WO2022262854A1 - Cly系列化合物及其制备方法和制备药物的用途 - Google Patents

Cly系列化合物及其制备方法和制备药物的用途 Download PDF

Info

Publication number
WO2022262854A1
WO2022262854A1 PCT/CN2022/099496 CN2022099496W WO2022262854A1 WO 2022262854 A1 WO2022262854 A1 WO 2022262854A1 CN 2022099496 W CN2022099496 W CN 2022099496W WO 2022262854 A1 WO2022262854 A1 WO 2022262854A1
Authority
WO
WIPO (PCT)
Prior art keywords
cly
group
alkyl
compound
skin
Prior art date
Application number
PCT/CN2022/099496
Other languages
English (en)
French (fr)
Inventor
陈敏
吴永平
Original Assignee
南京韦尔优众医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京韦尔优众医药有限公司 filed Critical 南京韦尔优众医药有限公司
Priority to KR1020247001632A priority Critical patent/KR20240022602A/ko
Priority to EP22824326.7A priority patent/EP4357345A1/en
Publication of WO2022262854A1 publication Critical patent/WO2022262854A1/zh
Priority to CN202310688735.7A priority patent/CN116803391A/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the invention belongs to the field of medicinal chemistry, and in particular relates to a CLY series compound, a preparation method thereof and an application for preparing medicine.
  • Chloasma is an acquired pigmentation disorder that occurs frequently in middle-aged and young women. Its pathogenesis is extremely complex and there are many influencing factors. The deposition of skin melanin caused by various reasons is the direct cause of chloasma. Tyrosinase produces melanin after a series of oxidation reactions. Tyrosine is oxidized to dopa in melanosomes under the action of tyrosinase, and dopa is further oxidized to dopaquinone by dopa oxidase, and dopaquinone is finally oxidized by tyrosinase to form melanin.
  • a series of oxidation and antioxidant reaction disturbances in this process may cause and promote the occurrence and development of chloasma, and the increase of tyrosine is the main material basis for the onset of chloasma.
  • SOD superoxide dismutase
  • MDA malondialdehyde
  • Chloasma is prone to recurring and difficult to treat. There are few products on the market that can really solve chloasma, and the spots are easy to recur.
  • Hydroquinone is the earliest and most widely used whitening agent, but due to uneven distribution of skin pigments, strong irritation, and even carcinogenicity, its application in the treatment of whitening and chloasma has been restricted.
  • Arbutin is one of the most widely used whitening agents in clinical practice, but its effect is limited.
  • Scar is the damage of physical, biological, chemical and other factors on human skin and soft tissue, resulting in serious damage to the skin and soft tissue, which cannot be completely repaired normally by itself, and is replaced by fibrous tissue to repair the symptoms that affect both appearance and function. Scars bring huge physical pain and mental pain to patients, especially the scars left behind by burns, scalds, and severe trauma. The treatment of scars is quite difficult. At present, it can only make red and hard scars soft and shallow, wide scars narrow, thick scars thinner, and scars cannot be completely eliminated. Therefore, it is very important to start intervention in the early stage of wound healing, which can effectively reduce scar formation, improve appearance, correct deformity and restore function.
  • glucocorticoids have obvious anti-fibrosis effects, but have many side effects.
  • Retinoic acid is an intermediate product of vitamin A metabolism in the body, which can reduce local inflammation, promote epithelial cell growth, reduce collagen synthesis, reduce DNA synthesis of fibroblasts, and inhibit cell growth.
  • concentration of retinoic acid drugs the more obvious the inhibitory effect on growth. But the curative effect of retinoic acid is limited, and the systemic application has many toxic and side effects, and the skin irritation is obvious when applied externally, and increases with the increase of the concentration.
  • Alopecia areata is a non-scarring hair loss with generally normal skin. Usually it is a sudden hair loss patch, which can involve the entire scalp in severe cases. At this time, it is called alopecia totalis (AT). universalis, AU), it is easy to have a serious impact on the appearance and psychology of the patient. At present, the etiology is not yet fully understood, and autoimmune dysfunction or instability, neuropsychiatric factors are considered to be important related factors.
  • the cure rate of alopecia areata is high, but the cure rate of alopecia areata caused by different etiologies is very different. Some patients with alopecia areata can recover naturally, and some patients with alopecia areata can last for several years.
  • Minoxidil is a common topical drug for the treatment of alopecia areata, which can promote the dilation of blood vessels in the skin, improve local blood circulation, and promote hair growth.
  • Glucocorticoids commonly used in severe alopecia areata mainly include prednisolone, compound betamethasone, etc., which can be taken orally, externally or intradermally.
  • immunosuppressants can be used for patients who are not suitable for glucocorticoids.
  • Common drugs include cyclosporine, methotrexate, glucocorticoids, and immunosuppressants. These drugs have many side effects.
  • AGA Androgenetic alopecia
  • seborrheic alopecia is a kind of androgen-dependent hereditary hair loss, is common disease, frequently-occurring disease. Usually it occurs in men aged 20-30. Hair loss is mainly on the top of the head. It usually starts from the hairline on both sides of the forehead, and some people start from the top. The hair loss area gradually expands upwards, and the hair becomes thinner and thinner gradually. Eventually, most or all of the hair on the top of the head falls off, but the hair on the back of the occipital and on both sides of the top of the temporo remains, showing a horseshoe-shaped appearance. The hair in this band-shaped area remains normal.
  • the skin at the hair loss area is bright, the pores are shrunk or a little soft vellus hair remains.
  • the speed, extent and severity of hair loss are influenced by genetics and individuals. Generally, it develops fastest around the age of 30, and severe total baldness is rare. Women mostly have diffuse hair loss that occurs on the top of the head, and the hair on the top of the head becomes thinner.
  • the epidemiological survey in my country shows that the prevalence rate of male androgenetic alopecia is 21.3%, and that of female is 6.0%. The etiology and pathogenesis of androgenetic alopecia are still unclear.
  • Finasteride is a type II 5a-reductase selective inhibitor. In recent years, it has been found that finasteride is effective in treating AGA and can continuously improve hair growth.
  • finasteride has adverse reactions such as sexual dysfunction, transient reduction of sperm and abnormal gynecomastia, and has been found to have teratogenic effects in animal experiments, so it is not suitable for use in children and women of childbearing age.
  • Cimetidine needs to be taken continuously for 5 months or more, and the side effects are gynecomastia, impotence, and decreased libido.
  • Oral contraceptives mainly sogestorone, levonorgestrel (levonorgestrel), norgestrel, norethindrone, norgestimate (nogestrel), norethindrone diester Alcohol and Norethindrone Acetate etc. It is commonly used to treat AGA in women, and the hair will improve after 6 to 12 months of treatment.
  • Acne (being commonly called as youth bean) is the chronic inflammatory disease that is prone to occur in the pilosebaceous gland, and the sickness rate is about 9.4%.
  • the occurrence of acne is closely related to the physiological and pathological changes of adolescent skin.
  • Clinical manifestations mainly include acne, papules, pustules, nodules, cysts, scars, etc., which have a serious impact on the appearance and psychology of patients.
  • Acne is related to multiple pathogenesis. Abnormal keratinization of hair follicles forms comedones is an important basis for the pathogenesis of this disease. Inflammation and infection are the pathogenic factors of acne.
  • the sebaceous glands are larger, the secretion of sebaceous glands is increased, and the level of linoleic acid in sebum is relatively reduced, which affects the synthesis of fat, resulting in the lack of fatty acids in the follicular epithelium, which induces hyperkeratosis of the follicles, so that the epithelial cells cannot be shed normally, and the pilosebaceous gland mouth If it becomes too small, the sebum cannot be discharged smoothly, forming acne.
  • the mouth of the pilosebaceous gland is blocked to form a hypoxic environment in the pilosebaceous gland, causing anaerobic Propionibacterium acnes to multiply, decompose sebum, produce chemokines, and white blood cells gather to form papules.
  • a large number of neutrophils in the pilosebaceous glands gather, and an inflammatory reaction occurs after swallowing Propionibacterium acnes, causing a large number of pus cells to accumulate to form pustules and cysts, and pitted scars are easy to form after healing.
  • Elevated androgen levels are the key link to accelerate the occurrence of acne, causing abnormal skin keratosis to block the pilosebaceous ducts, resulting in bacterial retention, reproduction, and inflammation.
  • Diseases with a mechanism of abnormal keratosis similar to acne include ichthyosis, keratosis pilaris (also known as lichen pilaris), keratosis pilaris and porokeratosis, etc.
  • keratosis pilaris follicular openings are enlarged and there are Horn plugs, ichthyosis manifest as reduced sweat and sebaceous glands, and keratinous plugs within hair follicles.
  • the medicine for treating abnormal keratosis and eliminating horn plugs and acne is mainly retinoids.
  • Retinoids can inhibit keratinization, reduce sebum secretion, promote normal keratinization of keratinocytes, and have immune-regulating and anti-inflammatory effects, thereby reducing the formation of acne, papules and pustules, and are widely used clinically to treat Abnormal keratosis diseases such as acne, ichthyosis, perikeratosis pilaris, follicular keratosis and porokeratosis.
  • retinoids can irritate the skin, which can easily lead to redness, swelling, tingling, and aggravate the original skin lesions.
  • Long-term external use of retinoids can lead to thinning of the skin, photosensitivity, and damage to the skin barrier. There are adverse reactions such as liver damage and elevated blood lipids. Therefore, more drugs for the treatment of such diseases are clinically needed.
  • Psoriasis is a common chronic relapsing inflammatory skin disease. From a global perspective, the prevalence of psoriasis patients in the natural population is 0.1% to 3%. Patients with moderate to severe disease are at increased risk of metabolic syndrome and cardiovascular damage. Therefore, early diagnosis and early treatment of psoriasis are of great significance for controlling and improving symptoms and preventing complications. Topical treatment is the first choice for mild to moderate psoriasis.
  • glucocorticoids External application of glucocorticoids is effective, but it is not suitable for long-term and continuous large-scale application; external application of retinoic acid is effective in the treatment of plaque psoriasis, and attention should be paid to skin irritation; vitamin D3 derivatives such as calcipotriol also have good curative effects , but it is not suitable for use on the face and skin folds; calcineurin inhibitors (such as tacrolimus, pimecrolimus, etc.) can be used on the scalp, skin folds, genitals, etc.
  • Immunosuppressive drugs are mainly used for erythrodermic, pustular and arthritic psoriasis; patients with obvious infection or generalized pustular psoriasis need to use antibiotics; immunosuppressants can be used to treat moderate to severe patients, However, long-term use has many adverse reactions, including bone marrow suppression, liver and kidney function damage, and increased risk of infection.
  • Eczema (also known as: atopic dermatitis, atopic dermatitis) is a skin inflammatory reaction with severe itching caused by various internal and external factors, and it is easy to recur.
  • the etiology of eczema is complex. Mild to moderate eczema is mainly treated externally. Choose appropriate dosage forms and drugs according to the skin lesions.
  • Subacute and chronic eczema should be treated with suitable glucocorticoid cream, tar preparations or immunomodulators, such as tacrolimus ointment and pimecrolimus ointment.
  • glucocorticoids and immunosuppressants can be used systematically, but there are many adverse reactions, so long-term use is not suitable.
  • graft versus host disease is due to the T lymphocytes in the allogeneic donor graft undergoing a series of "cytokine storm" stimuli initiated by the recipient after transplantation, which significantly enhanced their ability to respond to recipient antigens.
  • the immune response is a cytotoxic attack targeting recipient target cells, with the skin, liver, and intestinal tract being the main targets.
  • the incidence of acute graft-versus-host disease is 30%-45%, and the incidence of chronic disease is lower than that of acute disease.
  • Allogeneic hematopoietic stem cell transplantation is an effective treatment for hematological tumors and hematopoietic disorders.
  • Acute graft-versus-host disease is its main serious complication, with high morbidity, high mortality, and high disability rate, and it is an important cause of non-relapse death in hematologic malignancies.
  • Steroid hormones are the first-line treatment for acute GVHD, but about 50% of patients develop steroid resistance, and the GVHD response cannot be controlled.
  • Pulmonary fibrosis is a diffuse lung disease with unknown etiology and complex pathogenesis. It is currently recognized as the result of excessive deposition of extracellular matrix caused by excessive repair of lung injury.
  • the pathogenesis of pulmonary fibrosis is unknown, but it is generally accepted that repeated injury and excessive repair of the alveolar epithelium are the key to the pathogenesis.
