CLY series compounds, methods for preparing them, 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] This invention relates to the field of medicinal chemistry, specifically to CLY series compounds, methods for preparing them, and the uses of the prepared drugs. [Background technology]

[0002] Melasma is an acquired pigmentation disorder that commonly develops in middle-aged and adolescent women. Its etiology is extremely complex and involves many influencing factors, but the direct cause of melanin deposition in the skin due to various causes is melanin. Tyrosinase produces melanin through a series of oxidation reactions. Tyrosine is oxidized to dopa under the action of tyrosinase within melanin bodies, dopa is further oxidized to dopaquinone by dopa oxidase, and dopaquinone is finally oxidized under the action of tyrosinase to form melanin. Disruptions in this series of oxidation and antioxidant reactions can cause and promote the development and progression of melasma, and an increase in tyrosine is the main material basis for the development of melasma. When the balance of these oxidation reactions is disrupted, oxygen free radicals are excessively produced in the body, while the activity of antioxidant enzymes such as superoxide dismutase (SOD) decreases, leading to peroxidation of membrane lipids and the production of lipid peroxides. Lipid peroxides are unstable and rapidly decompose to produce aldehydes, which in turn increase the final product, malondialdehyde (MDA). MDA rapidly attacks phospholipids and proteins, causing oxidative damage to pigment cells, accelerating tyrosinase oxidation reactions, increasing melanin production, and depositing it in the basal layer of the skin. Therefore, reducing the amount of MDA in skin tissue and increasing 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 tend to recur. Hydroquinone is the earliest and most widely used skin whitening agent, but its application to skin whitening and melasma treatment is restricted because it causes uneven distribution of skin pigmentation, is highly irritating, and may even be carcinogenic. Arbutin is one of the most widely used skin whitening agents in clinical practice, but its effects are limited.

[0003] Scarring is damage to human skin and soft tissue caused by physical, biological, chemical, or other factors, resulting in serious damage to the skin and soft tissue that cannot be completely repaired by the body. Scarring causes great physical and mental distress to patients, and scars remaining after burns, severe injuries, and other traumas are particularly serious. Treating scarring is difficult; currently, it is only possible to soften and lighten red, hard scars, narrow wide scars, and thin thick scars, but it is not possible to completely eliminate scarring. Therefore, intervention in the early stages of wound healing is important, as it can effectively suppress scar formation, improve appearance, correct deformities, and restore function. Currently, commonly used methods for treating scars include surgery, laser treatment, cryotherapy, and drug therapy. Commonly used drugs include glucocorticoids and retinoids. Glucocorticosteroids have clear anti-fibrotic effects, but they 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, suppresses collagen synthesis, inhibits fibroblast DNA synthesis, and inhibits cell proliferation. The higher the concentration of retinoids, the more pronounced the inhibitory effect on proliferation becomes. However, retinoic acid has limited efficacy and systemic application carries a significant risk of toxic side effects. Topical application of retinoic acid causes clear skin irritation, and the irritation becomes more severe with increasing concentrations.

[0004] Alopecia areata (AA) is a non-scarring form of hair loss where the affected area of ​​skin is nearly normal. It typically presents as sudden, patchy hair loss, but in severe cases, it can affect the entire scalp, known as alopecia totalis (AT). If it affects all hair, including underarm and pubic hair, it is called alopecia universalis (AU), and can have a serious impact on the patient's appearance and psychological state. The etiology is not yet fully understood, but abnormalities or instability in the autoimmune system and neuropsychological factors are considered important contributing factors. While alopecia areata has a high chance of complete recovery, the likelihood of recovery varies greatly depending on the etiology. Some patients recover spontaneously, while others experience symptoms for several years. Minoxidil is a commonly used topical medication for alopecia areata, promoting vasodilation of the skin, improving local blood circulation, and stimulating hair growth. Glucocorticosteroids, commonly used for severe alopecia areata, primarily include prednisolone and betamethasone complex, and can be administered orally, topically, or intradermally. For patients unsuitable for glucocorticoids, immunosuppressant therapy is available. Commonly used drugs include cyclosporine, methotrexate, glucocorticosteroids, and immunosuppressants, but these drugs have many side effects.

[0005] Androgenetic alopecia (AGA), also known as seborrheic alopecia, is a common androgen-dependent hereditary hair loss disorder. It most commonly develops in men around the age of 20-30. Hair loss primarily occurs on the crown of the head, often starting at the hairline on both sides of the forehead, although some cases begin on the crown as well. The hair loss gradually spreads upward, and the hair becomes progressively thinner and less dense. Eventually, the hair on the crown falls out almost completely, but the hair on the back of the head and the upper sides of the temples remains, creating a horseshoe-shaped appearance with normal hair in this band-like area. The affected area is lighter in color, with shrunken pores or a few fine, soft vellus hairs remaining. The rate, extent, and severity of hair loss are influenced by genetics and individual differences. Generally, progression is fastest around the age of 30, and severe total alopecia is rare. In women, diffuse alopecia of the crown is more common, resulting in thinning hair on the crown of the head. According to epidemiological surveys in China, the prevalence of male pattern baldness is 21.3% in men and 6.0% in women. The etiology and pathogenesis of androgenetic alopecia are still not fully understood, but it is generally believed that androgens and their receptors play an important role in the development of this disease, and that type II 5α-reductase is a key factor in its onset. 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, and it is converted to dihydrotestosterone by 5α-reductase, the latter which can change terminal hairs into vellus hairs and ultimately cause hair loss. Currently, there is no ideal treatment, which is a challenge facing the treatment of alopecia-related diseases. Minoxidil is a nonspecific treatment for alopecia, a topical medication approved by the FDA as a first-line treatment for alopecia, but its use can cause hirsutism of the face and limbs, and the therapeutic effect gradually disappears when its use is discontinued. Since androgens play a significant role in the onset of AGA, recent new treatments attempt to terminate the miniaturization of hair follicles through anti-androgen effects. Finasteride is a type II 5α-reductase selective inhibitor and has recently been found to be effective in treating AGA, with sustained improvement in hair growth observed.However, finasteride has side effects such as sexual dysfunction, transient sperm count reduction, and male breast development abnormalities, and teratogenicity was also found in animal studies, so it was not used in pediatrics or for women of childbearing age. Cimetidine needs to be taken for more than 5 months, and side effects include male breast development, impotence, and decreased libido. Oral contraceptives mainly include sogonolone, levonorgestrel (levomethylnorethindrone), progesterone, norethindrone (oximenorethindrone), bisester norethindrone, and vinpocetine. These are often used to treat female androgenetic alopecia, and hair can be improved to some extent with 6 to 12 months of treatment.

[0006] Acne (commonly known as pimples) is a chronic inflammatory disease that commonly develops in the sebaceous glands of hair follicles, with a prevalence of approximately 9.4%. The development of acne is closely linked to the physiological and pathological changes of the skin during puberty. Clinical symptoms mainly include pimples, papules, pustules, nodules, cysts, and scars, which have a serious impact on the patient's appearance and psychology. Acne is associated with several pathological conditions, with abnormal keratinization of the hair follicle opening being a key basis for the formation of acne, and inflammation and infection being contributing factors to acne development. Acne patients have enlarged sebaceous glands, increased sebaceous gland secretion, and relatively low levels of linoleic acid in sebum, which affects lipid synthesis. This leads to a deficiency of fatty acids in the hair follicle epithelium, inducing hyperkeratosis of the hair follicles, preventing the epithelium from shedding normally, and the opening of the hair follicle sebaceous gland is too small, preventing sebum from being smoothly expelled, resulting in acne formation. When the sebaceous glands of hair follicles become blocked, a hypoxic environment is created within them, allowing anaerobic acne bacteria to proliferate in large numbers, break down sebum, produce chemokines, and cause white blood cells to accumulate, forming papules. Numerous neutrophils gather in the sebaceous glands of hair follicles, engulfing Propionibacterium acnes and triggering an inflammatory response. As a result, numerous suppurative cells accumulate, forming pustules and cysts, and making it easier for depressed scars to form after healing. Elevated androgen levels are an important part of promoting acne development, causing abnormal keratinization of the skin, blocking the sebaceous gland ducts of hair follicles, leading to bacterial retention and proliferation, and causing inflammation. Diseases with keratinization abnormalities similar to acne include ichthyosis, pilarid keratosis (also called lichen), Darier's disease, and porokeratosis. Pilarid keratosis is seen as enlargement of the hair follicle opening with keratin plugs, while ichthyosis presents with a decrease in sweat glands and sebaceous glands and keratin plugs within the hair follicles. The above-mentioned diseases are prone to recurrence and are difficult to treat. The main drugs used to treat keratinization disorders and remove comedones and acne are retinoids. Retinoids suppress keratinization, inhibit sebum secretion, promote normal keratinization of keratinocytes, and have immunomodulatory and anti-inflammatory effects, so they can suppress the formation of acne, papules, and pustules, and are widely used clinically to treat keratinization disorders such as acne, ichthyosis, pilarid keratosis, Darier's disease, and porokeratosis.However, topical retinoids tend to irritate the skin, causing redness, swelling, pain, and worsening of existing lesions. Long-term topical use of retinoids can lead to thinning of the skin, photosensitivity, and damage to the skin barrier. Oral retinoids have side effects such as liver damage and elevated blood lipids. Therefore, there is a clinical need for more medications to treat these conditions.

[0007] Psoriasis is a common chronic, relapsing inflammatory skin disease with a global prevalence of 0.1–3% in the natural population. Patients with moderate to severe psoriasis are at high risk of developing metabolic syndrome and cardiovascular complications. Therefore, early diagnosis and treatment of psoriasis are crucial for symptom control and improvement, as well as prevention of complications. Topical treatment is the first-line treatment for mild to moderate psoriasis. Topical glucocorticoids are more effective but not suitable for long-term, continuous, and widespread use; topical vitamin A acid treatment is more effective for plaque-type psoriasis, but care must be taken regarding skin irritation; vitamin D3 derivatives such as calcipotriol also have better efficacy but are not suitable for use on the face or skin folds; calcium-modulating nerve phosphatase inhibitors (tacrolimus, pimecrolimus, etc.) can be used on the scalp, skin folds, and genitals, but long-term, widespread use may increase the risk of lymphoma and skin cancer; various keratin-producing agents (e.g., tar preparations, anthraline ointment, 10-15% camptothecin ointment, salicylic acid ointment) can also be applied topically, but their effects are 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 moderately to severely ill patients, but long-term use often leads to side effects, including bone marrow suppression, liver dysfunction, kidney dysfunction, and an increased risk of infection.

