomycin (4 mg / capsule, Tianjin Taihe Pharmaceutical Co., Ltd), mouse anti-mouse MMP monoclonal antibody (NEO Mark-ers), mouse anti-mouse TIMP-1 polyclonal antibody (Wuhan Boster Biological Technology., LTD), enzyme-linked immunosorbent assay (ELISA) kit (R&D, USA), Quantscript RT Kit reverse transcription kit (Dalian TaKaRa). (2) Experimental animals: SPF grade Wistar rats, half male and half female, 51-55 days old, weighing (180±21) g, were obtained from the Animal Center of Nanjing Medical University.

[0237] 1.2 Animal grouping and modeling The rats were numbered according to their body weight and divided into blank control, model and CLY series treatment groups using a randomized allocation table method, with 12 rats in each group, half male and half female. Each group was anesthetized with 2% sodium pentobarbital (120 mg / kg) by intraperitoneal injection, fixed on the operating table and injected through a cervical tracheotomy. The blank control group was injected with saline (1.25 ml / kg), while the model and CLY treatment groups were each injected with 5 U / mL bleomycin solution (5 mg / kg) once a day for 14 consecutive days. Starting one week after modeling, each treatment group was injected with the corresponding CLY compound solution (3 mg / kg.d) via the tail vein, while the blank control and model groups were injected with the same amount of saline once a day for 14 consecutive days via the tail vein.

[0238] 1.3 Observation indices and test methods Peripheral venous blood was collected from the tail vein after modeling and 14 days after treatment in each group, and the concentrations of superoxide dismutase (SOD) and catalase (CAT) in peripheral blood were measured. After blood collection in each group, the rats were sacrificed twice (after modeling and 14 days after treatment), and the right lung tissue was collected for VEGF measurement and stored in a refrigerator at -4°C, while the left lung tissue was routinely paraffin-embedded and sliced, and the expression of MMP isoforms and TIMP-1 was measured in the lung tissue of the rats by immunohistochemical staining. The right lung tissue was collected and polished, tissue homogenized, and the supernatant was collected after high-speed centrifugation at 3000 r / min, and the VEGF protein in the lung tissue was measured by ELISA, and the VEGF-mRNA expression was measured by reverse transcription polymerase chain reaction assay.

[0239] 2. Experimental Results 2.1 Effect of CLY series compounds on MMP in rat lung tissue TIMP-1 and MMP isoforms were both expressed in small amounts in the lung tissues of rats in the blank control group. The expression of MMP-2 and MMP-9 in rats in the model group increased after modeling, and the expression of TIMP-1 decreased, and the differences were statistically significant compared with the blank corresponding group (P<0.05), indicating that the modeling was successful. In the CLY series compound group, the expression of MMP-2 and MMP-9 decreased, and the expression of TIMP-1 increased, and the differences were all statistically significant compared with the model group (all P<0.05). See Table 15.

[0240] [Table 15]

[0241] 2.2 Effect of CLY series compounds on VEGF in lung tissue The expression levels of VEGF protein and VEGF-mRNA in the lung tissue of rats in each group are shown in Table 16. The expression levels of VEGF protein and VEGF-mRNA in the model group were significantly decreased, and the difference was statistically significant (P<0.05) compared with the blank control group, indicating that the modeling of the pulmonary fibrosis model was successful. The expression levels of VEGF protein and VEGF-mRNA in the CLY series compound group were all statistically significant (P<0.05) compared with the model group.

[0242] [Table 16]

[0243] 2.3 Effects of CLY series compounds on SOD and CAT enzyme activities in peripheral blood The amount of SOD and CAT enzymes in the peripheral blood of rats in each group is shown in Table 17. The SOD and CAT enzyme activities in the peripheral blood of rats in the model group were decreased, and the difference was statistically significant compared with the p-brass control group; the SOD and CAT enzyme activities in the peripheral blood of rats in the CLY series compound group were increased, and the difference was statistically significant compared with the model group (P<0.05).

[0244] [Table 17]

[0245] 3. Experimental Conclusion Compounds CLY-1, CLY-2, CLY-8, CLY-14 and CLY-36 all significantly reduced the levels of MMP-2 and MMP-9, increased the levels of TIMP-1 and VEGF in the lung tissue of pulmonary fibrosis model mice, and also increased the levels of SOD and CAT enzymes in the peripheral blood, demonstrating an inhibitory effect on pulmonary fibrosis.