  • the pathological feature is that long-term chronic lung inflammation and persistent alveolar damage lead to the accumulation of extracellular matrix metalloproteinases (MMP), especially the abnormal increase of MMP-2 and MMP-9, and the decrease of tissue inhibitor of metalloproteinase-1 (TIMP-1).
  • MMP extracellular matrix metalloproteinases
  • VEGF vascular endothelial growth factor
  • autoimmune diseases at least hundreds of millions of patients worldwide, including rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, lupus erythematosus, dermatomyositis, scleroderma, dry Syndrome, etc.; when these diseases are severe, multiple organs can be involved, resulting in damage to organs such as the heart, liver, kidney, blood vessels, lungs, joints, and brain, and the mortality rate is high, second only to malignant tumors.
  • Rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, and Crohn's disease share some common pathogenesis pathways.
  • the etiology and pathogenesis of these diseases are quite complex, and there is still no cure for them, and long-term medication is needed to control the progression of the disease.
  • the commonly used clinical drugs are mainly glucocorticoids and immunosuppressants, etc., but the effective rate of these drugs is only about 50%, and because of their large adverse reactions, including bone marrow suppression, liver and kidney function damage, osteoporosis, easily Induce infection and tumor, etc., which limit the long-term application.
  • the current new biological agents also have immunosuppressive effects, have the risk of inducing infection and tumors, and are expensive, which limits their wide and long-term application.
  • the current treatment methods are far from meeting the clinical needs, and more new drugs with good curative effect, less side effects and low price need to be found to control the progression of the disease and reduce the occurrence of recurrence and complications.
  • the object of the present invention is to provide CLY series compounds with pharmaceutical value or pharmaceutically acceptable salts thereof.
  • Another 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 compounds.
  • the present invention provides a compound having a structure of formula I, its tautomer, its solvate or a pharmaceutically acceptable salt thereof,
  • R 1 is a substituted or unsubstituted 5-6 membered heterocycle containing at least one of N, O, and S, a benzene ring and the heterocycle (such as: isoquinolinyl) or a combination of at least two such heterocycles Ring, here the combined ring refers to the combined ring formed by two adjacent ring structures, such as pyrrolopyridine; the substituent is H, halogen or (C1-C4) alkyl;
  • R is H, halogen, hydroxy, methoxy, ethoxy, amino, methyl or ethyl ;
  • R is H, halogen, hydroxy, methoxy, ethoxy, amino, (C1-C3) alkyl or the following groups:
  • R 4 is a substituted or unsubstituted 5-6 membered cycloalkyl group or a 4-7 membered heterocyclic ring having 1-3 heteroatoms selected from N or O, and the substituents are selected from H, -NH 2 , - OH, (C1-C4) alkyl, (C1-C4) alkoxy, amino, (C1-C4) alkylamino;
  • R4 is
  • R 4 is H, halogen, hydroxyl, methoxy, amino, methyl or the following groups: , then R 4 is
  • R 6 and R 8 are each independently H, methyl, halogen or (C1-C4) alkyl, and R 6 and R 8 are not halogen at the same time; preferably, R 6 is H or methyl, and R 8 is H;
  • R 7 is hydroxyl, (C1-C4) alkoxy, (C1-C4) alkoxycarbonyloxy (C1-C4) alkyl or (C1-C4) alkylcarbonyloxy (Cl-C4) alkyl ;
  • R 10 and R 11 are each independently H, (C1-C4) alkyl or (C3-C6) cycloalkyl;
  • R 12 is selected from H, halogen, -OH, -NH 2 or (C 1 -C 3 ) alkyl;
  • R 13 is H, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy (C1-C4) alkyl or (C1-C4) alkoxycarbonyloxy (Cl-C4) alkyl;
  • R 14 is H, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy (C1-C4) alkyl or (C1-C4) alkoxycarbonyloxy (Cl-C4) alkyl;
  • R 15 is hydroxyl, tetrazolyl, (C1-C2) alkylsulfonyl or trifluoromethylsulfonyl
  • R 16 is H, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy (C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl.
  • R1 is isoquinolin- 1 -yl, and R1 is optionally monosubstituted with chloro or methyl.
  • R 2 is H, hydroxy, or methyl.
  • R 1 is substituted or unsubstituted pyridyl or pyrrolopyridyl; said substituent is H, chloro or methyl.
  • R 12 is selected from H, halogen, -OH, -NH 2 , or methyl; in some embodiments, R 12 is selected from H.
  • the present invention provides a compound having a structure of formula I-a, its tautomer, its solvate or a pharmaceutically acceptable salt thereof,
  • R is selected from any one of H, halogen, hydroxyl, methoxy, amino, methyl or substituted or unsubstituted following groups: R4 is
  • R 6 , R 7 , R 8 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 16 are consistent with those in any paragraph above.
  • R 4 is selected from H, halogen, hydroxy or methoxy, amino, methyl or the following substituted or unsubstituted groups: , then R 4 is
  • R 6 and R 8 are each independently H, methyl, halogen or (C1-C4) alkyl, and R 6 and R 8 are not halogen at the same time; preferably, R 6 is H or methyl, and R 8 is H;
  • R 7 is hydroxyl, (C1-C4) alkoxy, (C1-C4) alkoxycarbonyloxy (C1-C4) alkoxy or (C1-C4) alkylcarbonyloxy (Cl-C4) alk Oxygen;
  • R 10 and R 11 are each independently H, (C1-C4) alkyl or (C1-C4) cycloalkyl;
  • R 12 is selected from H, halogen, -OH, -NH 2 or (C 1 -C 3 ) alkyl;
  • R 13 is H, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy (C1-C4) alkyl or (C1-C4) alkoxycarbonyloxy (Cl-C4) alkyl;
  • R 14 is H, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy (C1-C4) alkyl or (C1-C4) alkoxycarbonyloxy (Cl-C4) alkyl;
  • R 15 is hydroxyl, tetrazolyl, (C1-C2) alkylsulfonyl or trifluoromethylsulfonyl
  • R 16 is H, (C1-C4) alkyl, (C1-C4) alkylcarbonyloxy (C1-C4)alkyl or (C1-C4)alkoxycarbonyloxy(Cl-C4)alkyl.
  • R6 is H
  • R8 is H
  • R 12 is selected from H, halogen, -OH, -NH 2 or methyl; In some embodiments, R 12 is selected from H.
  • R 11 is H
  • R 10 is H or methyl
  • R 13 is
  • the present invention provides a compound having a structure of formula I-b, its tautomer, its solvate or a pharmaceutically acceptable salt thereof,
  • R is H, halogen, hydroxyl, methoxy, amino, methyl or the following substituted or unsubstituted following groups: R4 is
  • R 6 , R 8 , R 10 , R 11 , R 12 , and R 13 are consistent with the definitions in any paragraph above.
  • R4 is H, halogen, hydroxyl, methoxy, amino, methyl or the following substituted or unsubstituted groups: Then R4 is
  • R 6 , R 8 , R 10 , R 11 , R 12 , and R 13 are consistent with the definitions in any paragraph above.
  • R 12 is selected from H, halogen, -OH, -NH 2 or methyl; In some embodiments, R 12 is selected from H.
  • the present invention provides the following compounds, their tautomers, their solvates or their pharmaceutically acceptable salts, but not limited to the scope of the following compounds:
  • the present invention also provides the preparation method of the compound shown in formula (I):
  • R 1 , R 2 , R 3 , and R 4 are as defined above.
  • the present invention also provides a pharmaceutical composition, which is composed of the compound of the present invention or a pharmaceutically acceptable salt thereof as an active component or a main active component, supplemented by a pharmaceutically acceptable carrier.
  • the present invention also provides the application of the compound of the present invention in the preparation of medicines for treating and/or preventing diseases.
  • the present invention provides the compounds of the present invention in the preparation of treatment and/or prevention of chloasma, scar, androgenetic alopecia, seborrheic alopecia, alopecia areata, acne, pulmonary fibrosis, psoriasis, eczema Or the application of drugs for diseases such as atopic dermatitis.
  • the compounds or compositions of the present invention can be prepared in any pharmaceutically acceptable dosage form, such as oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal , intranasal, inhalation, vaginal, intraocular, topical, topical, subcutaneous, intrafat, intraarticular, intraperitoneal or intrathecal formulations for any administration.
  • pharmaceutically acceptable dosage form such as oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal , intranasal, inhalation, vaginal, intraocular, topical, topical, subcutaneous, intrafat, intraarticular, intraperitoneal or intrathecal formulations for any administration.
  • the dosage form of the present invention is gel, emulsion, ointment, liniment, lotion, spray, solution, tablet, electuary, oral liquid, capsule, dropping pill, enema formulations, films or injections.
  • C 5 -C 6 monohydric alcohol means a saturated aliphatic hydrocarbon group containing 5 or 6 carbon atoms substituted by a hydroxyl group, including straight chain and branched chain groups.
  • Heterocycle means a saturated cyclic group of 4 to 7 ring atoms, of which one or two or three ring atoms are selected from N, O or S(O)m (where m is an integer from 0 to 2) The remaining ring atoms are C, wherein one or two C atoms may optionally be replaced by a carbonyl group.
  • the rings of a heterocyclyl group may be optionally substituted independently with one, two or three substituents.
  • Alkyl means a saturated aliphatic group of 1-20 carbon atoms, including straight-chain and branched-chain groups (the numerical ranges mentioned in this application, such as "1-4", refer to this group, here When it is an alkyl group, it can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms or 4 carbon atoms.
  • the alkyl group can be substituted or unsubstituted.
  • Cycloalkyl means an all carbon monocyclic or fused ring ("fused" ring means that each ring in the system shares adjacent pairs of carbon atoms with other rings in the system) group in which one or multiple rings that do not have a fully attached ⁇ -electron system
  • examples of cycloalkyl include without limitation cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane alkanes and cycloheptatriene.
  • Alkoxy means -O- (unsubstituted alkyl) or -O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • Alkylamino means -NH-(unsubstituted alkyl), -NH-(unsubstituted cycloalkyl), -N-(unsubstituted alkyl) 2 or -N-(unsubstituted cycloalkyl ) 2 .
  • Representative examples include, but are not limited to, methylamino, ethylamino, propylamino, butylamino, cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, and the like.
  • (C1-C4)alkoxycarbonyloxy(C1-C4)alkyl means (C1-C4)alkyl-O-C(O)-O-(C1-C4)alkyl-.
  • CLY series can significantly reduce the tyrosinase level in the skin and blood of the chloasma mouse model, reduce the skin Hepatocyte factor (SCF) and C-kit protein expression can inhibit the pigment formation of chloasma; it can obviously promote skin wound healing and reduce scar formation; it can obviously promote the hair growth of androgenetic alopecia mouse model and reduce the effect of androgen on Destruction of hair follicles; CLY series can significantly inhibit the inflammatory response of psoriasis and eczema mouse models; CLY series compounds can significantly improve the survival time of acute GVHD mice, relieve clinical symptoms, and have a therapeutic effect on acute GVHD; CLY series compounds can Significantly reduce the levels of MMP-2 and MMP-9 in the lung tissue of mouse models of pulmonary fibrosis, increase the levels of TIMP-1 and VEGF, and at the same time increase the levels of SOD and CAT enzymes in peripheral blood, showing that it has an inhibitory
  • Figure 1 CLY series compounds can significantly promote hair growth in alopecia model mice.
  • Figure 2 CLY series compounds can significantly reduce the psoriasis-like inflammatory response in mice.
  • Ligand RuPhos heated to 100°C, reacted for 7 hours, stopped the reaction, poured into water, extracted with ethyl acetate in stages, combined the ethyl acetate layer, washed with water 3 times, and recovered the ethyl acetate layer to obtain the residual
  • the paste was mixed with silica gel and subjected to silica gel column chromatography to obtain (R)-3-((3-chloropyridin-2-yl)amino)piperidine-1-carboxylic acid tert-butyl ester.
  • Step 1 as in Example 3, can obtain (R)-3-(N-(3-chloropyridin-2-yl)-3-cyanobenzamido)pyrrolidine-1-carboxylic acid tert-butyl ester.
  • Step 2 4.2 grams of (R)-3-(N-(3-chloropyridin-2-yl)-3-cyanobenzamido)pyrrolidine-1-carboxylic acid tert-butyl ester, sodium azide 1 gram, 2 grams of triethylamine hydrochloride, added to 50ml of N,N-dimethylformamide, stirred and reacted at 100°C for 20 hours, cooled, poured into 200ml of water, added dropwise with concentrated hydrochloric acid to pH2-3, filtered to obtain The solid was washed with water and dried.