[0008] Eczema (also known as atopic dermatitis or dystopic dermatitis) is an inflammatory skin reaction caused by various internal and external factors, characterized by intense itching and a tendency to recur. The causes of eczema are complex. Mild to moderate eczema is primarily treated with topical therapy. The appropriate dosage form and medication are used depending on the skin lesion. For subacute and chronic eczema, appropriate glucocorticoid creams, tar-based preparations, or immunomodulatory agents such as tacrolimus ointment or pimecrolimus ointment are used. Glucocorticoids and immunosuppressants can be used systemically in severely ill 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 allogeneic graft after transplantation. A series of cytokine storms initiated by the recipient significantly enhances the recipient's immune response to antigens, initiating cytotoxic attacks on target cells in the recipient, primarily the skin, liver, and intestines. The incidence of acute graft-versus-host disease is 30-45%, while the incidence in chronic cases is lower. Allogeneic hematopoietic stem cell transplantation is an effective treatment for curable hematological malignancies and hematopoietic dysfunctions. Acute graft-versus-host disease (GVHD) is a major complication, with high morbidity, mortality, and disability, and is a significant cause of non-relapse death in malignant hematological diseases. Teroids are the first-line treatment for acute GVHD, but approximately 50% of patients are hormonal resistant, making it difficult to control the GVHD response. Other second-line treatments for GVHD reactions, such as monoclonal antibodies against T cell surface antigens (CD3, CD25, etc.) and chemotherapeutic agents (mortimechloride, tacrolimus, etc.), demonstrate reliable efficacy against GVHD. However, subsequent immunodeficiency and opportunistic infections significantly diminish the benefits of these drugs, ultimately failing to extend patient survival. Therefore, finding new therapeutic methods to effectively control hormone-resistant acute GVHD is urgently needed.

[0010] Pulmonary fibrosis is a diffuse lung disease with an unknown cause and complex pathology, and is now recognized as a 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 key to its development. This condition is characterized by the aggregation of metalloproteinases (MMPs) in the extracellular matrix due to long-term chronic pneumonia and persistent damage to the alveoli, with MMP-2 and MMP-9 being abnormally increased and tissue metalloproteinase inhibitor-1 (TIMP-1) decreasing, disrupting the balance and leading to massive aggregation of the extracellular matrix in the lungs, histiocyte remodeling, and excessive collagen deposition. At the same time, it can inhibit the expression of vascular endothelial growth factor (VEGF) in tissues, reduce the permeability of pulmonary microvessels, inhibit the division and proliferation of vascular endothelial cells and vascular regeneration, worsen lung tissue damage, and ultimately lead to pulmonary fibrosis, a diffuse interstitial lung disease. Currently, there are no effective antifibrotic drugs, and glucocorticoid anti-inflammatory drugs are commonly used to suppress the fibrotic process, but their effects are limited and they have many side effects. Current treatments are far from meeting clinical needs, and there is a need to find more new drugs that are highly effective, have fewer side effects, and can slow disease progression, reduce relapses 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, systemic lupus erythematosus, dermatomyositis, scleroderma, Sjogren's syndrome, etc. When these diseases become severe, they invade multiple organs, causing disorders in the heart, liver, kidneys, blood vessels, lungs, joints, brain, etc., and showing a mortality rate second only to malignant tumors. Rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, and Crohn's disease have a common pathogenesis pathway. The etiology and pathogenesis of these diseases are very complex, and currently, they cannot be completely cured, and long-term drug therapy is required to suppress the progression of the disease. Commonly used therapeutic drugs in clinical practice mainly include glucocorticoids and immunosuppressive agents, etc. However, the effective rate of these drugs only stops at about 50%, and there are side effects such as bone marrow suppression, liver and kidney dysfunction, osteoporosis, induction of infectious diseases and tumors, etc., so long-term use is restricted. Current new biological preparations also have an immunosuppressive effect, have a risk of inducing infectious diseases and tumors, and are expensive, so long-term general use is restricted.

[0012] Regarding the above-mentioned diseases, the current treatment methods are far from meeting the clinical needs. In order to suppress the progression of the diseases and suppress the occurrence of recurrence and complications, it is necessary to further find new drugs with high efficacy, few side effects, and low cost.

Summary of the Invention

Problems to be Solved by the Invention

[0013] The object of the present invention is to provide a CLY series compound having medicinal efficacy or a pharmaceutically acceptable salt thereof. A further object of the present invention is to provide a preparation method of the above compound. A further object of the present invention is to provide the use of the above compound. The object of the present invention can be achieved by the following means: The present invention provides a compound having the structure shown in Formula I, its tautomer, its solvate or a pharmaceutically acceptable salt,

[0014]

Chemical formula

[0015] however: R1 is a substituted or unsubstituted 5-6 membered heterocycle, benzoheterocycle (e.g., isoquinolinyl), or dicyclic ring having at least two of the aforementioned heterocycles, where parallel rings refer to dicyclic rings where the two ring structures are formed by two adjacent atoms, e.g., pyrrolopyridine. The substituents are hydrogen, halogen, or (C1-C4)alkyl; R2 is hydrogen, halogen, hydroxyl, methoxy, ethoxy, amino, methyl, or ethyl; R3 is hydrogen, halogen, hydroxyl, methoxy, ethoxy, amino, (C1-C3) alkyl, or one of the following groups:

[0016] [ka]

[0017] and; R4 is a substituted or unsubstituted 5-6 membered cycloalkyl group, or a 4-7 membered heterocycle having 1-3 heteroatoms selected from N or O, wherein the substituent is selected from hydrogen, -NH2, -OH, (C1-C4)alkyl, (C1-C4)alkoxy, amino, and (C1-C4)alkylamino; Preferably, R4 is

[0018] [ka]

[0019] That is the case.

[0020] Preferably, R3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl, or one of the following groups:

[0021] [ka]

[0022] If so, R4

[0023] [ka]

[0024] And R3 is

[0025] [ka]

[0026] If so, R4

[0027] [ka]

[0028] and; however: R6 and R8 are each independently hydrogen, methyl, halogen, or (C1-C4)alkyl, provided that neither R6 nor R8 is a halogen at the same time. Preferably, R6 is hydrogen or methyl, and R8 is hydrogen; R7 is hydroxy, (C1-C4)alkoxy, (C1-C4)alkoxycarbonyloxy(C1-C4)alkyl, or (C1-C4)alkylcarbonyloxy(Cl-C4)alkyl. R l0 and R 11 Each of these is independently hydrogen, (C1-C4) alkyl, or (C3-C6) cycloalkyl; R 12 This 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 14is 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 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, and R l is optionally mono-substituted with chlorine or methyl.

[0030] In some embodiments, R2 is hydrogen, hydroxy, or methyl.

[0031] In some embodiments, R l is substituted or unsubstituted pyridinyl or pyrrolopyridinyl, and the substituent is hydrogen, chloro, or methyl.

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

[0033] The present invention provides a compound having the structure shown in Formula I-a, its tautomer, its solvate or a pharmaceutically acceptable salt,

[0034]

Chemical formula

[0035] wherein R3 is H, halogen, hydroxyl, methoxy, amino, methyl, or in the case of the following substituted or unsubstituted groups:

[0036] [ka]

[0037] R4 was selected from among them.

[0038] [ka]

[0039] and; However, R6, R7, R8, R l0 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 This limitation is the same as any one-level limitation mentioned above. Preferably, R3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl, or one of the following substituted or unsubstituted groups:

[0040] [ka]

[0041] If so, R4

[0042] [ka]

[0043] and; R3 is

[0044] [ka]

[0045] In this case, R4 is

[0046] [ka]

[0047] And, Here, R6 and R8 are independently hydrogen, methyl, halogen, or (C1-C4) alkyl, provided that neither R6 nor R8 is a halogen at the same time. Preferably, R6 is hydrogen or methyl, and R8 is hydrogen; R7 is hydroxyl, (C1-C4)alkoxy, (C1-C4)alkoxycarbonyloxy(C1-C4)alkoxy, or (C1-C4)alkylcarbonyloxy(Cl-C4)alkoxy. R l0 and R 11 Each of these is 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 R16 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 examples, the compound having the structure shown in formula Ia, its tautomer, its solvate, or pharmaceutically acceptable salt is such that R6 is H and R8 is H; In some examples, a compound having the structure shown in formula Ia, its tautomer, its solvate, or a pharmaceutically acceptable salt is R 12 R is selected from hydrogen, halogen, -OH, -NH2, or methyl; in some examples, R 12 It is selected from hydrogen.

[0049] In some examples, a compound having the structure shown in formula Ia, its tautomer, its solvate, or a pharmaceutically acceptable salt is R 11 H is R 10 is H or methyl, R 13 teeth

[0050] [ka]

[0051] That is the case.

[0052] This invention provides compounds having the structure shown in formula Ib, tautomers thereof, solvates thereof, or pharmaceutically acceptable salts thereof.

[0053] [ka]

[0054] However, if R3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl, or one of the following substituted or unsubstituted groups:

[0055] [ka]

[0056] And R4 is

[0057] [ka]

[0058] And R6, R8, Rl0 , R 11 , R 12 , R 13 This limitation is the same as any one-level limitation mentioned above.

[0059] Preferably, R3 is hydrogen, halogen, hydroxyl, methoxy, amino, methyl, or one of the following substituted or unsubstituted groups:

[0060] [ka]

[0061] If so, R4

[0062] [ka]

[0063] and; R3 is

[0064] [ka]

[0065] In this case, R4 is

[0066] [ka]

[0067] That is the case.

[0068] Here, R6, R8, R l0 , R 11 , R 12 , R 13 This limitation is the same as any one-level limitation mentioned above.