[0246] Example 16: Improvement of joint symptoms and inflammation indices in rheumatoid arthritis mice by CLY series compounds

[0247] 1. Experimental materials and methods (1) Experimental materials Freund's Adjuvant Complete (FCA, purchased from Sigma, USA); IL-10 ELISA kit (Multisciences (Lianke) Biotech, Co., Ltd); IL-17 ELISA kit (Raybiotech, USA).

[0248] (2) Grouping and treatment of experimental animals Eighteen 5-week-old female Wistar rats were randomly divided into a control group, an RA model group, and a CLY series compound treatment group, with six rats in each group. In the control group, the right toes of the rats were disinfected with 75% alcohol, and 0.15 mL of saline was subcutaneously injected into the right metatarsal of the rats. In the RA model group, on the first day of the experiment, each rat was disinfected as usual, and 0.15 mL of FCA was subcutaneously injected into the right metatarsal of the rats. In the CLY series compound treatment group, 0.15 mL was subcutaneously injected into the right sole of the rats from the first day of the experiment, and from 7 days later, the CLY series compound was orally administered intragastrically once a day at a dose of 10 mg / kg. The RA model group and the control group were orally administered intragastrically saline (10 mg / kg.d) for 21 consecutive days. On the 21st day after the intervention with the CLY series compounds, 3 mL of blood was collected from the rat's heart, serum was separated, and IL-10 and IL-17 levels in the serum were measured by ELISA.

[0249] (3) Observation indicators After 21 days of intervention, the rats in each group were scored for arthritis and the swelling of the right toe joints in each group were measured. The severity of the ankle was scored on a scale of 0 to 4 according to the inflammatory response of the RA rats. The severity of the ankle was scored on a scale of 0 to 4: normal, 0 points; mild redness and mild swelling in the ankle, 1 point; erythema and mild swelling from the ankle to the metatarsophalangeal joint or metacarpophalangeal joint, 2 points; erythema and moderate swelling from the ankle to the metacarpophalangeal joint or metatarsophalangeal joint, 3 points; severe redness and swelling from the ankle to the toe joint, 4 points. The scores of the limbs of each rat were summed up to form the arthritis score, with a maximum score of 16 points.

[0250] II. Experimental results (1) General description of rats Compared with the control group, the rats in the RA model group showed anorexia, depression, limited activity, and mild swelling of the left and right toes. Intervention with CLY series compounds was performed, and the symptoms improved compared with the other groups after 21 days.

[0251] (2) Body weight, joint swelling and arthritis scores of rats After 21 days of drug intervention, the body weight of the control group was higher than that of the other groups, and the difference was statistically significant (both P<0.05); the swelling degree and arthritis score of the right toe joint of the RA model group were higher than that of the control group, and the difference was statistically significant (P<0.05); the swelling degree and arthritis score of the joint of the CLY series compound treatment group were lower than that of the RA model group, and the difference was statistically significant (see Table 18).

[0252] [Table 18]

[0253] (3) Comparison of serum IL-17 and IL-10 concentrations in rats from each group The serum IL-10 concentrations of rats in the control group and the CLY series compound-treated group were higher than that of the RA model group (both P<0.05), and the serum IL-17 concentrations of rats in the control group and the CLY series compound-treated group were lower than that of the RA model group, and the differences were statistically significant (both P<0.05) (see Table 19).

[0254] [Table 19]

[0255] 3. Experimental conclusion The CLY series compounds can improve arthritis symptoms in rheumatoid arthritis mice by lowering IL-17 levels in peripheral blood and increasing inflammatory indicators such as IL-10.