  • Step 1 Synthesis of (R)-3-chloro-N-(tetrahydro-2H-pyran-3-yl)pyridin-2-amine.
  • Example 5 1-(5-(4-(4-((3-chloropyridin-2-yl)((R)-pyrrolidin-3-yl)carbamoyl)phenyl)-1-methyl Synthesis of -1H-pyrazol-5-yl)-2H-tetrazol-2-yl) ethyl isobutyrate (abbreviated as CLY-44).
  • step 1
  • Step 3 (R)-3-(N-(3-chloropyridin-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborinane- Preparation of 2-yl)benzamide)pyrrolidine-1-carboxylate tert-butyl ester
  • Step 4 1-(5-(4-(4-((3-Chloropyridin-2-yl)((R)-pyrrolidin-3-yl)carbamoyl)phenyl)-1-methyl- Synthesis of ethyl 1H-pyrazol-5-yl)-2H-tetrazol-2-yl)isobutyrate
  • reaction solution was lowered to room temperature, it was diluted with 200ml of ethyl acetate, and the ethyl acetate layer was separated, washed with 100ml*3 of water, dried over anhydrous sodium sulfate, and the ethyl acetate was recovered under reduced pressure to obtain a residue.
  • Embodiment 6 Rat Pharmacokinetic Properties Research
  • progesterone (20mg/ml) was purchased from Shanghai General Pharmaceutical Co., Ltd.
  • arbutin ointment was purchased from Shanghai New Pioneer Pharmaceutical Co., Ltd.
  • tyrosine, malondialdehyde (MDA), superoxide Dismutase (SOD) kit was purchased from Nanjing Jiancheng Bioengineering Institute.
  • excipient matrix components include methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), stearyl alcohol ( 5%), glycerin (20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%) ), distilled water (about 31-55%), respectively mixed with an appropriate amount of CLY series compounds to form a 0.25% mixed emulsion.
  • the cream base used in this example refers to the base ingredients of the cream from which the active ingredients are removed.
  • model control group smear cream base
  • blank control group smear cream base
  • CLY-1 treatment group skin smear 0.25% CLY-1 cream
  • CLY- 2 Treatment group 0.25% CLY-2 cream applied to the skin
  • CLY-3 treatment group 0.25% CLY-3 cream applied to the skin
  • CLY-4 treatment group 0.25% CLY-4 cream applied to the skin
  • CLY-3 treatment group 0.25% CLY-3 cream applied to the skin
  • -5 treatment group skin application of 0.25% CLY-5 cream
  • CLY-11 treatment group skin application of 0.25% CLY-11 cream
  • CLY-19 treatment group skin application of 0.25% CLY-19 cream
  • CLY-36 treatment group 0.25% CLY-36 cream applied to the skin
  • positive treatment group 0.25% arbutin cream applied to the skin
  • guinea pigs in other groups were intramuscularly injected with 20 mg/ml progesterone injection (7.5 mg/kg) at the root of the hind legs, once a day, and continuously injected for 30 days to establish a chloasma model.
  • the skin on the back of the guinea pig model area showed uniform and stable dark brown spots with clear borders, indicating that the model was successfully replicated.
  • the model control group, the blank control group, each CLY series treatment group, and the positive treatment group were smeared with corresponding ointment on the back of the guinea pigs for intervention, once a day for 30 consecutive days.
  • Tyrosine was determined by high performance liquid chromatography, MDA was determined by thiobarbituric acid method, SOD was determined by xanthine oxidase method, and tyrosine, MDA content and SOD activity were detected according to the kit instructions.
  • SPSS16.0 software was used for statistics, and the measurement data were expressed as mean ⁇ standard deviation (x ⁇ s).
  • the comparison among multiple groups was performed by one-way analysis of variance, and the comparison between groups was performed by t test. P ⁇ 0.05 was considered statistically significant.
  • the test results of tyrosine, MDA content and SOD activity in guinea pigs in each group are shown in Table 3.
  • the guinea pig skin tyrosine and MDA contents in the model group were higher than those in the blank group, and the SOD activity was lower than that in the blank group, indicating that the skin chloasma model was successfully established; compared with the model group, the tyrosine in the skin of the CLY series treatment group and the positive treatment group , MDA content was lower, while SOD activity was higher, the difference was statistically significant (P ⁇ 0.05).
  • CLY-1, CLY-2, CLY-3, CLY-4, CLY-5, CLY-11, CLY-19 and CLY-36 can reduce the content of tyrosine and MDA by increasing the activity of SOD enzyme in skin tissue, Inhibit the tyrosinase activity of melanocytes and melanoma cells, enhance the redox reaction of skin cells, reduce the production of free radicals, and inhibit the formation of melanin, thereby treating chloasma.
  • the excipient base composition comprises methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), stearyl alcohol (5%) ), glycerin (20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%), Distilled water (about 31-55%) is mixed with an appropriate amount of CLY series compounds respectively to form a mixed emulsion.
  • the emulsion base as used in this example refers to the base ingredients of the emulsion from which the active ingredients are removed.
  • CLY-3 treatment group (0.5% CLY-3 cream applied to the skin
  • CLY-4 treatment group (0.5% CLY-4 cream applied to the skin
  • CLY-8 treatment group (0.5% CLY-3 cream applied to the skin) -8 cream
  • CLY-11 treatment group (0.5% CLY-11 cream applied to the skin
  • CLY-19 treatment group (0.5% CLY-19 cream applied to the skin
  • CLY-36 treatment group (0.5% CLY-19 cream applied to the skin) CLY-36 cream)
  • 6 rats in each group 6 rats in each group.
  • Each group of rats was anesthetized with 2% sodium pentobarbital (120 mg/kg) intraperitoneally and fixed on the operating table, then a piece of complete skin of 4 ⁇ 5 cm was selected on the left side of its back, and 8% sodium sulfide was used for depilation.
  • Tissue scissors cut a circular wound with a diameter of 2.4 cm deep to the myofascia at each hair removal site, destroying part of the muscle surface fascia. Animals were kept in separate cages to prevent rats from biting and licking. The wounds were routinely disinfected with 2% tincture of iodine every day, and the wound healing of rats was observed.
  • the wounds were routinely disinfected every day, and the wounds of the rats were observed on the 1d, 3d, 5d, 7d, 12d, and 20d. From the 5th day, the wound recovery speed of each CLY series treatment group was significantly faster than that of the model group, and the wound area gradually became smaller. On the 12th day, the wounds in the treatment groups had basically recovered, while the wounds in the model group still had a wound size of about 0.5 cm 2 . By the 20th day, the wounds in each group had recovered, and the model control group left obvious scars, while the treatment groups only left pigmentation in varying amounts.
  • CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-11, CLY-19 and CLY-36 can all significantly promote skin wound healing and reduce scar formation.
  • Wistar rats were randomly divided into negative control group (external application of 75% ethanol), model group (external application of 75% ethanol), positive control group (external application of 5% minoxidil tincture), and CLY-1 external application treatment group (external application of 5% ethanol) according to body weight.
  • each rat selected an area of 4cmx5cm on the back to remove the hair as the observation area. Except for the negative control group, the rats were subcutaneously injected with testosterone propionate injection [5ml/(kg ⁇ d)] in the back of the neck, once a day, for 60 consecutive days to establish the SA model. After 4 weeks of continuous subcutaneous injection of testosterone propionate, the rats gradually lost their hair. The remaining hair became fine and brittle, which proved that the hair loss model was successfully established.
  • the skin was smeared and administered, and 2 mL/(one ⁇ time) was applied to the observation area of the back of the corresponding drug group rats, twice a day, with an interval of 8 hours between administrations.
  • Intravenous administration once a day.
  • the negative control group and the model control group were smeared with 75% ethanol solution, 2 mL/(one per time), twice a day, for 60 consecutive days.
  • the grading criteria are as follows: Normal structure of skin dermal tissue cells and subcutaneous hair follicles and sebaceous glands is recorded as "one": no hyperplasia in the skin dermis, limited lesions of hair follicles and sebaceous glands, and no subcutaneous inflammation is recorded as " ⁇ ": no skin dermal tissue Significant hyperplasia, obvious cystic degeneration of hair follicles. No obvious hyperplasia of sebaceous glands, no subcutaneous inflammation, recorded as "+”: segmental hyperplasia of skin dermal tissue, not obvious, a small number of hair follicles have cystic degeneration, sebaceous glands have Mild hyperplasia and hypertrophy.
  • the hair length of the rats in each treatment group was longer than that of the model control group on the 15th, 30th, 45th, and 60th day of administration, and the difference was statistically significant (P ⁇ 0.01). Compared with the positive treatment group, the difference was statistically significant (P ⁇ 0.05). See Table 6
  • the skin dermal tissue cells, subcutaneous hair follicles and sebaceous gland lesions of the rats in each treatment group were alleviated to varying degrees.
  • the number of skin damaged hair follicles in each treatment group was significantly reduced, and the difference was statistically significant (P ⁇ 0.01).
  • the lesions of skin dermal tissue cells, subcutaneous hair follicles and sebaceous glands of rats in each treatment group were significantly alleviated, and the difference was statistically significant (P ⁇ 0.01).
  • the difference was statistically significant (P ⁇ 0.05). See Table 7.
  • CLY-1, CLY-2, CLY-3, CLY-4, CLY-5, CLY-11, CLY-19 and CLY-36 can significantly promote the hair growth of the rat alopecia model and relieve the Subcutaneous hair follicles and sebaceous glands were damaged, and no obvious adverse reactions were seen.
  • mice Animal room 12h-12h alternates day and night, keeps animals free to drink water and eat, temperature 23-25°C, experimental animals enter the animal room and enter the experiment after a week of adaptive feeding.
  • Experimental mice were divided into 11 groups, negative control group (external application of 60% ethanol), model group (external application of 60% ethanol), positive control group (external application of 2% minoxidil tincture), CLY-1 external application treatment group (external application of 2 % CLY-1 tincture), CLY-2 topical treatment group (topical 2% CLY-2 tincture), CLY-3 topical treatment group (topical 2% CLY-3 tincture), CLY-4 topical treatment group (topical 2% CLY-3 -4 tincture), CLY-8 topical treatment group (topical 2% CLY-8 tincture), CLY-11 topical treatment group (topical 2% CLY-11 tincture), CLY-19 topical treatment group (topical 2% CLY-19 tincture), CLY-36 topical treatment group (2% CLY-36
  • mice in other groups were subcutaneously injected with testosterone propionate injection [8ml/(kg ⁇ d)] once a day for 60 consecutive days to establish the SA model. Smear and administer at the same time as the model is established, and apply 1ml/(one per time) to the observation area on the back of the rats in the corresponding drug group, twice a day, with an interval of 2h between administrations.
  • the normal control group and the model control group were smeared with excipient (60% ethanol solution), 1 ml/(one per time), twice a day, for 60 consecutive days.
  • the grading criteria are as follows: Normal structure of skin dermal tissue cells and subcutaneous hair follicles and sebaceous glands is recorded as "one": no hyperplasia in the skin dermis, limited lesions of hair follicles and sebaceous glands, and no subcutaneous inflammation is recorded as " ⁇ ": no skin dermal tissue Significant hyperplasia, obvious cystic degeneration of hair follicles. No obvious hyperplasia of sebaceous glands, no subcutaneous inflammation, recorded as "+”: segmental hyperplasia of skin dermal tissue, not obvious, a small number of hair follicles have cystic degeneration, sebaceous glands have Mild hyperplasia and hypertrophy.
  • mice in each group were longer than that of the model control group on the 15th, 30th, 45th, and 60th day of administration, and the difference was statistically significant (P ⁇ 0.01). Compared with the positive treatment group, the difference was statistically significant ( P ⁇ 0.05). See Table 8. The hair growth of mice in each group on the 30th day of administration is shown in Figure 1.
  • the mice had different degrees of segmental thickening, and the mice had mild subcutaneous lymphocyte hyperplasia; some mice had obvious cystic changes in the subcutaneous hair follicles, and the hair follicles were of different sizes, and there were shedding angles in the enlarged hair follicle cavity.