[0069] In some examples, a compound having the structure shown in formula Ib, its tautomer, its solvate, or a pharmaceutically acceptable salt is R 12R is selected from hydrogen, halogen, -OH, -NH2, or methyl, and in some examples, 12 It is selected from hydrogen.

[0070] In some specific examples, the present invention provides, but is not limited to, the following compounds, their tautomers, their solvates, or pharmaceutically acceptable salts thereof.

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] [ka]

[0075] The present invention also provides a method for preparing the compound represented by formula (I):

[0076] [ka]

[0077] raw material

[0078] [ka]

[0079] By preparing

[0080] [ka]

[0081] A generation is created, and

[0082] [ka]

[0083] and

[0084] [ka]

[0085] This generates the final product I, Here, R1, R2, R3, and R4 conform to the definitions above.

[0086] The present invention also provides a drug composition comprising a compound described in the present invention or a pharmaceutically acceptable salt thereof as an active ingredient or principal active ingredient, and comprising a pharmaceutically acceptable carrier.

[0087] The present invention also provides the use of the compounds described in the present invention in the preparation of drugs for therapeutic and / or preventive purposes.

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

[0089] The compounds or compositions described in the present invention are suitable for any pharmaceutically acceptable dosage form, such as oral, parenteral, intraperitoneal, intravenous, intra-arterial, transdermal, sublingual, intramuscular, rectal, transbuccal, transnasal, inhalation, vaginal, intraocular, topical, parenteral, cutaneous, subcutaneous, intrafat, intra-articular, intraperitoneal, or intrathecal administration.

[0090] In preferred embodiments, the dosage forms described in the present invention include gels, emulsions, creams, topical preparations, lotions, sprays, solutions, tablets, GranulesThese are oral solutions, capsules, drops, enemas, films, or injections.

[0091] Definitions in the invention:

[0092] "C5-C6 monohydric alcohols" refer to saturated aliphatic hydrocarbon groups containing 5 or 6 carbon atoms substituted with one hydroxyl group, and include both linear and branched groups.

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

[0094] "Alkyl" refers to a saturated aliphatic hydrocarbon group having 1 to 20 carbon atoms, including both linear and branched groups. (The numerical range referred to in this application, e.g., "1 to 4," means an alkyl group, which may include groups with 1, 2, 3, or 4 carbon atoms. Alkyl groups may or may not be substituted.)

[0095] "Cycloalkyl" refers to a monocyclic or densely packed ring ("packed" means that each ring in the system shares a pair of adjacent carbon atoms with the other rings in the system) that does not have a fully linked π-electron system of one or more rings. 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). Typical examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy.

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

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

[0099] "(C1-C4)alkoxycarbonyloxy(C1-C4)alkyl" represents (C1-C4)alkyl-OC(O)-O-(C1-C4)alkyl-. [Effects of the Invention]

[0100] The CLY series of compounds or their pharmaceutically acceptable salts are applicable to the pharmaceutical field. According to animal models, all compounds in the CLY series significantly reduce tyrosinase levels in the skin and blood of melasma model mice, decrease the expression of hepatic cytokines (SCFs) and C-kit proteins in the skin, suppress the formation of melasma pigmentation, significantly promote wound healing, and reduce scar formation. They can significantly promote hair growth and reduce androgen-induced hair follicle destruction in male pattern baldness mouse models. The CLY series can significantly suppress inflammatory responses in mouse models of psoriasis and eczema. The CLY series compounds can significantly improve the survival time and alleviate clinical symptoms in acute GVHD mice, demonstrating therapeutic effects against acute GVHD. The CLY series compounds significantly reduce MMP-2 and MMP-9 levels and increase TIMP-1 and VEGF levels in lung tissue of a mouse model of pulmonary fibrosis, while simultaneously increasing SOD and CAT enzyme levels in peripheral blood, demonstrating an inhibitory effect on pulmonary fibrosis. The 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. Due to the common pathogenesis of many diseases, the efficacy of these compounds is not limited to the diseases mentioned above. These compounds can be used alone or in combination with other drugs, providing a new drug option for treating the aforementioned diseases. [Brief explanation of the drawing]

[0101] [Figure 1] The CLY series compounds can significantly promote hair growth in alopecia model mice. [Figure 2] The CLY series compounds can significantly suppress psoriasis-like inflammatory responses in mice. [Figure 3] CLY-2 nuclear magnetic field detection results. [Figure 4] CLY-8 nuclear magnetic field detection results. [Modes for carrying out the invention]

[0102] The present invention will be further described below using examples. The specific examples described herein are for illustrative purposes only and are not intended to limit the present invention. Furthermore, it should be understood that simple improvements to the preparation methods of the present invention, based on the concept of the present invention, also fall within the scope of protection of the present invention. Experimental methods in the following examples where no specific conditions are indicated shall generally follow methods known in the art. Unless otherwise specified, the test materials used in the following examples can be purchased from a regular biochemical reagent store.

[0103] Example 1: Method for preparing (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-N-(3-chloropyridine-2-yl)-N-(1-methylpiperidine-3-yl)benzamide (abbreviated as CLY-1) and (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-N-(3-chloropyridine-2-yl)-N-(1-ethylpiperidine-3-yl)benzamide (abbreviated as CLY-2): 1. Synthesis pathway

[0104] [ka]

[0105] 2. Specific implementation methods (1) Synthesis of (R)-3-((3-chloropyridine-2-yl)amino)piperidine-1-formate tertbutyl (R)-1-Boc-3-aminopiperidine, 2-bromo-3-chloropyridine, sodium tert-butoxide, and dioxane are added to a 250 ml container bottle, stirred under the protection of nitrogen gas, RuPhosPd G3 and ligand RuPhos are added, heated to 100°C, and reacted for 7 hours. After stopping the reaction, the mixture is added to water and extracted in several stages with ethyl acetate. After combining the ethyl acetate layers, the mixture is washed three times with water to recover the ethyl acetate layer. The remaining paste is obtained and mixed with silica gel, and (R)-3-((3-chloropyridine-2-yl)amino)piperidine-1-formate tert-butyl is obtained by silica gel column chromatography.

[0106] (2) Synthesis of piperidineamide Add 4-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)benzoic acid to a container bottle, heat and stir at 30°C until clarified with pre-dried toluene, N,N-dimethylformamide, and thionyl chloride, and collect the solvent under reduced pressure to obtain a residual solid. Add 50 ml of pre-dried tetrahydrofuran and (R)-tert-butyl 3-((3-chloropyridine-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 from the ice bath, stir for 2 hours, pour into water, extract with ethyl acetate, wash the ethyl acetate layer, collect the solvent, and obtain a paste under reduced pressure.

[0107] (3) Synthesis of piperidineamide Piperidineamide, dichloromethane, and trifluoroacetic acid are stirred overnight at room temperature. The mixture is poured into water, sodium bicarbonate is added to adjust the pH to 10-11, and the mixture is extracted with dichloromethane. After washing, the solvent is recovered under reduced pressure to obtain a paste-like residue, and piperidineamine is obtained by silica gel column chromatography.

[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 mixture is then subjected directly to silica gel column chromatography to obtain CLY-1.

[0109] The chemical formula for CLY-1 is C 23 H 22 It is ClN7O. 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 was dissolved in dichloromethane, iodoethane and silver carbonate were added, and the mixture was stirred at room temperature for 48 hours, away from light. The reaction mixture was then subjected to silica gel column chromatography to obtain CLY-2. The mass spectrum of CLY-2 is shown in Figure 3.

[0111] Example 2: Method for preparing (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-N-(3-chloropyridine-2-yl)-N-(pyrrolidine-3-yl)benzamide (abbreviation: CLY-8): 2. Synthesis pathway

[0112] [ka]

[0113] 2. Specific synthesis steps (1) Synthesis of ETH-1 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 are added to a 250ml three-necked flask. Stirring is started under the protection of nitrogen gas, and 0.1g of palladium acetate, 1,1'-bian-2-naphthol, and tris(dimethylamino)phosphine are added. The mixture is heated to 100°C. After reacting for 7 hours, the reaction is stopped, the mixture is poured into 500ml of water, and extracted in several batches with 1000ml of ethyl acetate. The ethyl acetate layers are combined and washed with 200ml of water three times. The ethyl acetate layer is recovered to obtain a residual paste-like residue, which is mixed with silica gel, and approximately 7g of ETH-1 is obtained by silica gel column chromatography.

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

[0115] (3) Synthesis of compound CLY-8 10 g of pyrrolidineamide, 50 ml of dichloromethane, and 10 ml of 4 mol / L methanol hydrochloride solution were stirred overnight at room temperature. The mixture was poured into 100 ml of water, sodium bicarbonate was added to adjust the pH to 10-11, and the mixture was extracted with 100 ml of dichloromethane. After washing, the solvent was recovered under reduced pressure to obtain a paste-like residue. 3.2 g of compound (R)-4-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)-N-(3-chloropyridine-2-yl)-N-(pyrrolidine-3-substrate)benzamide (abbreviation: CLY-8) was obtained by silica gel column chromatography. M+H = 420.1 (see Figure 4 for the results of the mass spectrum analysis). The compounds listed in Table 1 can also be synthesized using a similar method to the one described above.

[0116] [Table 1-1]

[0117] [Table 1-2]

[0118] [Table 1-3]

[0119] Example 3: Synthesis of (R)-N-(3-chloropyridine-2-yl)-N-(pyrrolidine-3-yl)-3-(1H-tetrazole-5-yl)benzenemethaneamine (abbreviation: CLY-14)

[0120] [ka]

[0121] (R)-3-(N-(3-chloropyridine-2-yl)-3-cyanobenzamide)pyrrolidine-1-carboxylate tert-butyl can be obtained in the same manner as in Step 1 and Example 3.