Claims

1. 1. Use of a compound having the structure shown in Formula I, a tautomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and / or prevention of melasma, scarring, male pattern baldness, or graft-versus-host disease. 【Chemistry 1】 (however: R 1 is 3-chloropyridinyl; R 2 is hydrogen; R 3 teeth 【Chemistry 2】 and R 4 is a substituted or unsubstituted 5- to 6-membered cycloalkyl group, or a 4- to 7-membered heterocycle having 1 to 3 heteroatoms selected from N and O, and the substituents are hydrogen, —NH 2 , —OH, (C1-C4) alkyl, (C1-C4) alkoxy, amino, (C1-C4) alkylamino; however; R 6 and R 8 are each independently hydrogen, halogen, or (C-C) alkyl, provided that R 6 and R 8 are not both halogens at the same time; R 7 is hydroxy, (C1-C4)alkoxy, (C1-C4)alkoxycarbonyloxy(C1-C4)alkyl, or (C1-C4)alkylcarbonyloxy(Cl-C4)alkyl; R l0 and R 11 are each independently hydrogen, (C1-C4) alkyl, or (C3-C6) cycloalkyl; R 12 are hydrogen, halogen, -OH, -NH 2 or (C1-C3) alkyl; R 13 is hydrogen, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy(C1-C4) alkyl or (C1-C4) alkoxycarbonyloxy(Cl-C4) alkyl; R 14 is hydrogen, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy(C1-C4) alkyl or (C1-C4) alkoxycarbonyloxy(Cl-C4) alkyl; R 15 is hydroxy, tetrazolyl, (C1-C2) alkylsulfonyl or trifluoromethylsulfonyl; R 16 is hydrogen, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy(C1-C4) alkyl, or (C1-C4) alkoxycarbonyloxy(Cl-C4) alkyl.

2. R 3 but 【Transformation 3】 If R 4 teeth 【Chemistry 4】 and R 3 but 【Transformation 5】 If R 4 but 【Transformation 6】 2. The use according to claim 1, wherein

3. 2. The use according to claim 1, wherein the compound has the structure shown in formula Ia. 【Transformation 7】 where R 3 but 【Transformation 8】 If selected from 4 but 【Chemistry 9】 and R 3 but 【Chemistry 10】 In the case of R 4 but 【Chemistry 11】 and where R 6 , R 7 , R 8 , R l0 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 The limitations are consistent with claim 1.

4. R 3 but 【Chemistry 12】 If R 4 but 【Chemistry 13】 and R 3 but 【Chemistry 14】 If R 4 but 【Chemistry 15】 and where R 6 , R 8 , R l0 , R 11 , R 12 , R 13 The use according to claim 1, wherein the limitations of claim 1 are the same as those of claim 1.

5. R 6 is hydrogen or methyl, and R 8 The use according to any one of claims 1 to 4, characterized in that is hydrogen.

6. R 12 are hydrogen, halogen, -OH, -NH 2 The use according to any one of claims 1 to 4, characterized in that the aryl group is selected from the group consisting of aryl, aryl methyl ...

7. The use according to any one of claims 1 to 4, characterized in that the compound is selected from the following compounds: 【Chemistry 16-1】 【Chemistry 16-2】 【Chemistry 16-3】

8. A compound selected from the following: a tautomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof. 【Chemistry 17-1】 【Chemistry 17-2】 【Chemistry 17-3】

9. A pharmaceutical composition comprising the compound according to claim 8, its tautomer, its solvate or a pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and further comprising a pharmaceutically acceptable carrier.

10. 10. Use of a compound according to claim 8, a tautomer thereof, a solvate thereof or a pharmaceutically acceptable salt thereof or a composition according to claim 9 in the preparation of a medicament for the treatment and / or prevention of a disease.

11. 11. The use according to claim 10, wherein the disease is melasma, scarring, male pattern baldness, seborrheic alopecia, acne, ichthyosis, porokeratosis, perifollicular keratosis, psoriasis, eczema, atopic dermatitis, graft-versus-host disease, pulmonary fibrosis or rheumatoid arthritis.

12. The use according to any one of claims 1 to 7, 10 and 11, characterized in that the drug is administered in a dosage form suitable for any of the following administration modes: oral, parenteral, intraperitoneal, intravenous, intraarterial, topical, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, inhalation, vaginal, intraocular, topical, subcutaneous, intraadipose, intraarticular, intraperitoneal or intrathecal.

13. 13. The use according to claim 12, wherein the dosage form comprises an ointment, gel, emulsion, liniment, lotion, solution, spray, tablet, granule, oral liquid, capsule, drop, enema, film, or injection.

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