  • There is mild fibrosis in the periphery the cells around the hair follicles disappear or the cell layers are significantly reduced, the cavity seems to have calcifications stained blue, the number of sebaceous glands increases, some glands have hypertrophy, the nucleus of hypertrophic glands is significantly reduced, and the number of normal hair follicles reduce.
  • the skin dermal tissue cells, subcutaneous hair follicles and sebaceous gland lesions of the mice in each treatment group were alleviated to varying degrees.
  • the number of skin damaged hair follicles in each treatment group was significantly reduced, and the difference was statistically significant (P ⁇ 0.01).
  • the lesions of skin dermal tissue cells, subcutaneous hair follicles and sebaceous glands of mice in each treatment group were significantly alleviated, and the difference was statistically significant (P ⁇ 0.01). See Table 9.
  • CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-11, CLY-19, and CLY-36 can significantly promote hair growth in mouse hair loss models, and reduce damage to subcutaneous hair follicles and sebaceous glands .
  • the preparation method of treatment emulsifiable paste comprises methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), stearyl alcohol ( 5%), glycerin (20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%) ), distilled water (about 31-55%), respectively mixed with an appropriate amount of CLY series compounds to form a mixed emulsion.
  • the emulsion base as used in this example refers to the base ingredients of the emulsion from which the active ingredients are removed.
  • New Zealand rabbits were numbered according to body weight, and were divided into model control group (smeared with cream base), blank control group (smeared with cream base), positive treatment group (skin smeared with Duffin), and CLY-1 topical treatment group by random permutation table method.
  • Histological judgment and grading standard of acne model grade 3 according to histological grade.
  • Level 0 "one" means only loose keratinized cells in the infundibulum, no acne formation
  • level 1 means skin redness on the surface of the rabbit ear, or a small amount of dense keratinized substance in the infundibulum of the hair follicle, and the infundibulum does not expand "+”
  • Grade 2 moderately dense keratinized substance seen in the infundibulum of the hair follicle, extending to the sebaceous gland, accompanied by hyperplasia of the sebaceous gland duct, and "2+” infundibulum expansion
  • grade 3 extensive keratinized substance in the hair follicle, caused by dense keratin embolism Severe expansion of hair follicles, obvious hyperplasia of sebaceous duct epithelium, raised skin, scars, and degeneration of some sebaceous glands "3+”.
  • the left ear of the model group showed thinner epidermis, visible hair follicles, and a clear junction between the dermis and the epidermis.
  • the epidermis was thickened, hyperkeratosis, the granular layer and spinous layer were thickened, the horn plug blocked the hair follicle opening, the hair follicle expanded and extended to the sebaceous gland, and the hair follicle infundibulum was filled with keratinized substances, making the hair follicle funnel thicken.
  • the head is enlarged in a pot shape; the capillaries in the superficial dermis are dilated, and scattered inflammatory cells infiltrate around the hair follicles, with a small amount of neutrophils; the number of sebaceous glands increases, and the volume of sebaceous glands increases.
  • Compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-9, CLY-11, CLY-19 and CLY-36 can significantly alleviate the symptoms of rabbit ear acne model, reduce pore blockage and Comedone formation, shown to have significant therapeutic effects on acne.
  • Example 12 Compound CLY series inhibits psoriasis-like inflammatory response in mice
  • glucocorticoid drug mometasone furoate cream (Aloson), product of Shanghai Schering-Plough Pharmaceutical Co., Ltd.
  • CLY cream preparation method matrix composition comprises methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), stearyl alcohol (5%), glycerin ( 20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%), distilled water (about 31 -55%), form a mixed emulsion with an appropriate amount of CLY series compound liquid.
  • the cream base used in this embodiment refers to the base components of CLY cream except active ingredients.
  • Vaseline was applied topically in the blank control group, and 62.5 mg of 5% imiquimod cream was regularly applied on the back of the model group, positive control group and CLY treatment group every day for 6 consecutive days. Photographs were taken every day for PASI scoring.
  • Example 13 CLY series compounds inhibit the inflammatory response of the mouse eczema model
  • Ovalbumin 20g/L in PBS, stored at -20°C.
  • Calcipotriol liniment (trade name: Dalux liniment): product of Danish Leo Pharmaceutical Co., Ltd.
  • glucocorticoid drug mometasone furoate cream (trade name: Aloson), product of Shanghai Schering-Plough Pharmaceutical Co., Ltd.
  • CLY cream preparation method matrix composition comprises methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), stearyl alcohol (5%), glycerin ( 20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%), distilled water (about 31 -55%), form a mixed emulsion with an appropriate amount of CLY series compound liquid.
  • the cream base used in this embodiment refers to the base components of CLY cream except active ingredients.
  • Modeling 14.3ul of 75% ethanol was smeared on both sides of the ears of the normal control group mice, and 14.3ul of 1nmoI/L calcipotriol liniment was first smeared on the ears of both sides of the model group, the positive drug group and each treatment group at regular intervals every day. After air-drying, apply 20g/L OVA 25ul, once a day, for 12 days to build a model.
  • the cream base was applied to the ear skin of the mice in the blank control group and the model group, and Eloxone was applied to the ear skin of the mice in the positive drug group, and the ear skin of the mice in each treatment group was respectively Apply the treatment cream, 2 times a day in the morning and evening, for 10 consecutive days, take pictures every day, and score.
  • mice The ear thickness of the mice was measured and recorded with an ear thickness measuring instrument before modeling and on the 14th day. After the measurement was completed on the 14th day, the mice were killed by neck dislocation, blood was collected, and serum was separated.
  • IL-4 antibody Coat the microtiter plate with rabbit anti-mouse interleukin (IL)-4 antibody, overnight at 4°C, perform staining and stop the reaction according to the instructions of the ELISA kit, and detect the serum IL-4 level. All ELISA kits were purchased from Raybiotech, USA.
  • Serum IL-4 concentration Before modeling, there was no significant difference in serum IL-4 concentration among the groups (P>0.05). After modeling, the levels of serum IL-4 in the peripheral blood of mice in each group are shown in Table 13. The model group was significantly different from other groups (P ⁇ 0.01), and the difference between the positive drug group and each treatment group was not statistically significant (P>0.05).
  • Compounds CLY-1, CLY-2, CLY-3, CLY-4, CLY-8, CLY-11, CLY-19 and CLY-36 can all inhibit the inflammatory response of eczema mouse model by reducing serum IL-4 levels.
  • mice 6-8 weeks old SPF grade male C57BL/6 mice (H-2b) and female BALB/c mice (H-2d), body weight 16-21g, all experimental mice were raised in SPF grade In the isolation cage, the feed and litter were sterilized, and the mice were bred for 1 week before the experiment.
  • Preparation of bone marrow cells sacrifice the donor mouse by neck dislocation, soak in 75% ethanol for 5 minutes, take out the two femurs and tibia of the mouse under aseptic conditions in an ultra-clean bench, and place them in RPMI1640 medium containing 2% fetal bovine serum middle. Rinse the bone marrow cavity with RPMI1640 medium, take out the bone marrow cells, pipette a sterile washing solution tube into a bone marrow suspension, filter through a 200-mesh filter, centrifuge at 1500r/min for 10min, collect the cell pellet, and add normal saline to resuspend. In addition, take a small amount of cell suspension, and count the amount of nucleated cells after removing red blood cells in the red blood cell lysate. And adjust the nucleated cell concentration of bone marrow suspension to 5 ⁇ 1010L-1.
  • Preparation of spleen mononuclear cells Cut the spleen capsule with sterile scissors, place it on a 200-mesh filter, soak it in RPMI1640 medium containing 2% fetal bovine serum by volume fraction, and gently grind it with the handle end of a sterile syringe For the spleen, wash the filter screen several times with medium, collect the filtered spleen cell suspension, centrifuge at 1500r/min for 10min, and collect the spleen cell pellet.
  • mice C57BL/6 mice were used as donor mice for bone marrow transplantation, and BABL/c mice were used as recipient mice.
  • BABL/c mice were numbered according to body weight, and were divided into control group, model group, and CLY series treatment group (each group was intravenously injected with the corresponding CLY compound at 5 mg/kg.d) by random permutation table method, with 6 mice in each group.
  • One week before transplantation BALB/c mice were fed sterile acidified water containing 200mg/L gentamicin, and mice were given 8.5Gy whole-body irradiation (cesium source) within 24 hours before transplantation.
  • the model group and each CLY series treatment group were injected with 0.5mL mixture of C57BL/6 mouse bone marrow nucleated cells (containing 1 ⁇ 107 cells) and spleen mononuclear cells (containing 5 ⁇ 106 cells) into the tail vein to establish a mouse model of acute GVHD , BABL/c mice in the control group were infused with 0.5 mL of normal saline through the tail vein.
  • the CLY series treatment group was intravenously injected with 5 mg/kg.d of each CLY series compound solution, and the other groups were injected with normal saline 5 mg/kg.d.
  • GVHD symptom score in mice Observe the general condition of the mice every day after transplantation, including body weight, fur, posture, activity, diarrhea and mental state, and judge whether the mice have acute GVHD. According to the clinical GVHD symptom scoring standard, the clinical GVHD symptoms of mice in each group were scored 11 days after transplantation.
  • mice in the control group were reduced by simple irradiation, but no GVHD symptoms appeared.
  • mice in the acute GVHD group began to exhibit arched backs, significant weight loss, decreased activity, tremors, and diarrhea symptoms, while the CLY series treatment group also developed GVHD symptoms, but the symptoms were significantly alleviated.
  • the results of clinical symptom score 11 days after transplantation showed that the acute GVHD group was 7.48 ⁇ 0.77, and the CLY-1 group, CLY-2 group, CLY-5 group, CLY-8 group, CLY-11 group, and CLY-36 group were 5.28 ⁇ 0.53 , 4.68 ⁇ 0.42, 4.91 ⁇ 0.46, 4.56 ⁇ 0.39, 5.06 ⁇ 0.41, 4.93 ⁇ 0.39, the clinical symptom scores of the CLY series treatment group were lower than those of the acute GVHD group (P ⁇ 0.01).
  • mice The survival curve was drawn by the kaplan-meier method, and the Log-rank analysis method was used to compare the differences in the survival rates of the mice in each group. The results showed that the acute GVHD group, the CLY-1 group, and the CLY-2 group , CLY-5 group, CLY-8 group, CLY-11 group, CLY-36 group the survival rates of mice were 12.13%, 61.26%, 68.36%, 58.79%, 63.13%, 62.60%, 67.75%, respectively. Compared with acute GVHD group, CLY series group can prolong the survival time of mice (P ⁇ 0.01).
  • the CLY series compounds CLY-1, CLY-2, CLY-5, CLY-8, CLY-11, and CLY-36 can significantly increase the survival time of acute GVHD mice, relieve clinical symptoms, and show a therapeutic effect on acute GVHD.
  • Reagent Bleomycin (4 mg/support, Tianjin Taihe Pharmaceutical Co., Ltd.), mouse anti-mouse MMP monoclonal antibody (NEO Mark-ers company), mouse anti-mouse TIMP-1 polyclonal antibody (Wuhan Boster company), enzyme-linked immunosorbent assay (ELISA) kit (US R&D Company), Quantscript RT Kit reverse transcription kit (Dalian TaKaRa Company).
  • the rats were numbered according to body weight, and were divided into blank control group, model group, and CLY series treatment group by random permutation table method, with 12 rats in each group, half male and half male. Rats in each group were anesthetized with 2% sodium pentobarbital (120 mg/kg) intraperitoneally, fixed on the operating table, and injected with tracheotomy in the neck.
  • the blank control group was injected with normal saline (1.25ml/kg), and the model group and CLY treatment group were injected with 5U/mL bleomycin solution (5mg/kg), once a day for 14 consecutive days.
  • each treatment group was injected with the corresponding CLY compound solution (3 mg/kg.d) through the tail vein, and the blank control group and the model group were injected with the same amount of normal saline through the tail vein, once a day for 14 consecutive days.
  • Peripheral venous blood was obtained from the tail vein after modeling and 14 days after treatment in each group, and the levels of superoxide dismutase (SOD) and catalase (CAT) in peripheral blood were detected. Rats in each group were sacrificed twice after blood collection (after modeling and treatment for 14 days).
  • the right lung tissue of the animal was stored in a -4°C refrigerator for detection of VEGF. Histochemical staining was used to detect MMP subtypes and TIMP-1 expression in rat lung tissue. The right lung tissue was ground and homogenized, centrifuged at 3000r/min and the supernatant was taken. The VEGF protein in the lung tissue was detected by ELISA, and the expression of VEGF-mRNA was measured by reverse transcription polymerase chain method.