[0122] [ka]

[0123] Step 2: Add 4.2 g of tert-butyl(R)-3-(N-(3-chloropyridine-2-yl)-3-cyanobenzamide)pyrrolidine-1-carboxylate, 1 g of sodium azide, and 2 g of triethylamine hydrochloride to 50 ml of N,N-dimethylformamide. Stir and react at 100°C for 20 hours, then cool. Pour into 200 ml of water, add concentrated hydrochloric acid dropwise until the pH reaches 2-3, filter to obtain a solid, wash with water, and dry. Dissolve 50 ml of dichloromethane in ml of methanol, add 10 ml of 4 mol / L hydrogen chloride dioxane solution, stir overnight at room temperature, concentrate under reduced pressure to obtain a solid, and obtain 2.7 g of (R)-N-(3-chloropyridine-2-yl)-N-(pyrrolidine-3-yl)-3-(1H-tetrazole-5-yl)benzamide by silica gel chromatography. MS(ES+): 370(M+H). Chemical formula: C17H16ClN7O Molecular weight: 369.81 Hydrogen spectrum data in CDCl3: 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 listed 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]pyridine-3-yl)-N-(3-chloropyridine-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)pyridine-2-amine.

[0134] [ka]

[0135] 10.1 g of (R)-tetrahydro-2H-pyran-3-amine, 14.0 g of 2-bromo-3-chloropyridine, 12.0 g of sodium tert-butoxide, and 100 ml of tetrahydrofuran were added to a 250 ml three-necked flask. Under the protection of nitrogen gas, stirring was started, and 0.1 g of palladium acetate, 1,1'-bian-2-naphthol, and tris(dimethylamino)phosphine were added. The mixture was heated to 65°C and reacted for 12 hours, after which the reaction was stopped. The mixture was poured into 500 ml of water and extracted in several batches with 1000 ml of ethyl acetate. The ethyl acetate layer was combined with the extracted mixture and washed with 200 ml of water three times. The ethyl acetate layer was recovered to obtain a paste-like residue. This residue was mixed with silica gel and obtained by silica gel column chromatography to obtain approximately 8 g of (R)-3-chloro-N-(tetrahydro-2H-pyran-3-yl)pyridine-2-amine.

[0136] Step 2:

[0137] [ka]

[0138] Dissolve 5g of 4-(3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl)benzoic acid in 50ml of dichloromethane, add 2.5ml of oxalyl chloride and 0.2ml of N,N-dimethylformamide catalyst, and react at room temperature until clarified. Collect the solvent under reduced pressure, add the residue to 50ml of tetrahydrofuran solvent, add 4g of the previous product (R)-3-chloro-N-(tetrahydro2H-pyran-3-yl)pyridine-2-amine in tetrahydrofuran solution, cool to below 5°C in an ice bath, add 20ml of bis(trimethylsilyl)aminolithium 1M solution, and react with stirring overnight at room temperature. Pour into water, extract with 100ml*3 of ethyl acetate, wash with water, and collect the ethyl acetate under reduced pressure. The residue is analyzed by column chromatography by (R)-4-(3H-[1,2,3 Approximately 6 g of triazolo[4,5-b]pyridine-3-yl)-N-(3-chloropyridine-2-yl)-N-(tetrahydro-2H-pyran-3-yl)benzamide was obtained. MS (ES+): 435 (M+H).

[0139] Refer to a similar method

[0140] [ka]

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

[0142] Example 5: Synthesis of ethyl 1-(5-(4-(4-(3-chloropyridine-2-yl)((R)-pyrrolidine-3-yl)carbamoyl)phenyl)-1-methyl-1H-pyrazole-5-yl)-2H-tetrazole-2-yl)isobutyrate (abbreviation: CLY-44).

[0143] Step 1:

[0144] [ka]

[0145] (1) Synthesis of (R)-3-(((3-chloropyridine-2-yl)amino)pyrrolidine-1-carboxylate tert-butyl. 11.8g of (R)-1-tert-butoxycarbonyl-3-aminopyrrolidine, 11.0g of 2-bromo-3-chloropyridine, 10.0g of tert-butoxide sodium, and 70ml of toluene were added to a 250ml three-necked flask. Under the protection of nitrogen gas, stirring was started, and 0.1g of palladium acetate, 1,1'-binaphthalene-2-phenol, and tris(dimethylamino)phosphine were added. The mixture was heated to 100°C and reacted for 7 hours, after which the reaction was stopped. The mixture was poured into 500ml of water and extracted in several batches with 1000ml of ethyl acetate. The ethyl acetate layer was combined with the extracted mixture and washed three times with 200ml of water. The ethyl acetate layer was recovered to obtain a paste-like residue. This residue was mixed with silica gel and obtained by silica gel column chromatography to obtain approximately 7g of tert-butyl(R)-3-(((3-chloropyridine-2-yl)amino)pyrrolidine-1-carboxylate.

[0146] Step 2:

[0147] [ka]

[0148] At 0°C, 5 g of tert-butyl(R)-3-((3-chloropyridine-2--2-yl)amino)pyrrolidine-1-carboxylate and 4 g of 4-bromobenzoyl chloride are added dropwise to 100 ml of tetrahydrofuran solution, and the mixture is stirred overnight at room temperature. After dilution with 300 ml of ethyl acetate and washing with 200 ml of water three times, the ethyl acetate layer is concentrated under reduced pressure, and the residue is obtained by column chromatography to obtain approximately 4 g of tert-butyl(R)-3-(4-bromo-N-(3-chloropyridine-2--2-yl)benzamide)pyrrolidine-1-carboxylate.

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

[0150] [ka]

[0151] 3 g of tert-butyl(R)-3-(4-bromo-N-(3-chloropyridine-2--2-yl)benzamide)pyrrolidine-1-carboxylate, 3 g of bis(pinacolato)diborone, and 2 g of potassium acetate were added to 30 ml of 1,4-dioxane and stirred. Under the protection of nitrogen gas, 0.6 g of palladium chloride was added, and the mixture was heated to 100°C and stirred for 5 hours. The residue was concentrated under reduced pressure and obtained by silica gel column chromatography to obtain 3.1 g of tert-butyl(R)-3-(N-(3-chloropyridine-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-chloropyridine-2-yl)((R)-pyrrolidine-3-yl)carbamoyl)phenyl)-1-methyl-1H-pyrazole-5-yl)-2H-tetrazole-2-yl) isobutyrate

[0153] [ka]

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

[0155] Refer to 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 to 12 rats used for each of the above test compounds. These 12 rats were further randomly divided into two groups. Six of these rats received intravenous administration (1 mg / kg), and blood samples were collected at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration, and plasma was separated. The other six rats received oral intragastric administration (5 mg / kg), and blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration, and plasma was separated. Plasma concentrations of each compound were measured by LC / MS / MS, and relevant pharmacokinetic parameters were calculated using Phocnix WinNonlin 6.2.1 software. Bioavailability in rats was calculated, and the pharmacokinetic characteristics of each compound were evaluated.

[0159] 2. Experimental Results In all of 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, and CLY-44 are male. The compounds showed good bioavailability in adult SD rats, with percentages of 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%, respectively. Therefore, the compounds exhibit good pharmacokinetic properties.

[0160] Example 7: Effect of compound CLY on a guinea pig melasma model 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 from Shanghai Asia Pioneer Pharmaceutical Co. Ltd., and the kit containing tyrosine, malondialdehyde (MDA), and superoxide dismutase (SOD) was purchased from Nanjing Jiancheng Bioengineering Institute.

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

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

[0163] 1.1.4 Method for preparing therapeutic cream: The excipient substrate composition consisted of 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%), and distilled water (approximately 31-55%). This was mixed with an appropriate amount of CLY series compound to form a 0.25% mixed emulsion. The cream substrate used in this example is the substrate composition of a cream from which the active ingredient has been removed.

[0164] 1.2 Animal Grouping and Modeling Participants were numbered according to their weight and randomized into the following groups: a model control group (applied cream substrate), a blank control group (applied cream substrate), a CLY-1 treatment group (applied 0.25% CLY-1 cream to the skin), a CLY-2 treatment group (applied 0.25% CLY-2 cream to the skin), a CLY-3 treatment group (applied 0.25% CLY-3 cream to the skin), and a CLY-4 treatment group (applied 0.25% CLY-4 cream to the skin). The guinea pigs were divided into four groups: a CLY-5 treatment group (0.25% CLY-5 cream applied to the skin), a CLY-14 treatment group (0.25% CLY-14 cream applied to the skin), a CLY-19 treatment group (0.25% CLY-19 cream applied to the skin), a CLY-36 treatment group (0.25% CLY-36 cream applied to the skin), and a positive treatment group (0.25% arbutin cream applied to the skin), with 6 guinea pigs in each group. Excluding the blank control group, all guinea pigs were injected with 20 mg / ml progesterone injection solution (7.5 mg / kg) once daily for 30 consecutive days at the base of their hind legs to establish a melasma model. Successful model reproduction was considered to have occurred when the skin in the model area on the back of the guinea pigs showed clearly defined, uniform, and stable dark brown spots. After modeling, the relevant cream was applied once daily to the backs of guinea pigs in the model control group, blank control group, CLY series treatment group, and positive treatment group for 30 consecutive days.

[0165] 1.3 Observational Indicators (1) Measurement of tyrosine, MDA content and SOD activity One piece of preliminary skin tissue was collected from each guinea pig, washed with pre-cooled saline, and after removing subcutaneous fat and other connective tissue, it was wiped dry. Each piece of skin tissue was then cut into 0.5 g portions and placed in five small test tubes containing 2.0 ml of pre-cooled saline. The samples were homogenized in a high-performance disperser at a speed of 10 r / min for 10 seconds, repeated once, and then centrifuged again at a speed of 3500 r / min for 15 minutes. The supernatant was collected. Tyrosine was measured by high-performance liquid chromatography, MDA by the thiobarbiturate method, and SOD by the xanthine oxidase method. The tyrosine and MDA content and SOD activity were measured according to the kit instructions.

[0166] (2) Pathological and morphological observation of cutaneous melanocytes One preliminary skin tissue sample of approximately 2 cm × 1 cm was taken from each guinea pig, fixed with 10% paraformaldehyde, and histopathological measurements were taken. Chemical staining was performed on the immunohistochemistry, and the staining and number of melanocytes were observed. Positive cells were determined according to the literature: none: 0 points, less than 15%: 0.5 points, less than 30%: 1 point, 30% or more: 2 points. Each section was observed in 5 fields of view, and after finding positive targets in the cytoplasm of epidermal cells and associated epithelial cells of the skin that showed a brown reaction, the data was quantitatively analyzed using the BX50F4 Beihuang pathology image analysis system to determine the average area of ​​melanin-positive targets in 5 fields of view from each guinea pig, the ratio of targets to the area of ​​the statistical field of view (surface density), the ratio of the number of targets to the area of ​​the statistical field of view (count density), the average grayscale, the average optical density, and the integrated optical density.