  • TIMP-1 and MMP subtypes were slightly expressed in the lung tissue of the rats in the blank control group.
  • the expression of MMP-2 and MMP-9 in the model group increased after modeling, and the expression of TIMP-1 decreased. Compared with the blank control group, the difference was statistically significant (P ⁇ 0.05), indicating that the model was successfully established.
  • the expressions of MMP-2 and MMP-9 in the CLY series compound group were decreased, and the expression of TIMP-1 was up-regulated, and the differences were statistically significant compared with the model group (all P ⁇ 0.05). See Table 15
  • Table 16 shows the expression of VEGF protein and VEGF-mRNA in lung tissue of rats in each group.
  • the expression of VEGF protein and VEGF-mRNA in the model group decreased significantly, and the difference was statistically significant compared with the blank control group (P ⁇ 0.05), indicating that the pulmonary fibrosis model was successfully established.
  • VEGF protein (pg/ml) VEGF-mRNA Model control group 37.23 ⁇ 4.16 0.52 ⁇ 0.19 Blank control group 54.26 ⁇ 3.95* 0.83 ⁇ 0.13* CLY-1 group 52.16 ⁇ 3.61* 0.76 ⁇ 0.11* CLY-2 group 51.18 ⁇ 3.69* 0.71 ⁇ 0.12* CLY-8 group 53.16 ⁇ 3.62* 0.79 ⁇ 0.13* CLY-11 group 49.86 ⁇ 3.58* 0.76 ⁇ 0.13* CLY-36 group 53.87 ⁇ 3.73* 0.83 ⁇ 0.13*
  • Compounds CLY-1, CLY-2, CLY-8, CLY-11, and CLY-36 can significantly reduce the levels of MMP-2 and MMP-9 in the lung tissue of a mouse model of pulmonary fibrosis, increase the levels of TIMP-1 and VEGF, At the same time, it can increase the levels of SOD and CAT enzymes in peripheral blood, showing that it has an inhibitory effect on pulmonary fibrosis.
  • Example 16 CLY series compounds can improve joint symptoms and inflammatory indicators in mice with rheumatoid arthritis (RA)
  • FCA Freund's Adjuvant Complete
  • RA model group Eighteen 5-week-old female Wistar rats were randomly divided into control group, RA model group, and CLY series compound treatment group, with 6 rats in each group.
  • Control group 75% alcohol was used to disinfect the right toes of the rats, and 0.15 mL of normal saline was injected subcutaneously into the right paw of the rats.
  • RA model group On the first day of the experiment, each rat was routinely disinfected, and 0.15 mL of FCA was injected subcutaneously on the right paw of the rat.
  • CLY series compound treatment group On the first day after the start of the experiment, each rat was routinely disinfected, and FCA 0.15mL was injected subcutaneously on the right paw of the rat, and 7 days later, the CLY series compound was administered orally every day at a dose of 10mg/ kg, once a day, the RA model group and the control group were gavaged with normal saline (10 mg/kg.d) every day for 21 consecutive days. On the 21st day after the intervention of the CLY series compounds, 3 mL of heart blood was collected from the rats, and the serum was separated, and the levels of IL-10 and IL-17 in the rat serum were detected by ELISA.
  • the rats in each group were scored for arthritis, and the joint swelling of the right toe of the rats in each group was measured.
  • the severity of the ankle joint is scored on a scale of 0-4. 0 points, normal; 1 point, the ankle joint has slight redness and swelling; 2 points, there is erythema from the ankle joint to the metatarsal joint or palm joint and mild swelling; 3 points, erythema and moderate swelling from the ankle joint to the metatarsophalangeal joint; 4 points, severe redness and swelling from the ankle joint to the toe joint.
  • the scores of the four limbs of each rat were added together as the arthritis score, and the highest score was 16 points.
  • the rats in the RA model group had anorexia, listlessness, limited activity, and gradual swelling of the left and right toes. After 21 days of intervention with CLY series compounds, the results were better than those in other groups.
  • the body weight of the control group was higher than that of the other groups, and the difference was statistically significant (all P ⁇ 0.05); the swelling degree of the right toe joint and the arthritis score of the RA model group were higher than those of the control group, and the difference was statistically significant (P ⁇ 0.05), the joint swelling degree and arthritis score of the CLY series compound treatment group were lower than those of the RA model group, and the difference was statistically significant (see Table 18).
  • the levels of IL-10 in the serum of the rats in the control group and the CLY series compound treatment group were higher than those in the RA model group (both P ⁇ 0.05); the levels of IL-17 in the serum of the rats in the control group and the CLY series compound treatment group were lower than those in the RA model group, The difference was statistically significant (all P ⁇ 0.05) (see Table 19).
  • CLY series compounds can improve the symptoms of arthritis in rheumatoid arthritis mice by reducing the level of IL-17 in peripheral blood and increasing inflammatory indicators such as IL-10.

Abstract

本发明公开一种CLY系列化合物及其制备方法和制备药物的用途，所述CLY系列化合物为具有式I结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，动物模型实验表明，化合物CLY系列均能明显减少黄褐斑鼠模型皮肤和血中的酪氨酸酶水平，减少皮肤中肝细胞因子(SCF)、C-kit蛋白表达，抑制黄褐斑的色素形成；能明显促进皮肤创面愈合，减少疤痕形成；能够明显促进脱发鼠模型的毛发生长，减少对毛囊的破坏；能明显减轻兔耳痤疮模型症状，减少毛孔堵塞和粉刺形成；CLY系列化合物能明显提高急性GVHD小鼠生存时间，减轻临床症状，显示对急性GVHD具有治疗作用；能明显降低肺纤维化小鼠模型肺组织中MMP-2、MMP-9水平，增加TIMP-1和VEGF水平，同时可提高外周血中SOD、CAT酶水平；CLY系列化合物能通过降低外周血中IL-17水平和升高IL-10等炎症指标来改善类风湿关节炎小鼠的关节炎症状。该化合物可以单独使用也可以联合其他药物使用，为以上疾病的治疗提供了新的药物选择。

Description

CLY系列化合物及其制备方法和制备药物的用途 技术领域
本发明属于药物化学领域，具体涉及一种CLY系列化合物及其制备方法和制备药物的用途。
背景技术
黄褐斑是一种好发于中、青年女性的获得性色素障碍性疾病，其发病机制极为复杂，影响因素众多，各种原因所致的皮肤黑色素的沉积是引起黄褐斑直接原因。酪氨酸酶经过系列氧化反应后生成黑色素。酪氨酸在酪氨酸酶的作用下在黑素小体内被氧化成多巴，多巴进一步被多巴氧化酶氧化成多巴醌，多巴醌最终在酪氨酸酶的做一下氧化形成黑色素。在此过程中出现的系列氧化与抗氧化反应紊乱都可能引起和促进黄褐斑的发生和发展，酪氨酸增多是黄褐斑发病的主要物质基础。当系列氧化反应平衡紊乱时，体内生成氧自由基过量，同时超氧化物歧化酶(SOD)等抗氧化酶活性降低，引起膜脂质过氧化，形成过氧化脂质，过氧化脂质不稳定，会迅速分解产生醛类，其终产物丙二醛(MDA)相应增多，并迅速攻击磷脂及蛋白质，导致色素细胞的氧化性损伤，促进了酪氨酸酶的氧化反应，使黑色素增多并沉积于皮肤基底层。因此降低皮肤组织MDA量，提高抗氧化酶SOD活性，对黄褐斑的预防及治疗有着重要意义。黄褐斑容易反复出现，治疗困难。市场上真正能够解决黄褐斑的产品很少，而且色斑容易复发。氢醌是最早并最广泛应用的美白剂，但因皮肤色素分布不均，而且刺激性较强，甚至可能致癌等原因，在美白和黄褐斑的治疗中已被限制应用。熊果苷是目前临床应用最为广泛的美白剂之一，但是效果有限。
疤痕是物理、生物、化学等因素的损害作用于人体皮肤软组织，导致皮肤软组织的严重损伤而不能完全自行正常修复，转由纤维组织替代修复留下的即影响外观又影响功能的症状。疤痕给患者带来的是巨大的肉体痛苦和精神痛苦，尤其是烧伤、烫伤、严重外伤后遗留的疤痕。对于瘢痕的处理难度较大，目前只能做到使发红、发硬的疤痕变软、变浅，宽的疤痕变窄，粗的疤痕变细，还不能完全彻底消除疤痕。因此在伤口愈合早期即开始进行干预非常重要，可以有效减少疤痕的形成，改善外观，纠正畸形，恢复功能。目前常用来治疗疤痕方法有：手术治疗、激光治疗、冷冻治疗和药物治疗等。常用的药物主要有：糖皮质激素和维甲酸。糖皮质激素有明显的抗组织纤维化的效应，但毒副作用多。维甲酸是维生素A在体内代谢的中间产物，能减轻局部炎症，促进上皮细胞生长，减少胶原合成，使成纤维细胞的DNA合成减少，抑制细胞生长。维甲酸类药物浓度越大，抑制生长作用越明显。但维甲酸疗效有限，系统应用毒副作用也不少，外用对皮肤刺激明显，并随着浓度增加而增加。
斑秃(alopecia areata，AA)是一种非瘢痕性脱发，局部皮肤大体正常。通常情况下为突发的脱发斑，严重时可累及整个头皮，此时称为全秃(alopecia totalis，AT)，当累及包括腋毛、阴毛在内的全身所有毛发时，称为普秃(alopecia universalis，AU)，容易给患者的容貌和心理带来严重的影响。目前病因尚未完全明了，自身免疫功能异常或不稳定、神经精神因素被认为是重要相关因素。斑秃的治愈机率较高，但不同病因引起的斑秃，治愈概率有很大区别。部分斑秃患者可以自然痊愈，还有部分斑秃患者可持续数年。米诺地尔是一种治疗斑秃常见的外用药物，可促进皮肤血管扩张、改善局部血液循环、促进毛发生长。严重斑秃常用的糖皮质激素，主要包括泼尼松龙、复方倍他米松等等，可以口服、外用或皮内注射。对于不适用糖皮质激素类药物的患者，可采用免疫抑制剂治疗，常见的药物有环孢素、甲氨蝶呤、糖皮质激素和免疫抑制剂，这些药物副作用较多。
雄激素源性秃发(AGA)又名脂溢性脱发，是一种雄激素依赖性的遗传性毛发脱落，是常见病、多发病。多为20～30岁左右的男性发病。脱发主要在头顶部，多先从前额两侧发际开始，也有自顶部开始者。脱发区逐渐向上扩延，头发也渐变得稀少纤细，终而头顶部头发大部或全部脱落，但枕后及双侧颞上方头发依存，呈马蹄形外观，此带形区内头发保持正常。脱发处皮肤光亮，毛孔缩小或残留少许细软毳毛。脱发的速度、范围和严重程度，受遗传和 个体影响。一般30岁左右发展最快，严重全秃者少见。女性多为发生于头顶的弥漫性脱发，头顶头发变稀疏。我国流行病学调查显示男性雄激素性脱发患病率为21.3％，女性为6.0％。雄激素性脱发的病因及发病机制尚不明确，一般认为雄激素及其受体在本病的发生中起关键作用，Ⅱ型5a-还原酶是其发病的重要因素。正常生理状态下，雄激素在体内对毛发的生长发育起一定的刺激促进作用，但在某些特定部位能诱导毛发脱失；睾酮是体内主要的雄激素，它经5a-还原酶转化为二氢睾酮，后者可引起终毛向毳毛转变，最终导致脱发。目前尚无理想治疗方法，是脱发性疾病难治的类型。米诺地尔是一种非特异性治疗脱发的药物，是FDA批准用于治疗脱发的一线外用药物，但使用过程中可能引起面部和四肢多毛症，停用后治疗效果逐渐消失。由于雄激素在发病中起很大作用，因而近年来的新治疗方法企图通过抗雄激素的效应来终止毛囊的变小。非那雄胺是一种Ⅱ型5a-还原酶选择性抑制剂，近年发现非那雄胺治疗AGA有效，可持续改善头发的生长情况。但非那雄胺存在引起性功能异常，精子一过性减少和男性乳房发育异常等不良反应，在动物实验中发现有致畸作用，故不宜用于小儿和育龄妇女。西咪替丁需连服5个月或更久，副作用为男性乳房发育、阳痿、性欲降低等。口服避孕药：主要有的索高诺酮、左炔诺孕酮(左旋甲基炔诺孕酮)、炔诺孕酮、炔诺酮、诺孕酯(肟炔诺酯)、双酯炔诺醇和醋炔诺酮等。常用来治疗女性AGA，治疗6～12个月后头发会有所改善。
痤疮(俗称青春豆)是好发于毛囊皮脂腺的慢性炎症性疾病，发病率约9.4％。痤疮的产生与青春期皮肤的生理病理变化密切相关。临床表现主要有粉刺、丘疹、脓疱、结节、囊肿、疤痕等，给患者的容貌和心理带来严重的影响。痤疮与多个发病机制相关，毛囊口角化异常形成粉刺是本病发病的重要基础，炎症和感染是痤疮的发病因素。痤疮患者的皮脂腺较大，皮脂腺分泌增加，皮脂中亚油酸水平相对减少，影响脂肪的合成，导致滤泡上皮缺乏脂肪酸，从而诱导滤泡过度角化，使上皮细胞不能正常脱落，毛囊皮脂腺口过度变小，皮脂不能顺畅排出，形成粉刺。毛囊皮脂腺口被堵塞而形成毛囊皮脂腺内缺氧的环境，造成厌氧性的痤疮丙酸杆菌大量繁殖，分解皮脂，产生化学趋化因子，白细胞聚集形成丘疹。毛囊皮脂腺内的大量中性粒细胞聚集，吞咽痤疮丙酸杆菌后发生炎症反应，使得大量脓细胞堆积而形成脓疱、囊肿，愈后易形成凹陷性疤痕。雄激素水平升高是加速痤疮产生的关键环节，使皮肤角化异常堵塞毛囊皮脂腺导管，导致细菌滞留、繁殖，产生炎症。与痤疮具有类似角化异常机制的疾病包括鱼鳞病、毛周角化病(又名毛发苔藓)、毛囊角化病和汗孔角化症等，毛周角化病见毛囊口扩大，内有角栓，鱼鳞病表现为汗腺和皮脂腺减少，毛囊内出现角质性栓塞。以上疾病容易反复出现，治疗困难。治疗角化异常，消除角栓和粉刺的药物主要是维甲酸类药物。维甲酸类药物可抑制角化，减少皮脂分泌，促进角质形成细胞的正常角化，并具有调节免疫和抗炎作用，从而减少粉刺、丘疹和脓疱的形成，在临床上被广泛应用来治疗痤疮、鱼鳞病、毛周角化病、毛囊角化病和汗孔角化症等角化异常性疾病。但维甲酸类药物外用对皮肤有刺激，容易导致红肿、刺痛，加重原有皮损，长期外用维甲酸类药物可导致皮肤变薄，光敏和皮肤屏障受损等，而口服维甲酸类药物有肝损和血脂升高等不良反应。因此，临床上需要更多治疗此类疾病的药物。
银屑病是一种常见的慢性复发性炎症性皮肤病，从世界范围看，银屑病患者在自然人群中的患病率为0.1％～3％。中重度患者伴发代谢综合征和心血管损害的风险增加。因此银屑病疾病早诊断、早治疗对于控制改善症状、预防并发症有重要意义。轻中度银屑病首选外用治疗。糖皮质激素外用效果较好，但不宜长期、连续大面积使用；维A酸外用治疗斑块型银屑病效果较好，需要注意皮肤刺激；维生素D3衍生物如钙泊三醇也有较好疗效，但不宜用于面部和皮肤皱褶部；钙调神经磷酸酶抑制剂(如他克莫司、吡美莫司等)可用于头皮、皮肤褶皱、生殖器等部位，但长期、大面积使用可增加淋巴癌和皮肤癌的发生风险；各种角质促成剂(如焦油制剂、蒽林软膏、10％～15％喜树碱软膏及水杨酸软膏等)也可外用，但效果有限。免疫抑制药主要用于红皮病型、脓疱型及关节病型银屑病；感染明显或泛发性脓疱型银屑病患者需使用抗菌药物；免疫抑制剂可用于治疗中重度患者，但长期使用不良反应多，常见有 骨髓抑制，肝肾功能损伤、感染风险增加等。
湿疹(又名：特应性皮炎、异位性皮炎)是由多种内外因素引起的瘙痒剧烈的一种皮肤炎症反应，易反复发作。湿疹病因复杂。轻中度湿疹主要以外用治疗为主。根据皮损情况选用适当剂型和药物。亚急性、慢性湿疹应用合适的糖皮质激素霜剂、焦油类制剂或免疫调节剂，如他克莫司软膏、匹美莫司软膏。严重患者可系统用糖皮质激素和免疫抑制剂，但不良反应多，不宜长期使用。
移植物抗宿主病(graft versus host disease，GVHD)是由于移植后异体供者移植物中的T淋巴细胞，经受者发动的一系列“细胞因子风暴”刺激，显著增强了其对受者抗原的免疫反应，以受者靶细胞为目标发动细胞毒攻击，其中皮肤、肝及肠道是主要的靶目标。急性移植物抗宿主病的发生率为30％-45％，慢性者发生率低于急性。异基因造血干细胞移植是可治愈血液系统肿瘤和造血功能异常性疾病的一种有效治疗措施。急性移植物抗宿主病是其主要的严重并发症，发病率高，致死率高，致残率高，为恶性血液病非复发死亡的重要原因。类固醇激素为治疗急性GVHD的一线治疗药物，但约50％的患者出现激素耐药，GVHD反应无法得到控制。其他针对GVHD反应的二线治疗药物如针对T细胞表面抗原的单抗(CD3、CD25等)、化疗药物(吗替麦考酚酯、他克莫司等)，虽然对GVHD具有可靠的治疗效果，但随之而来的免疫力低下、机会性感染，严重削弱了这些药物的优势，最终并没有延长患者的生存时间。因此急需寻找一种新的有效控制激素耐药型急性GVHD的治疗措施。
肺纤维化(pulmonary fibrosis)是一种病因未明、发病机制复杂的弥漫性肺部疾病，目前公认是肺损伤后过度修复导致细胞外基质过度沉积的结果。肺纤维化的发病机制不明，目前公认为肺泡上皮反复损伤与过度修复是发病的关键。病理特点是长期慢性肺部炎症及肺泡持续性损伤导致细胞外基质金属蛋白酶(MMP)聚集，特别是MMP-2、MMP-9异常增多，金属蛋白酶组织抑制剂-1(TIMP-1)降低，使其平衡关系破坏，导致肺大量细胞外基质聚集，组织细胞重构和胶原过度沉积。同时可能抑制组织中血管内皮生长因子(VEGF)表达，降低肺微小静脉血管的通透性，抑制血管内皮细胞分裂增殖及血管再生，加重肺组织损伤，并最终导致弥漫性肺间质疾病一肺纤维化。目前无有效的抗纤维化药物，常用糖皮质激素抗炎以降低抗纤维化进程，但疗效有限，不良反应多。目前的治疗方法远不能满足临床需求，需要寻找更多疗效好、副作用少的新药来控制疾病进展，减少复发和并发症，降低死亡率。
自身免疫性疾病发病率高，全世界至少有数亿患者，其中包括类风湿性关节炎、强直性脊柱炎、克隆恩病、溃疡性结肠炎、红斑狼疮、皮肌炎、硬皮病、干燥综合征等；这些疾病严重时可以累及多器官，导致心、肝、肾、血管、肺、关节和脑等器官损伤，死亡率高，仅次于恶性肿瘤。类风湿性关节炎、强直性脊柱炎、溃疡性结肠炎和克隆恩病具有一些共同的发病机制通路，该类疾病的病因和发病机制相当复杂，目前仍无法根治，需要长期用药控制病情进展。临床常用的治疗药物主要是糖皮质激素和免疫抑制剂等，但是这类药物的有效率仅为50％左右，并且由于其不良反应大，包括骨髓抑制、肝肾功能损伤、骨质疏松、易诱发感染和肿瘤等，限制了长期应用。目前的新型生物制剂也具有免疫抑制作用，有诱发感染和肿瘤的风险，而且价格昂贵，限制了其广泛长期应用。
对于上述疾病，目前的治疗方法远不能满足临床需求，需要寻找更多疗效好、副作用少、价格便宜的新药来控制疾病进展，减少复发和并发症的发生。
发明内容
本发明的目的在于提供具有药用价值的CLY系列化合物或其药学上可接受的盐。
本发明的另一目的在于提供上述化合物的制备方法。
本发明的进一步的目的在于提供上述化合物的用途。
本发明的目的可以通过以下措施达到：
本发明提供具有式I结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，
Figure PCTCN2022099496-appb-000001
其中：
R 1是取代或未取代的含有N、O、S中至少一种的5～6元的杂环、苯环并该杂环(如：异喹啉基)或者至少两个该杂环的并环，此处的并环指2两个环结构通过通用相邻2个原子形成的并环，如吡咯并吡啶；所述取代基为H、卤素或(C1-C4)烷基；
R 2是H、卤素、羟基、甲氧基、乙氧基、氨基、甲基或乙基；
R 3是H、卤素、羟基、甲氧基、乙氧基、氨基、(C1-C3)烷基或以下基团：
Figure PCTCN2022099496-appb-000002
Figure PCTCN2022099496-appb-000003
R 4是取代或未取代的5～6元的环烷基或具有选自N或O的1～3杂原子的4～7元杂环，所述取代基选自H、-NH 2、-OH、(C1-C4)烷基、(C1-C4)烷氧基、氨基、(C1-C4)烷氨基；
优选地，R 4是
Figure PCTCN2022099496-appb-000004
Figure PCTCN2022099496-appb-000005
优选地，如果R 3为H、卤素、羟基、甲氧基、氨基、甲基或以下基团：
Figure PCTCN2022099496-appb-000006
Figure PCTCN2022099496-appb-000007
时，则R 4为
Figure PCTCN2022099496-appb-000008
Figure PCTCN2022099496-appb-000009
如果R 3为
Figure PCTCN2022099496-appb-000010
Figure PCTCN2022099496-appb-000011
时，则R 4为
Figure PCTCN2022099496-appb-000012
Figure PCTCN2022099496-appb-000013
其中：
R 6和R 8各自独立地是H、甲基、卤素或(C1-C4)烷基，且R 6和R 8不同时是卤素；优选地，R 6是H或甲基，且R 8是H；
R 7是羟基、(C1-C4)烷氧基、(C1-C4)烷氧基羰基氧基(C1-C4)烷基或(C1-C4)烷基羰基氧基(Cl-C4)烷基；
R l0和R 11各自独立地是H、(C1-C4)烷基或(C3-C6)环烷基；
R 12选自H、卤素、-OH、-NH 2或(C 1-C 3)烷基；
R 13是H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基；
R 14是H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基；
R 15是羟基、四唑基、(C1-C2)烷基磺酰基或三氟甲基磺酰基，且R 16是H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基。