[0167] 1.4 Statistical methods SPSS 16.0 software was used for statistics. Measured values ​​were expressed as mean ± standard deviation (x ± s). One-way ANOVA was performed for comparisons between multiple groups, and t-tests were used for comparisons between groups. A statistically significant difference was considered to exist if P < 0.05.

[0168] 2. Experimental Results (1) Tyrosine, MDA content and SOD activity in guinea pigs of each group Table 3 shows the results for tyrosine, MDA content, and SOD activity in guinea pigs from each group. The tyrosine and MDA content in the skin of the model group guinea pigs was higher than in the blank group, and SOD activity was lower than in the blank group, indicating successful establishment of a melasma model. The tyrosine and MDA content in the skin of the CLY series treatment group and the positive treatment group were lower than in the model group, and SOD activity was increased, showing a statistically significant difference (P<0.05).

[0169] [Table 3]

[0170] (2) Area, number, and intensity of melanocytes in guinea pigs in each group Tables 4 and 5 show the area, number, and color of melanocytes in guinea pigs from each group. Compared to the blank group, the model group guinea pigs showed an increase in melanin deposition area, melanocyte number, optical density, and color. Compared to the model group, the CLY series treatment group and the positive treatment group guinea pigs showed a decrease in melanin deposition area, a decrease in melanocyte number, 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 enzymes in skin tissue, reducing the content of tyrosine and MDA, inhibiting tyrosinase activity in melanophores and melanoma cells, enhancing redox reactions in skin cells, reducing free radical production, and suppressing melanin production.

[0174] Example 8: Effects of the CLY series compounds on a rat scar model 1. Experimental Method 1.1 Method for preparing therapeutic cream: The excipient substrate composition consisted of 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%), and distilled water (approximately 31-55%). This was mixed with an appropriate amount of CLY series compound to form a mixed emulsion. The emulsion substrate used in this example is the substrate composition of the emulsion from which the active ingredient has been removed.

[0175] 1.2 Grouping and Modeling of Experimental Animals: SPF-grade male rats, body weight (210 ± 28) g, were obtained from the Animal Center of Nanjing Medical University. The animals were numbered according to their weight and randomized into groups of 6 each: a model control group (applied cream substrate), a CLY-1 treatment group (0.5% CLY-1 cream applied to the skin), a CLY-2 treatment group (0.5% CLY-2 cream applied to the skin), a CLY-3 treatment group (0.5% CLY-3 cream applied to the skin), a CLY-4 treatment group (0.5% CLY-4 cream applied to the skin), a CLY-8 treatment group (0.5% CLY-8 cream applied to the skin), a CLY-14 treatment group (0.5% CLY-14 cream applied to the skin), a CLY-19 treatment group (0.5% CLY-19 cream applied to the skin), and a CLY-36 treatment group (0.5% CLY-36 cream applied to the skin). Each group of rats was anesthetized with 2% pentobarbital sodium (120 mg / kg) by intraperitoneal injection and fixed to an operating table. A 4 x 5 cm intact skin piece was selected from the left side of the central part of the back, and after depilation with 8% sodium sulfide, a circular incision with a diameter of 2.4 cm to a depth of fascia was made in each depilated area with tissue scissors, partially destroying the surface fascia. To prevent the rats from biting or licking the wounds, the animals were housed in separate cages. The wounds were disinfected daily with 2% iodine tincture, and the wound healing of the rats was observed.

[0176] 2. Experimental Results 2.1 Observation results of rat wounds The wounds were disinfected regularly every day, and the rats' wounds were observed on days 1, 3, 5, 7, 12, and 20. From day 5 onward, the wound healing rate in each CLY series treatment group was significantly faster than in the model group, and the wound area gradually decreased. By day 12, the wounds in each treatment group had mostly healed, but the wounds in the model group were still about 0.5 cm. 2 On day 20, the wounds in all groups had healed, but the model group was left with obvious scarring, while the treatment group was left with only varying amounts of hyperpigmentation.

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

[0178] Example 9 Influence of the CLY series on the rat hair removal model 1. Experimental method 1.1 Materials (1) Preparation method of the tincture agents of compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-14, CLY-19, and CLY-36: Appropriate amounts of the above compounds were mixed in 75% ethanol to prepare tincture agents with different concentrations. (2) Positive therapeutic drug: Minoxidil 5% tincture (trade name: Mandy, 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 negative control group (75% ethanol topical application), model group (75% ethanol topical application), positive control group (5% minoxidil tincture topical application), CLY-1 topical group (5% CLY-1 tincture topical application), CLY-2 topical group (5% CLY-2 tincture topical application), and CLY-3 topical group (5% CLY-3 tincture topical application). CLY-4 topical application group (5% CLY-4 tincture topical application), CLY-5 topical application group (5% CLY-5 tincture topical application), CLY-14 topical application group (5% CLY-14 tincture topical application), CLY-19 topical application group (5% CLY-19 tincture topical application), CLY-36 topical application 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 ( Rats were randomly divided into two groups of 10: one receiving 2 mg / kg.d) of testosterone propionate, the other 2 mg / kg.d) of CLY-36 intravenous injection, and the third receiving CLY-36. Before the experiment, a 4 cm x 5 cm area was selected on the back of each rat, and hair removal was performed to create an observation site. Excluding the negative control group, rats were subcutaneously injected with testosterone propionate injection [5 ml / (kg-d)] once daily for 60 consecutive days to establish an SA model. After 4 weeks of subcutaneous injection of testosterone propionate, the rats gradually showed hair loss. The remaining hairs became thin and brittle, demonstrating the successful establishment of an alopecia model. Simultaneously, topical administration was performed, with testosterone applied to the observation site on the back of rats in the corresponding drug group at a dose of 2 mL / (rat / dose) twice daily, with an 8-hour interval. The negative control group and model group were administered 75% ethanol aqueous solution at a dose of 2 mL / (rat / dose) twice daily for 60 days.

[0180] 1.3 Observation Indicators and Test Methods Every 15 days after drug administration, 10 hairs were plucked from the observation site on the back of each rat, and the hair length was measured with a vernier caliper. Sixty days after drug administration, the skin of the experimental observation site was collected, and routine tissue dehydration, paraffin embedding, HE staining, and optical microscopy were performed to observe the pathological histological changes of hair follicles and sebaceous glands in rat skin. Semi-quantitative analysis of the lesions in each group was performed. The grading criteria are as follows: The normal skin dermis tissue cells and the subcutaneous hair follicles and sebaceous gland structures are recorded as "—". If the skin dermis is hyperplastic, no lesions are found in the hair follicles and sebaceous glands, and no subcutaneous inflammation is found, it is recorded as "±". The skin dermis tissue has obvious hyperplasia, and the hair follicles and sebaceous glands are clearly cystically degenerated. Obvious cystic changes are observed in the hair follicles. If no obvious hyperplasia or subcutaneous inflammation is found in the sebaceous glands, it is recorded as "+". Segmental hyperplasia is observed in the skin dermis tissue, which is not obvious. Follicular lesions are observed in a small part of the hair follicles, and mild hyperplasia and hypertrophy are observed in the sebaceous glands. There is no obvious inflammation subcutaneously and it is recorded as "++": There is varying degrees of segmental hyperplasia in the skin tissue cells of the dermis. Cystic changes are observed in some hair follicles. The performance of the hair follicles is not uniform in size, and the periphery is acellular. There is hyperplasia in the sebaceous glands. There are few nuclei in the hyperplastic glands. Mild inflammatory hyperplasia is observed subcutaneously in individual rats and it is recorded as "+++".

[0181] 2. Experimental Results 2.1 Effect of CLY on Hair Growth in Rats On the 15th, 30th, 45th, and 60th days after drug administration, the hair lengths of the rats in each treatment group were longer than those in the model group, and the difference was statistically significant (P < 0.01). Compared with the positive treatment group, the difference was statistically significant (P < 0.05). Refer to Table 6.

[0182]

Table 6

[0183] 2.2 Effect of CLY on the Hair Follicle Morphology of the Superficial Dermis of the Skin Tissue at the Observation Site in Rats Some dermal tissue cells in the model rats showed varying degrees of segmental thickening, and mild lymphocytosis was observed subcutaneously. Some hair follicles in the rats' subcutaneous tissue showed clear cystic degeneration, with varying follicle sizes, and shedding of keratinized material was observed in the lumen of the hypertrophied follicles. Mild fibrosis was observed in the periphery, cells around the hair follicles were absent or clearly reduced in cellularity, some calcified material in the lumen appeared to stain blue, the number of sebaceous glands was increased, some sebaceous glands were hypertrophied, the nuclei of the hypertrophied sebaceous glands were clearly reduced, and the number of normal hair follicles was decreased. Lesions in the dermal tissue cells and subcutaneous hair follicles and sebaceous glands of rats in all treatment groups showed varying degrees of reduction compared to the model group. The number of damaged hair follicles in the skin of rats in each treatment group was significantly reduced compared to the model control group, and the difference was statistically significant (P<0.01). Compared to the model control group, all treatment groups showed a significant reduction in dermal tissue cells, subcutaneous hair follicles, and sebaceous gland lesions in rats, with statistically significant differences (P<0.01). Compared to 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 in rat alopecia models, both through topical application and systemic use, while reducing damage to subcutaneous hair follicles and sebaceous glands and showing no significant side effects.

[0186] Example 10: Effects of the CLY series on a 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: A 2% tincture was prepared by mixing 60% ethanol with the above compounds. (2) Positive treatment drug: 2% minoxidil tincture (manufactured by the Institute of Dermatology, Chinese Academy of Medical Sciences) (3) Experimental animals: Healthy SPF-grade C57BL / 6 male mice, half male and half female, 6-8 weeks old, weighing 20-25g, were provided by GemPharmatech (Chengdu) Co., Ltd.