在一些实施例中，R l是异喹啉-1-基，且R l任选地被氯或甲基单取代。
在一些实施例中，R 2是H、羟基或甲基。
在一些实施例中，R 1是取代或未取代的吡啶基或吡咯并吡啶基；所述取代基为H、氯或甲基。
在一些实施例中，R 12选自H、卤素、-OH、-NH 2或甲基；在一些实施例中，R 12选自H。
本发明提供具有式I-a结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，
Figure PCTCN2022099496-appb-000014
其中，R 3选自H、卤素、羟基、甲氧基、氨基、甲基或者取代或未取代的以下基团中的任意一个：
Figure PCTCN2022099496-appb-000015
Figure PCTCN2022099496-appb-000016
Figure PCTCN2022099496-appb-000017
R 4为
Figure PCTCN2022099496-appb-000018
Figure PCTCN2022099496-appb-000019
其中，R 6、R 7、R 8、R l0、R 11、R 12、R 13、R 14、R 15、R 16的限定与上述任意一段的限定一致。
优选地，如果R 3选自H、卤素、羟基或甲氧基、氨基、甲基或以下取代或未取代的以下基团：
Figure PCTCN2022099496-appb-000020
时，则R 4为
Figure PCTCN2022099496-appb-000021
Figure PCTCN2022099496-appb-000022
如果R 3为
Figure PCTCN2022099496-appb-000023
Figure PCTCN2022099496-appb-000024
时，则R 4为
Figure PCTCN2022099496-appb-000025
Figure PCTCN2022099496-appb-000026
其中R 6和R 8各自独立地是H、甲基、卤素或(C1-C4)烷基，且R 6和R 8不同时为卤素；优选地，R 6是H或甲基，且R 8是H；
R 7是羟基、(C1-C4)烷氧基、(C1-C4)烷氧基羰基氧基(C1-C4)烷氧基或(C1-C4)烷基羰基氧基(Cl-C4)烷氧基；
R l0和R 11各自独立地是H、(C1-C4)烷基或(C1-C4)环烷基；
R 12选自H、卤素、-OH、-NH 2或(C 1-C 3)烷基；
R 13是H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基；
R 14是H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基；
R 15是羟基、四唑基、(C1-C2)烷基磺酰基或三氟甲基磺酰基，且R 16是H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基。
在一些实施例中，具有式I-a结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，R 6是H，且R 8是H；
在一些实施例中，具有式I-a结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，R 12选自H、卤素、-OH、-NH 2或甲基；在一些实施例中，R 12选自H。
在一些实施例中，具有式I-a结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，R 11为H，且R 10是H或甲基，R 13为
Figure PCTCN2022099496-appb-000027
本发明提供具有式I-b结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，
Figure PCTCN2022099496-appb-000028
Figure PCTCN2022099496-appb-000029
其中，R 3为H、卤素、羟基、甲氧基、氨基、甲基或以下取代或未取代的以下基团：
Figure PCTCN2022099496-appb-000030
Figure PCTCN2022099496-appb-000031
R 4为
Figure PCTCN2022099496-appb-000032
Figure PCTCN2022099496-appb-000033
其中，R 6、R 8、R l0、R 11、R 12、R 13的限定与上述任意一段的限定一致。
优选地，如果R 3为H、卤素、羟基、甲氧基、氨基、甲基或以下取代或未取代的以下基团：
Figure PCTCN2022099496-appb-000034
则R 4为
Figure PCTCN2022099496-appb-000035
Figure PCTCN2022099496-appb-000036
如果R 3为
Figure PCTCN2022099496-appb-000037
R 4为
Figure PCTCN2022099496-appb-000038
Figure PCTCN2022099496-appb-000039
其中，R 6、R 8、R l0、R 11、R 12、R 13的限定与上述任意一段的限定一致。
在一些实施例中，具有式I-b结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，R 12选自H、卤素、-OH、-NH 2或甲基；在一些实施例中，R 12选自H。
在一些具体实施例中，本发明提供以下化合物，其互变异构体，其溶剂化物或其药学上 可接受的盐，但不局限于以下化合物范围：
Figure PCTCN2022099496-appb-000040
Figure PCTCN2022099496-appb-000041
Figure PCTCN2022099496-appb-000042
Figure PCTCN2022099496-appb-000043
本发明还提供式(I)所示化合物的制备方法：
Figure PCTCN2022099496-appb-000044
由原料
Figure PCTCN2022099496-appb-000045
制备生成
Figure PCTCN2022099496-appb-000046
然后，
Figure PCTCN2022099496-appb-000047
与
Figure PCTCN2022099496-appb-000048
生产终产物I；
其中，R 1、R 2、R 3、R 4定义如上所述。
本发明还提供一种药物组合物，以本发明所述化合物或其药学上可接受的盐为活性成分或主要活性成分，辅以药学上可接受的载体组成。
本发明还提供本发明所述化合物在制备治疗和/或预防疾病药物中的应用。
在一些实施例中，本发明提供本发明所述化合物在制备治疗和/或预防黄褐斑、疤痕、雄性激素性脱发、脂溢性脱发、斑秃、痤疮、肺纤维化、银屑病、湿疹或特应性皮炎等疾病的药物中的应用。
本发明所述的化合物或组合物可制备为药学上允许的任何剂型，例如为适于口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、眼内、局部、皮肤外用、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
在一种优选的实施方式中，本发明所述的剂型为凝胶、乳剂、膏剂、搽剂、洗剂、喷雾剂、溶液、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂或注射剂。
说明书中的定义：
“C 5～C 6一元醇”表示被一个羟基取代的含有5或6个碳原子的饱和的脂烃基，包括直链和支链基团。
“杂环”表示4到7个环原子的饱和环状基团，其中一个或两个或三个环原子是选自N、O或S(O)m(其中m是0至2的整数)的杂原子，其余环原子是C，其中一个或两个C原子可以可选地被羰基代替。杂环基的环可以可选地独立地被一个、两个或三个取代基取代。
“烷基”表示1-20个碳原子的饱和的脂烃基，包括直链和支链基团(本申请书中提到的数字范围，例如“1-4”，是指该基团，此时为烷基，可以含1个碳原子、2个碳原子、3个碳原子或4个碳原子。烷基可以是取代的或未取代的。
“环烷基”表示全部为碳的单环或稠合的环(“稠合”环意味着系统中的每个环与系统中的其它环共享毗邻的一对碳原子)基团，其中一个或多个环不具有完全连接的π电子系统，环烷基的实例包括不限于环丙烷、环丁烷、环戊烷、环戊烯、环己烷、金刚烷、环己二烯、环庚烷和环庚三烯。
“烷氧基”表示-O-(未取代的烷基)或-O-(未取代的环烷基)。代表性实例包括但不限于甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基等。
“烷氨基”表示-NH-(未取代的烷基)、-NH-(未取代的环烷基)、-N-(未取代的烷基) 2或-N-(未取代的环烷基) 2。代表性实例包括但不限于甲氨基、乙氨基、丙氨基、丁氨基、环丙氨基、环丁氨基、环戊氨基、环己氨基等。
“(C1-C4)烷氧基羰基氧基(C1-C4)烷基”表示(C1-C4)烷基-O-C(O)-O-(C1-C4)烷基-。
“(C1-C4)烷基羰基氧基(Cl-C4)烷基”-表示(C1-C4)烷基C(O)-O-(C1-C4)烷基-。本发明的化合物CLY系列或其药学上可接受的盐可应用于制药领域，动物模型实验表明，化合物CLY系列均能明显减少黄褐斑鼠模型皮肤和血中的酪氨酸酶水平，减少皮肤中肝细胞因子(SCF)、C-kit蛋白表达，抑制黄褐斑的色素形成；能明显促进皮肤创面愈合，减少疤痕形成；能够明显促进雄激素性脱发鼠模型的毛发生长，减少雄激素对毛囊的破坏；CLY系列能够明显抑制银屑病和湿疹小鼠模型的炎症反应；CLY系列化合物能明显提高急性GVHD小鼠生存时间，减轻临床症状，显示对急性GVHD具有治疗作用；CLY系列化合物能明显降低肺纤维化小鼠模型肺组织中MMP-2、MMP-9水平，增加TIMP-1和VEGF水平，同时可提高外周血中SOD、CAT酶水平，显示对肺纤维化具有抑制作用；CLY系列化合物能通过降低外周血中IL-17水平和升高IL-10等炎症指标来改善类风湿关节炎小鼠的关节炎症状。由于许多疾病的具有共同的发病通路，该化合物的药效包括但不限于以上疾病。该化合物可以单独使用也可以联合其他药物使用，为以上疾病的治疗提供了新的药物选择。
附图说明
图1 CLY系列化合物能够明显促进脱发模型小鼠的毛发生长。
图2 CLY系列化合物能够明显减轻小鼠银屑病样炎症反应。
图3 CLY-2核磁检测结果。
图4 CLY-8核磁检测结果。
具体实施方式
下面结合实施例对本发明做进一步说明，应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，凡在本发明的构思前提下对本发明制备方法的简单改进都属于本发明的保护范围之内。下面实施例未注明具体条件的实验方法，通常按照本领域的公知手段。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
实施例1制备(R)-4-(3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-N-(3-氯吡啶-2-基)-N-(1-甲基哌啶-3-基)苯甲酰胺(简称CLY-1)和(R)-4-(3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-N-(3-氯吡啶-2-基)-N-(1-乙基哌啶-3-基)苯甲酰胺(简称CLY-2)的方法：
1、合成线路
Figure PCTCN2022099496-appb-000049
2、具体实施方式：
(1)(R)-3-((3-氯吡啶-2-基)氨基)哌啶-1-甲酸叔丁酯的合成
(R)-1-叔丁氧羰基-3-氨基哌啶、2-溴-3-氯吡啶、叔丁醇钠，二氧六环加入到250ml容器瓶中，氮气保护，开启搅拌，加入RuPhosPd G3、配体RuPhos，加热至100℃，反应7小时，停止反应，倒入水中，用乙酸乙酯分次萃取，合并乙酸乙酯层后，用水3次洗涤，回收乙酸乙酯层后得到残留膏状物，用硅胶拌样，经硅胶柱层析得到(R)-3-((3-氯吡啶-2-基)氨基)哌啶-1-甲酸叔丁酯。
(2)哌啶酰胺的合成
4-(3H-[1,2,3]三氮唑[4,5-b]吡啶-3-基)苯甲酸加入到容器瓶中，加入预干燥的甲苯，N,N- 二甲基甲酰胺，氯化亚砜于30℃加热搅拌至澄清，减压回收溶剂得到残留固体。加入预干燥四氢呋喃50ml，(R)-3-((3-氯吡啶-2-基)氨基)哌啶-1-甲酸叔丁酯，搅拌至澄清，置冰水浴，加入双三甲基硅基胺基锂，搅拌1小时，撤去冰水浴，搅拌2小时，倒入水中，用乙酸乙酯萃取，洗涤乙酸乙酯层后，减压回收溶剂得到膏状物。
(3)哌啶胺的合成
哌啶酰胺，二氯甲烷，三氟乙酸，室温搅拌过夜。倒入水中，加碳酸氢钠调节pH＝10～11，用二氯甲烷萃取，洗涤后，减压回收溶剂，得到膏状残留，经硅胶柱层析得到哌啶胺。
(4)CLY-1的合成
取哌啶胺于二氯甲烷中溶解，加入碘甲烷，碳酸银室温避光搅拌48小时，反应液直接上硅胶柱层析得到CLY-1。
CLY-1的Chemical Formula:C 23H 22ClN 7O
氢谱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)。
(5)CLY-2的合成
取哌啶胺于二氯甲烷中溶解，加入碘乙烷，碳酸银室温避光搅拌48小时，反应液直接上硅胶柱层析得到CLY-2。CLY-2质谱见图3。
实施例2制备(R)-4-(3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-N-(3-氯吡啶-2-基)-N-(吡咯烷-3-基)苯甲酰胺(简称CLY-8)的方法：