[0187] 1.2 Animal Grouping and Modeling The animal barn operated on a 12-hour day-night shift system, allowing animals free access to food and drink, and maintaining a temperature of 23-25°C. The experimental animals were acclimatized in the barn for one week before being subjected to experiments. The experimental mice were divided into 11 groups: a negative control group (60% ethanol topical application), a model group (60% ethanol topical application), a positive control group (2% minoxidil tincture topical application), a CLY-1 topical application group (2% CLY-1 tincture topical application), a CLY-2 topical application group (2% CLY-2 tincture topical application), a CLY-3 topical application group (2% CLY-3 tincture topical application), a CLY-4 topical application group (2% CLY-4 tincture), a CLY-8 topical application group (2% CLY-8 tincture), a CLY-14 topical application group (2% CLY-11 tincture), a CLY-19 topical application group (2% CLY-19 tincture), and a CLY-36 topical application group (2% CLY-36 tincture). Each group consisted of 6 mice, with an equal number of males and females. A 4cm x 5cm area was selected from the back of experimental mice, and hair removal was performed. The hairless area was then colored yellow with a 3% picric acid solution as the observation area for the hair removal experiment. Excluding the negative control group, the other groups of mice were subcutaneously injected with testosterone propionate injection [8ml / (kg·d)] once daily for 60 consecutive days to establish the SA model. Drug administration was performed simultaneously with the modeling. The drug was applied twice daily at a rate of 1ml / (animal / dose) with a 2-hour interval to the observation site on the back of the rats in the drug group. The normal control group and the model group were applied twice daily at a rate of 1ml / (animal / dose) with a 2-hour interval for 60 consecutive days to the excipient (60% ethanol solution).

[0188] 1.3 Observation Indicators and Test Methods Ten hairs were extracted from the observation site on the back of each mouse every 15 days after drug administration, and their length was measured with calipers. 60 days after drug administration, skin samples were taken from the observation site, and the usual tissue dehydration, paraffin embedding, HE staining, and light microscopy were performed to observe the histopathological changes in the hair follicles and sebaceous glands of the mouse skin. The lesions in each group were analyzed semi-quantitatively. The grading criteria are as follows: A "one" is recorded for normal dermal histiocytes, subcutaneous hair follicles, and sebaceous gland structures. A "±" is recorded for no dermal hyperplasia, limited lesions in hair follicles and sebaceous glands, and no subcutaneous inflammation. A "+" is recorded for no obvious dermal hyperplasia, obvious cystic degeneration in hair follicles, no obvious sebaceous gland hyperplasia, and no subcutaneous inflammation. If segmental hyperplasia of the dermal tissue is present but not clearly evident, follicular changes are seen in a few hair follicles, mild hyperplasia and hypertrophy of the sebaceous glands are observed, and there is no obvious inflammation in the subcutaneous tissue, it is recorded as "++". If segmental hyperplasia of varying degrees is seen in the dermal tissue cells of the skin, cystic changes are seen in some hair follicles, the size of the hair follicles is uneven, there are no cells around the hair follicles, the sebaceous glands are hyperplastic, the hyperplastic glands have few nuclei, and mild inflammatory hyperplasia is seen in some subcutaneous tissue, it is recorded as "+++".

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

[0190] [Table 8]

[0191] 2.2 Effects of CLY on hair follicle morphology in the epidermal layer of skin tissue in mice In the model group, segmental thickening with varying degrees was observed in the dermal tissue cells of a part of the mouse skin, and mild lymphocyte hyperplasia was seen in the subcutaneous skin. Clear cystic degeneration was observed in some of the hair follicles under the mouse skin, with different sizes of hair follicles, and shedding of keratinized substances was seen in the lumen of the enlarged hair follicles. Mild fibrosis was seen in the peripheral part, the cells around the hair follicles disappeared or the cell level was significantly reduced, some of the calcified substances in the lumen seemed to be stained blue, the number of sebaceous glands increased, some sebaceous glands were enlarged, the nuclei of the enlarged sebaceous glands were significantly reduced, and the number of normal hair follicles decreased. The lesions of the dermal tissue cells of the skin and the subcutaneous hair follicles and sebaceous glands in the mice of all treatment groups were reduced to varying 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 lesions of the dermal tissue cells of the skin, subcutaneous hair follicles, and sebaceous glands in the mice of the treatment groups were significantly reduced, and the difference was statistically significant (P<0.01). Refer to Table 9.

[0192]

Table 9

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

[0194] Example 11 Influence of the CLY series on the rabbit ear acne model 1. Experimental method 1.1 Materials (1) Method for preparing the therapeutic cream: The excipient substrate composition consisted of 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%), and distilled water (approximately 31-55%). This was mixed with an appropriate amount of CLY series compound to form a mixed emulsion. The emulsion substrate used in this example is the substrate 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) Laboratory 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 New Zealand rabbits were numbered according to their weight and randomized into the following groups: a model control group (cream application), a blank control group (cream application), a positive treatment group (Daphne applied to the skin), a CLY-1 topical treatment group (0.25% CLY-1 cream applied to the skin), a CLY-2 topical treatment group (0.25% CLY-2 cream applied to the skin), a CLY-3 topical treatment group (0.25% CLY-3 cream applied to the skin), a CLY-4 topical treatment group (0.25% CLY-4 cream applied to the skin), a CLY-8 topical treatment group (0.25% CLY-4 cream applied to the skin), a CLY-8 topical treatment group (0.25% CLY-2 cream applied to the skin), a CLY-3 topical treatment group (0.25% CLY-3 cream applied to the skin), and a CLY-4 topical treatment group (0.25% CLY-4 cream applied to the skin). (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 The cream was applied to the skin; the CLY-1 intravenous injection group (1 mg / kg.d), CLY-2 intravenous injection group (1 mg / kg.d), CLY-3 intravenous injection group (1 mg / kg.d), CLY-4 intravenous injection group (1 mg / kg.d), CLY-8 intravenous injection group (1 mg / kg.d), CLY-9 intravenous injection group (1 mg / kg.d), CLY-14 intravenous injection group (1 mg / kg.d), CLY-19 intravenous injection group (1 mg / kg.d), and CLY-36 intravenous injection group (1 mg / kg.d) consisted of 10 animals each. The inner right ear of rabbits that had undergone hair removal treatment was used as the observation site. 95% alcohol was applied to the inner left ear of all rabbits as a self-negative control. In both the model group and the treatment group, 2% coal tar was uniformly applied to the inner right ear (2% coal tar solution prepared with 95% alcohol, manufactured by Alfa Aesar China). Using a sterile cotton swab, 0.5 mL was uniformly applied once a day to the inner ear canal opening of the rabbits in an area of ​​approximately 2 cm x 2 cm. The previous application site was wiped with lukewarm water, and the application was continued for 14 consecutive days to establish an acne microacne model. Local skin changes, including ear thickness, hardness, roughness, and the presence or absence of black keratin plugs at the hair follicle openings, were observed with the naked eye.Eighteen hours after the final application, the subjects were killed, a 5mm hole was punched in the application area, and skin tissue was collected. This tissue was fixed with 10% formaldehyde, embedded in paraffin, sectioned, and then stained with HE before histopathological observation and analysis.

[0196] 1.3 Observational Indicators There are three grades for histological assessment of acne models. Grade 0 ("I") has loose keratinized cells in the infundibulum, but no acne has formed. Grade 1 ("+") has redness of the skin on the surface of the rabbit's ear, or a small amount of keratinized material densely packed in the infundibulum of the hair follicle, with no infundibulary dilation. Grade 2 ("2+") has a moderate amount of dense keratinized material in the infundibulum of the hair follicle, extending toward the sebaceous gland, with infundibulary dilation accompanied by sebaceous duct hyperplasia. Grade 3 ("3+") has extensive keratinized material within the hair follicle, severe dilation of the hair follicle due to dense keratin embolism within the hair follicle, marked hyperplasia, elevation, and scarring of the sebaceous duct epithelium, and degenerative changes in the sebaceous gland.

[0197] Pathological changes were observed under a microscope, and the epidermal thickness at five locations on one section was measured using the Biomias99 image analysis system and the average value was calculated. The area of ​​two hair follicles and the diameter of four sebaceous glands were measured in the four sections with the best structural preservation at the same location, and the average values ​​for each were calculated. By subtracting the data from the left and right external auditory canals of each group of rabbits, the difference in epidermal thickness, hair follicle area, and sebaceous gland diameter between the left and right ears of each group of rabbits was determined.

[0198] 1.4 Statistical methods For statistical analysis, SPSS 16.0 software was used. Paired t-tests were performed for comparisons between left and right sides of the same model, and t-tests were used for comparisons between groups. A statistically significant difference was considered to exist if P < 0.05.

[0199] 2. Experimental Results Visual observation: Fourteen days after coal tar application, the skin of the left ears of all rabbits was soft, the hair follicle openings in the external auditory canal were orderly, and no acne, pimples, or pustules were observed. In the model control group, the right ears of rabbits after coal tar application were thickened, the skin hardened, the surface was rough, and most hair follicle openings showed darkening or pimples, were hard to the touch, and some had fused together into a single piece. The right ears of each topical treatment group showed mild skin roughness and thickening, and a small amount of darkening. In all intravenous injection groups, most of the hair follicle papules in the right ears of rabbits subsided, the skin became thin and soft, acne was significantly reduced, pores were significantly reduced, and there was no desquamation; basically, the condition was close to that of a normal rabbit ear. In the positive treatment group, the right ears of rabbits showed less redness of the skin compared to the left ears, with some peeling and a small amount of acne.

[0200] Tissue section observation: In the left ear of the model group, the epidermis was thin, hair follicles were visible, and the junction between the dermis and epidermis was clear. In the right ear of the model group, after the model was established, thickening of the epidermis, hyperkeratosis, and thickening of the granular and spinous layers were observed, keratin plugs blocked the opening of the hair follicles, the hair follicles were enlarged and extended to the sebaceous glands, the hair follicle infundibulum was filled with keratinized material and was enlarged into a pot shape; in the superficial layer of the dermis, capillaries were dilated, inflammatory cells were scattered and infiltrated around the hair follicles, and a small number of neutrophils were observed; the number of sebaceous glands had increased, and the volume of the sebaceous glands had expanded.

[0201] Histological assessment of experimental acne under a microscope in each group (see Table 10): The difference between the right ear (experimental control) and the left ear (blank control) of rabbits in the model group was statistically significant (P<0.05), indicating the success of the rabbit ear acne model; the difference between the right ear of rabbits in each treatment group and the right ear of rabbits in the model group was also statistically significant (both P<0.05), indicating that the positive control group and each treatment group were able to improve acne acne skin damage.

[0202] Table 10 Histological grades of acne in each group As shown in Table 11, the epidermal thickness of the right ear of the rabbits in the model group (experimental control), the area of ​​hair follicle visualization, and

[0203] [Table 10]

[0204] The sebaceous gland diameter showed a statistically significant difference (P<0.05) compared to the left ear (blank control), indicating successful replication of the rabbit ear acne model; the epidermal thickness, hair follicle area, and sebaceous gland diameter of the right ears of rabbits in each treatment group were all reduced compared to the right ears of rabbits in the model group, showing a statistically significant difference (P<0.05), suggesting that the positive control group and each treatment group were able to improve the pathological damage of the acne-prone 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 reduced acne symptoms, inhibited pore clogging and acne formation, and demonstrated remarkable therapeutic effects against acne in a rabbit ear acne model.

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

[0208] 1, Material: Positive drug (glucocorticoid): Mometasone furoate cream (Elozone), a product of Schering-Plough Ltd (Shanghai). Animals: SPF-grade healthy purebred mice (C57BL / 6); 8 weeks old. Preparation of CLY Cream: The substrate 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 (approximately 31-55%), and an appropriate amount of CLY series compound solution to form a mixed emulsion.

[0209] The cream substrate used in this embodiment is a substrate composition of a cream 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 a blank control group, a model group, a positive control group (topical application of Erosone cream), a CLY-1 treatment group (topical application of 0.5% CLY-1 cream), a CLY-2 treatment group (topical application of 0.5% CLY-2 cream), a CLY-8 treatment group (topical application of 0.5% CLY-8 cream), a CLY-14 treatment group (topical application of 0.5% CLY-14 cream), a CLY-19 treatment group (topical application of 0.5% CLY-19 cream), and a CLY-36 treatment group (topical application of 0.5% CLY-36 cream), with each group consisting of five mice. The mice were anesthetized by intraperitoneal injection of pentobarbital sodium 80 mg / kg, their backs were shaved to an area of ​​approximately 2 cm x 3 cm, and they were housed in individual cages for one day. (2) The blank control group was treated with petrolatum, while the model group, positive control group, and CLY treatment group were treated with 5% imiquimod cream 62.5 mg applied to their backs at a fixed time each day for 6 consecutive days. Photographs were taken daily and PASI scoring was performed. (3) On day 1 of modeling, the blank control group and the model group were treated with a cream substrate twice daily, while the treatment group was treated with 0.5% CLY series cream twice daily.

[0211] 3. Experimental results: (1) As shown in Figure 2, after applying 5% imiquimod cream for 6 consecutive days, the application sites on the backs of the model group mice all showed clear erythema, scaling, and infiltration. However, the erythema, scaling, and infiltration on the backs of the mice in each CLY treatment group were significantly milder than in the model group, and the erythema, scaling, and infiltration in the treatment groups were similar to those in the positive drug treatment group. Therefore, this indicates that the CLY series compounds can significantly suppress the inflammatory response in psoriasis-like model mice.

[0212] Example 13: Suppression of inflammatory response in a mouse eczema model by CLY series compounds. 1. Experimental materials: Ovalbumin (OVA): Prepare to 20 g / L in PBS and store at -20°C. Calcipotriol ointment (product name: Darex ointment): A product of Leo Pharmaceutical Products. Positive drug (glucocorticoid): Mometasone furoate cream (Elozone), a product of Schering-Plough Ltd (Shanghai). Preparation of CLY Cream: The substrate 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 (approximately 31-55%), and an appropriate amount of CLY series compound solution to form a mixed emulsion. The cream substrate used in this example is the substrate composition of a cream from which the active ingredient has been removed. Animals: SPF-grade healthy purebred 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 one of the following groups: a blank control group, a model group, a positive drug group, a CLY-1 treatment group (0.1% CLY-1 cream topical application), a CLY-2 treatment group (0.1% CLY-2 cream topical application), a CLY-3 treatment group (0.1% CLY-3 cream topical application), a CLY-4 treatment group (0.1% CLY-4 cream topical application), a CLY-8 treatment group (0.1% CLY-8 cream topical application), a CLY-14 treatment group (0.1% CLY-8 cream topical application), a CLY-14 treatment group (0.1% CLY-3 cream topical application), a CLY-19 treatment group (0.1% CLY-19 topical application), and a CLY-36 treatment group (0.1% CLY-36 topical application). Each group consisted of six mice.

[0214] Modeling: Normal control mice were treated with 14.3 µl of 75% ethanol applied to both ears. Model mice, positive drug groups, and each treatment group were treated with 1 nmoI / L calcipotriol 14.3 µl applied to both ears at a fixed time each day, followed by air drying. Then, 25 µl of 20 g / L OVA was applied once daily, and modeling was performed for 12 consecutive days.

[0215] Four days after the start of modeling, a cream substrate was applied to the ear skin of mice in the blank control group and the model group, Eloxone was applied to the ear skin of mice in the positive drug group, and the therapeutic cream was applied to the ear skin of mice in each treatment group twice a day, morning and evening, for 10 consecutive days. Photographs were taken daily and scored.

[0216] The thickness of the mouse ears was measured using an ear thickness meter before modeling and again on day 14, and the thickness of the mouse's outer ear was recorded. On day 14 after measurement, the mice were killed by occlusion, and blood was collected and serum was separated.

[0217] Rabbit anti-mouse interleukin (IL)-4 antibody was loaded onto enzyme-labeled plates, left overnight at 4°C, stained according to the instructions on the ELISA kit, the reaction was terminated, and serum IL-4 levels were detected. All ELISA kits were purchased from Raybiotech, Inc., USA.

[0218] 3. Experimental results: (1) Comparison of mouse ear thickness: Before modeling, there was no statistically significant difference in ear thickness between the groups (P>0.05). After modeling, the ear thickness of the mice in each group is shown in Table 12. The model group had significantly higher ear thickness than each treatment group, the positive drug group, and the blank control group (all P<0.01), and there was no statistically significant difference between the positive drug group and each treatment group (all P>0.05).

[0219] [Table 12]

[0220] (2) Serum IL-4 concentration: Before modeling, there was no statistically significant difference in serum IL-4 concentration between the groups (P>0.05). Table 13 shows the peripheral blood serum IL-4 concentrations of mice in each group after modeling. The model group showed a significant difference compared to the other groups (all P<0.01), and there was no statistically significant difference between the positive drug group and each treatment group (P>0.05).

[0221] [Table 13]

[0222] 4. Experimental conclusion: The compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-14, CLY-19, and CLY-36 all suppressed the inflammatory response in a mouse model of eczema by lowering serum IL-4 levels.

[0223] Example 14: Effects of CLY series compounds on a mouse graft-versus-host disease model. 1. Experimental Method 1.1 Experimental animals: 6-8 week old SPF grade male C57BL / 6 mice (H-2b) and female BALB / c mice (H-2d), weighing 16-21g. ​​All experimental mice were housed in SPF grade isolation cages, provided with sterilized feed and bedding, and kept for one week before being used in 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 one tibia were then removed from the mice under sterile conditions on a super-clean table and placed in RPMI1640 medium containing 2% fetal bovine serum by volume. The bone marrow cavity was washed with RPMI1640 medium, bone marrow cells were removed, and the bone marrow suspension was blown in using a sterile washer tube. The suspension was filtered through a 200-mesh filter, and the cell precipitate was collected by centrifugation at 1,500 r / min for 10 minutes. The cells were then resuspended in physiological saline. A small amount of cell suspension was taken, red blood cells were removed with erythrocyte lysate, and 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 spleen mononuclear cells: The spleen precipitum was cut with sterile scissors, placed on a 200-mesh filter, and immersed in RPMI1640 medium containing 2% by volume of fetal bovine serum. The spleen was lightly crushed with the tip of a sterile syringe, the filter was rinsed several times with the medium, and the filtered spleen cell suspension was collected. The suspension was centrifuged at 1500 r / min for 10 minutes, and the spleen cell precipitate was collected. After resuspending in PBS, it was blown into a single-cell suspension and gently placed on top of the mouse spleen lymphocyte isolate. The suspension was centrifuged at 1800 r / min for 20 minutes, and the cells of the leukocyte layer were collected. The cells were washed twice with PBS, cell counting was performed, and the cell density was adjusted to 5 × 10¹⁴ L⁻¹. The suspension was then stored in a refrigerator at 4°C.

[0226] C57BL / 6 mice were used as bone marrow donor mice, and BABL / c mice were used as recipient mice. The BABL / c mice were numbered according to their body weight and randomly divided into a control group, a model group, and a CLY series treatment group (each group received intravenous injection of the corresponding CLY compound at a dose of 5 mg / kg.d), with each group consisting of 6 mice. One week before transplantation, the BABL / c mice were given sterile acidic water containing 200 mg / L of gentamicin, and within 24 hours before transplantation, they received 8.5 Gy of whole-body irradiation (cesium source). To construct an acute GVHD model mouse, the model group and each CLY series treatment group were injected via tail vein with 0.5 mL of a mixture of C57BL / 6 mouse bone marrow nucleated cells (containing 1 × 10⁷ cells) and spleen mononucleated cells (containing 5 × 10⁶ cells), while the control group of BABL / c mice were injected via tail vein with 0.5 mL of physiological saline. Simultaneously, the CLY series treatment group received 5 mg / kg / d intravenous injection of CLY series compound solutions, while the other groups received 5 mg / kg / d intravenous injection of physiological saline.

[0227] 1.3 Observation Indicators and Test Methods 1.3.1 Mouse GVHD Symptom Score: To determine whether or not mice developed acute GVHD, their general condition, including body size, fur, posture, mobility, diarrhea, and mental state, was observed daily after transplantation. Clinical GVHD symptoms were scored in each group of mice on day 11 post-transplantation according to the clinical GVHD symptom scoring criteria.

[0228] [Table 14]

[0229] 1.3.2 Mouse survival time analysis: Survival data for each group of mice was collected 3 weeks after transplantation, survival curves were plotted using the Kaplan-Meier method, and the differences in survival rates among the groups of mice were compared using log-rank analysis.

[0230] 1.3.3 Histopathological examination: Two weeks after transplantation, three mice that had developed GVHD were collected from each group. Dorsal skin tissue, liver tissue, and small intestine tissue were collected, fixed with 40 g / L paraformaldehyde, embedded in paraffin, stained with hematoxylin-eosin, and the pathological changes in the tissue were observed under a light microscope.

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

[0232] 2.1.1 Clinical Symptoms: Control mice that received radiation showed weight loss and decreased activity, but did not develop GVHD symptoms. In the acute GVHD group, symptoms such as lordosis, significant weight loss, decreased activity, tremors, and diarrhea began to appear in mice from day 6 after transplantation. GVHD symptoms also occurred in the CLY series treatment group, but the symptoms were significantly reduced. The clinical symptom scoring results on day 11 post-transplant were 7.48±0.77 for 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 for the CLY-1, CLY-2, CLY-5, CLY-8, CLY-11, and CLY-36 groups, respectively. The clinical symptom scoring for the CLY series treatment groups was lower than that of the acute GVHD group (P<0.01).

[0233] 2.1.2 Mouse Survival Status: Survival curves were plotted using the Kaplan-Meier method, and the difference in survival rates among the mice in each group was compared using the Log-rank analysis method. 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 to the acute GVHD group, all CLY series groups showed an extension of mouse survival time (P < 0.01).

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

[0236] Example 15 Effects 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 (US R&D company), 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, obtained from the Animal Center of Nanjing Medical University.

[0237] 1.2 Animal Grouping and Modeling Rats were numbered according to their body weight and divided into a blank control group, a model group, and a CLY series treatment group using a randomized placement table. Each group consisted of 12 rats, with an equal number of males and females. Rats in each group were anesthetized with 2% pentobarbital sodium (120 mg / kg) via intraperitoneal injection, then fixed to an operating table and injected via cervical tracheostomy. The blank control group was injected with physiological saline (1.25 ml / kg), while the model group and the CLY treatment group were each injected with 5 U / mL bleomycin solution (5 mg / kg) once daily for 14 consecutive days. One week after modeling, each treatment group was injected with the corresponding CLY compound solution (3 mg / kg.d) via tail vein, while the blank control group and the model group received the same amount of physiological saline once daily via tail vein for 14 consecutive days.

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

[0239] 2. Experimental Results 2.1 Effects of CLY series compounds on MMPs in rat lung tissue Both TIMP-1 and MMP isoforms were expressed at low levels in the lung tissue of rats in the blank control group. In the model group rats, the expression of MMP-2 and MMP-9 increased after modeling, while the expression of TIMP-1 decreased. These differences were statistically significant compared to the blank control group (P<0.05), indicating successful modeling. In the CLY series compound group, the expression of MMP-2 and MMP-9 decreased, while the expression of TIMP-1 increased. These differences were all statistically significant compared to the model group (all P<0.05). See Table 15.

[0240] [Table 15]

[0241] 2.2 Effects of CLY series compounds on VEGF in lung tissue Table 16 shows the VEGF protein and VEGF-mRNA expression levels in rat lung tissue from each group. The VEGF protein and VEGF-mRNA expression levels in the model group were significantly reduced, and this difference was statistically significant compared to the blank control group (P<0.05), indicating successful modeling of the pulmonary fibrosis model. All VEGF protein and VEGF-mRNA expression levels in the CLY series compounds were statistically significant compared to the model group (P<0.05).

[0242] [Table 16]

[0243] 2.3 Effects of CLY series compounds on SOD and CAT enzyme activity in peripheral blood Table 17 shows the levels of SOD and CAT enzymes in the peripheral blood of rats from each group. SOD and CAT enzyme activity in the peripheral blood of rats in the model group was decreased, and this difference was statistically significant compared to the p-brass control group; SOD and CAT enzyme activity in the peripheral blood of rats in the CLY series compound group was increased, and this difference was statistically significant compared to 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, and also increased the levels of SOD and CAT enzymes in peripheral blood in pulmonary fibrosis model mice, demonstrating an inhibitory effect on pulmonary fibrosis.

[0246] Example 16: Improvement of joint symptoms and inflammation indicators in rheumatoid arthritis mice using 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 processing of experimental animals Eighteen five-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 physiological saline was subcutaneously injected into the metatarsal bones of the right feet of the rats. In the RA model group, each rat was disinfected as usual on the first day of the experiment, and 0.15 mL of FCA was subcutaneously injected into the metatarsal bones of the right feet of the rats. In the CLY series compound treatment group, 0.15 mL was subcutaneously injected into the soles of the right feet of the rats from the first day of the experiment, and from the 7th day onward, the CLY series compound was administered orally and intragastricly once daily at a dose of 10 mg / kg. The RA model group and the control group received oral intragastric administration of physiological saline (10 mg / kg.d) for 21 consecutive days. 21 days after intervention with CLY series compounds, 3 mL of blood was collected from the heart of rats, serum was separated, and IL-10 and IL-17 levels in the rat serum were measured by ELISA.

[0249] (3) Observational indicators Twenty-one days after intervention, arthritis scores were assigned to rats in each group, and the degree of joint swelling in the right toes of each rat was measured. The severity of ankle inflammation in RA rats was scored on a scale of 0 to 4. Ankle severity was scored on a scale of 0 to 4. 0 points indicated normal condition; 1 point indicated mild redness and mild swelling of the ankle; 2 points indicated erythema and mild swelling from the ankle to the metatarsophalangeal or metacarpophalangeal joints; 3 points indicated erythema and moderate swelling from the ankle to the metacarpophalangeal or metatarsophalangeal joints; and 4 points indicated severe redness and swelling from the ankle to the phalangeal joints. The scores for each rat's limbs were totaled to determine the arthritis score, with a maximum score of 16 points.

[0250] 2. Experimental results (1) General expression of rat Compared to the control group, rats in the RA model group exhibited loss of appetite, depression, limited activity, and mild swelling of both toes. Intervention with CLY series compounds resulted in improvement after 21 days compared to the other groups.

[0251] (2) Rat body weight, joint swelling, and arthritis score Twenty-one days after drug intervention, the control group had a higher body weight than the other groups, and the difference was statistically significant (P<0.05 in all cases); the RA model group had a higher degree of swelling of the right toe joints and a higher arthritis score than the control group, and the difference was statistically significant (P<0.05); and the CLY series compound treatment group had a lower degree of joint swelling and an arthritis score than the RA model group, and the difference was statistically significant (see Table 18).

[0252] [Table 18]

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

[0254] [Table 19]

[0255] 3. Experimental conclusions 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. Use of compounds having the structure shown in Formula I, their tautomers, their solvates, or pharmaceutically acceptable salts in the preparation of drugs for the treatment and / or prevention of melasma, scarring, male pattern baldness, or graft-versus-host disease. 【Chemistry 1】 (however: R 1 It is 3-chloropyridinyl; R 2 is hydrogen; R 3 teeth 【Chemistry 2】 And; R 4 The substituent is a substituted or unsubstituted 5-6 membered cycloalkyl group, or a 4-7 membered heterocycle having 1-3 heteroatoms selected from N or O, wherein the substituent is hydrogen, -NH 2 Selected from -OH, (C1-C4)alkyl, (C1-C4)alkoxy, amino, and (C1-C4)alkylamino, however; R 6 and R 8 each independently is hydrogen, halogen, or (C1-C4) alkyl, provided that R 6 and R 8 are not both halogen simultaneously; R 7 These are hydroxy, (C1-C4)alkoxy, (C1-C4)alkoxycarbonyloxy(C1-C4)alkyl, or (C1-C4)alkylcarbonyloxy(Cl-C4)alkyl. R l0 and R 11 Each of these is independently hydrogen, (C1-C4) alkyl, or (C3-C6) cycloalkyl; R 12 These are hydrogen, halogens, -OH, and -NH. 2 or selected from (C1-C3) alkyl groups; R 13 is hydrogen, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy(C1-C4) alkyl, or (C1-C4) alkoxycarbonyloxy(Cl-C4) alkyl; R 14 These are hydrogen, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy(C1-C4) alkyl, or (C1-C4) alkoxycarbonyloxy(Cl-C4) alkyl. R 15 These are hydroxy, tetrazolyl, (C1-C2) alkylsulfonyl, or trifluoromethylsulfonyl; R 16 (These are 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】 The use according to claim 1, characterized in that it is the same as the use described in claim 1.

3. The use according to claim 1, characterized in that the compound has the structure shown in formula Ia. 【Transformation 7】 Here, R 3 but 【Transformation 8】 If selected from, R 4 but 【Chemistry 9】 And; R 3 but 【Chemistry 10】 In the case of R 4 but 【Chemistry 11】 And; Here, R 6 , R 7 , R 8 , R l0 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 The limitation is the same as in 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; Here, R 6 , R 8 , R l0 , R 11 , R 12 , R 13 The limitation is the use according to claim 1, which is consistent with claim 1.

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

6. R 12 Hydrogen, halogens, -OH, -NH 2 The use according to any one of claims 1 to 4, characterized in that it is selected from 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, its tautomer, its solvate, or a pharmaceutically acceptable salt thereof. 【Chemistry 17-1】 【Chemistry 17-2】 【Chemistry 17-3】

9. A drug composition characterized by containing the compound described in claim 8, a tautomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient or main active ingredient, and further containing a pharmaceutically acceptable carrier.

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

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, or 11, characterized in that the drug is administered in a dosage form suitable for one of the following administration methods: oral, parenteral, intraperitoneal, intravenous, intraarterial, topical, transdermal, sublingual, intramuscular, rectal, transchucral, nasal, inhalation, vaginal, intraocular, topical, subcutaneous, intrafat, intraarticular, intraperitoneal, or intrasacral.

13. The use according to claim 12, characterized in that the dosage form includes an ointment, gel, emulsion, topical preparation, lotion, solution, spray, tablet, granule, oral liquid, capsule, drop, enema, film, or injection.

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