2、合成线路
Figure PCTCN2022099496-appb-000050
2、具体合成步骤：
(1)ETH-1的合成
(R)-1-叔丁氧羰基-3-氨基吡咯烷11.8克、2-溴-3-氯吡啶11.0克、叔丁醇钠10.0克，甲苯70ml加入到250ml三口瓶中，氮气保护，开启搅拌，加入0.1克乙酸钯、1，1‘-联-2-萘酚，三(二甲氨基)膦，加热至100℃，反应7小时，停止反应，倒入500ml水中，用乙酸乙酯 1000ml分次萃取，合并乙酸乙酯层后，用水200*3洗涤，回收乙酸乙酯层后得到残留膏状物，用硅胶拌样，经硅胶柱层析得到ETH-1约7克。
(2)吡咯烷酰胺的合成
4-(3H-[1,2,3]三氮唑[4,5-b]吡啶-3-基)苯甲酸在溶剂中，加入草酰氯和催化剂N,N-二甲基甲酰胺，在25～55℃反应至澄清，减压回收溶剂后，残留物加入溶剂，加入(R)-3-((3-氯吡啶-2-基)氨基)吡咯烷-1-羧酸叔丁酯，置冰水浴降温至5℃以下，加入双(三甲基硅基)胺基锂，得到吡咯烷酰胺，即(R)-3-(4-(3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-N-(3-氯吡啶-2-基)苯甲酰胺)吡咯烷-1-羧酸叔丁基酯。
(3)化合物CLY-8的合成
吡咯烷酰胺10克，二氯甲烷50ml，4mol/L盐酸甲醇溶液10ml，室温搅拌过夜。倒入100ml水中，加碳酸氢钠调节pH＝10～11，用100ml二氯甲烷萃取，洗涤后，减压回收溶剂，得到膏状残留，经硅胶柱层析得到3.2克化合物(R)-4-(3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-N-(3-氯吡啶-2-基)-N-(吡咯烷-3-基)苯甲酰胺(简称CLY-8)，M+H＝420.1(质谱检测结果见图4)。
参照以上同样的方法还可以合成以下表1化合物：
表1
Figure PCTCN2022099496-appb-000051
Figure PCTCN2022099496-appb-000052
Figure PCTCN2022099496-appb-000053
实施例3：(R)-N-(3-氯吡啶-2-基)-N-(吡咯烷-3-基)-3-(1H-四唑-5-基)苯甲胺(简称CLY-14)的合成
Figure PCTCN2022099496-appb-000054
步骤1，同实施例3可以得到(R)-3-(N-(3-氯吡啶-2-基)-3-氰基苯甲酰胺基)吡咯烷-1-羧酸叔丁酯。
Figure PCTCN2022099496-appb-000055
步骤2，将(R)-3-(N-(3-氯吡啶-2-基)-3-氰基苯甲酰胺基)吡咯烷-1-羧酸叔丁酯4.2克，叠氮钠1克，三乙胺盐酸盐2克，加入到50mlN,N-二甲基甲酰胺中，于100℃搅拌反应20小时后冷却，倒入200ml水中，滴加浓盐酸至pH2-3，过滤得到固体，用水洗涤后，烘干。使用二氯甲烷50ml，甲醇ml溶解后，加入浓度为4mol/L的氯化氢二氧六环溶液10ml室温搅拌过夜，减压浓缩得到固体经硅胶层析得到(R)-N-(3-氯吡啶-2-基)-N-(吡咯烷-3-基)-3-(1H-四唑-5-基)苯甲酰胺2.7克，MS(ES+):370(M+H)。
Chemical Formula:C17H16ClN7O
Molecular Weight:369.81
氢谱CDCl 3中数据：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)。
参照同样的方法还可以合成以下表2化合物：
表2
Figure PCTCN2022099496-appb-000056
Figure PCTCN2022099496-appb-000057
Figure PCTCN2022099496-appb-000058
Figure PCTCN2022099496-appb-000059
Figure PCTCN2022099496-appb-000060
Figure PCTCN2022099496-appb-000061
Figure PCTCN2022099496-appb-000062
实施例4：(R)-4-(3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-N-(3-氯吡啶-2-基)-N-(四氢-2H-吡喃-3-基)苯甲酰胺(简称CLY-42)的合成。
步骤1：(R)-3-氯-N-(四氢-2H-吡喃-3-基)吡啶-2-胺的合成。
Figure PCTCN2022099496-appb-000063
(R)-四氢-2H-吡喃-3-胺10.1克、2-溴-3-氯吡啶14.0克、叔丁醇钠12.0克，四氢呋喃100ml加入到250ml三口瓶中，氮气保护，开启搅拌，加入0.1克乙酸钯、1，1‘-联-2-萘酚，三(二甲氨基)膦，加热至65℃，反应12小时，停止反应，倒入500ml水中，用乙酸乙酯1000ml分次萃取，合并乙酸乙酯层后，用水200*3洗涤，回收乙酸乙酯层后得到残留膏状物，用硅胶拌样，经硅胶柱层析得到(R)-3-氯-N-(四氢-2H-吡喃-3-基)吡啶-2-胺约8克。
步骤2：
Figure PCTCN2022099496-appb-000064
4-(3H-[1,2,3]三氮唑[4,5-b]吡啶-3-基)苯甲酸5克在50ml二氯甲烷中，加入草酰氯2.5ml和催化剂N,N-二甲基甲酰胺0.2ml，在室温反应至澄清，减压回收溶剂后，残留物加入四氢呋喃50ml溶剂，加入上一步产物(R)-3-氯-N-(四氢-2H-吡喃-3-基)吡啶-2-胺4克的四氢呋喃溶液，置冰水浴降温至5℃以下，加入双(三甲基硅基)胺基锂1M溶液20ml，室温搅拌反应过夜，倒入水中，用乙酸乙酯萃取100ml*3，水洗涤后减压回收乙酸乙酯后得到残留物经柱层析，得到(R)-4-(3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-N-(3-氯吡啶-2-基)-N-(四氢-2H-吡喃-3-基)苯甲酰胺约6克，MS(ES+):435(M+H)。
参照同样的方法还可以合成
Figure PCTCN2022099496-appb-000065
实施例5：1-(5-(4-(4-((3-氯吡啶-2-基)((R)-吡咯烷-3-基)氨基甲酰基)苯基)-1-甲基-1H-吡唑-5-基)-2H-四唑-2-基)异丁酸乙酯(简称CLY-44)的合成。
步骤1：
Figure PCTCN2022099496-appb-000066
(1)(R)-3-(((3-氯吡啶-2--2-基)氨基)吡咯烷-1-羧酸叔丁酯的合成。
(R)-1-叔丁氧羰基-3-氨基吡咯烷11.8克、2-溴-3-氯吡啶11.0克、叔丁醇钠10.0克，甲苯70ml加入到250ml三口瓶中，氮气保护，开启搅拌，加入0.1克乙酸钯、1，1‘-联-2-萘酚，三(二甲氨基)膦，加热至100℃，反应7小时，停止反应，倒入500ml水中，用乙酸乙酯1000ml分次萃取，合并乙酸乙酯层后，用水200*3洗涤，回收乙酸乙酯层后得到残留膏状物，用硅胶拌样，经硅胶柱层析得到(R)-3-(((3-氯吡啶-2--2-基)氨基)吡咯烷-1-羧酸叔丁酯 约7克
步骤2：
Figure PCTCN2022099496-appb-000067
在0℃，向(R)-3-(((3-氯吡啶-2--2-基)氨基)吡咯烷-1-羧酸叔丁酯5克和4-溴苯甲酰氯4克在100ml四氢呋喃溶液中滴加1M双(三甲基硅基)胺基锂溶液20ml，室温搅拌过夜，用乙酸乙酯300ml稀释后，用水200ml*3洗涤，将乙酸乙酯层减压浓缩得到残留物柱层析，得到(R)-3-(4-溴-N-(3-氯吡啶-2--2-基)苯甲酰胺基)吡咯烷-1-羧酸叔丁酯约4克。
步骤3：(R)-3-(N-(3-氯吡啶-2-基)-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苯甲酰胺)吡咯烷-1-羧酸叔丁酯的制备
Figure PCTCN2022099496-appb-000068
将(R)-3-(4-溴-N-(3-氯吡啶-2--2-基)苯甲酰胺基)吡咯烷-1-羧酸叔丁酯3克，双(频那醇合)二硼3克，醋酸钾2克加入到1，4-二氧六环30ml中搅拌，氮气保护，加入氯化钯0.6克，加热到100℃搅拌5小时，减压浓缩，残留物通过硅胶柱层析得到3.1克(R)-3-(N-(3-氯吡啶-2-基)-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苯甲酰胺)吡咯烷-1-羧酸叔丁酯。
步骤4：1-(5-(4-(4-((3-氯吡啶-2-基)((R)-吡咯烷-3-基)氨基甲酰基)苯基)-1-甲基-1H-吡唑-5-基)-2H-四唑-2-基)异丁酸乙酯的合成
Figure PCTCN2022099496-appb-000069
将(R)-3-(N-(3-氯吡啶-2-基)-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苯甲酰胺)吡咯烷-1-羧酸叔丁酯3克，1-(5-(4-碘-1-甲基-1H-吡唑-5-基)-2H-四唑-2-基)异丁酸乙酯2.5克，磷酸三钾5克，甲烷磺酸(2-二环己基膦基-2',6'-二甲氧基-1,1'-联苯基)(2'-氨基-1,1'-联苯-2-基)钯(II)0.1g加入到50％1，4-二氧六环水溶液中，在氮气保护下，于100℃反应5小时。反应液降到室温后，用200ml乙酸乙酯稀释，分取乙酸乙酯层，用水100ml*3洗涤后，无水硫酸钠干燥，减压回收乙酸乙酯得到残留物。将残留物用30ml二氯甲烷溶解，加入4mol/L,1，4-二氧六环氯化氢溶液4ml，搅拌2小时，减压回收溶剂至干，用硅胶层析分离得到1-(5-(4-(4-((3-氯吡啶-2-基)((R)-吡咯烷-3-基)氨基甲酰基)苯基)-1-甲基-1H-吡唑-5-基)-2H-四唑-2-基)异丁酸乙酯约0.5克。MS(ES+):565(M+H)。
参照同样的方法还可以合成
Figure PCTCN2022099496-appb-000070
实施例6大鼠药代动力学性质研究
1.实验方法：
购买雄性SD大鼠随机分配，将以上每个化合物试验用的12只大鼠再随机分成两组，其中6只大鼠分别采用静脉注射给药(1mg/kg)，给药后0.0833、0.25、0.5、1、2、4、6、8和24h分别采血并分离出血浆，另外6只大鼠采用灌胃给药(5mg/kg)，给药后0.25、0.5、1、2、4、6、8和24h分别采血并分离血浆。采用LC/MS/MS法测定每种化合物在血浆中的浓度，并用Phocnix WinNonlin 6.2.1软件计算相关药代动力学参数，计算其在大鼠中的生物利用度，评价每种化合物的药代动力学性质。
2.实验结果
以上所有化合物中，化合物CLY-1、CLY-2、CLY-3、CLY-4、CLY-5、CLY-8、CLY-11、CLY-13、CLY-17、CLY-18、CLY-19、CLY-21、CLY-26、CLY-28、CLY-30、CLY-32、CLY-35、CLY-36、CLY-37、CLY-44在雄性SD大鼠中生物利用度较好，分别为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％、50.6％，说明以上化合物具有良好的药代动力学性质。
实施例7化合物CLY对豚鼠黄褐斑模型的影响
1.实验方法
1.1实验材料
1.1.1试剂：黄体酮(20mg/ml)购自上海通用药业股份有限公司，熊果苷软膏购自上海新先锋药业有限公司，酪氨酸、丙二醛(MDA)、超氧化物歧化酶(SOD)试剂盒购自南京建成生物工程研究所。
1.1.2仪器：DY89-Ⅱ型电动玻璃匀浆机购自宁波新兰生物科技股份有限公司，系统生物显微镜(Image-Pro Plus 6.0)购自美国Media Cybernetics公司。
1.1.3实验动物：SPF级健康纯系雌性豚鼠，体重(230±30)g，来源于上海斯莱克实验动物技术有限公司。
1.1.4治疗乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量CLY系列化合物混匀形成0.25％混合乳剂。本实施例所用的乳膏基质是指乳膏除去活性成分的基质成分。
1.2动物分组与造模
按体重编号，采用随机排列表法分为模型对照组(涂抹乳膏基质)、空白对照组(涂抹乳膏基质)、CLY-1治疗组(皮肤涂抹0.25％CLY-1乳膏)、CLY-2治疗组(皮肤涂抹0.25％CLY-2乳膏)、CLY-3治疗组(皮肤涂抹0.25％CLY-3乳膏)、CLY-4治疗组(皮肤涂抹0.25％CLY-4乳膏)、CLY-5治疗组(皮肤涂抹0.25％CLY-5乳膏)、CLY-11治疗组(皮肤涂抹0.25％CLY-11乳膏)、CLY-19治疗组(皮肤涂抹0.25％CLY-19乳膏)、CLY-36治疗组(皮肤涂抹0.25％CLY-36乳膏)、阳性治疗组(皮肤涂抹0.25％熊果苷乳膏)，每组6只。除了空白对照组外，其余各组豚鼠均于后腿根部肌内注射20mg/ml黄体酮注射液(7.5mg/kg)，每天1次，连续注射30d建立黄褐斑模型。豚鼠背部模型区域皮肤呈均一稳定、边界清晰的深褐色色斑为模型复制成功。造模后，模型对照组、空白对照组、各CLY系列治疗组、阳性治疗组豚鼠背部涂抹相应乳膏进行干预，每天1次，连续30天。
1.3观察指标
(1)酪氨酸、MDA含量，SOD活性测定
所有豚鼠取备用皮肤组织1块用预冷生理盐水冲洗，除去皮下脂肪及其他结缔组织后拭干，每块皮肤组织各切取0.5g，再分别放入盛有2.0ml预冷生理盐水的5根小试管内，高速分散机以10r/min的速度匀浆，持续10s，重复1次，再以3500r/m速度离心15min，取上清液。酪氨酸测定采用高效液相法，MDA测定用硫代巴比妥酸法，SOD测定用黄嘌呤氧化酶法，按试剂盒说明书检测酪氨酸、MDA含量及SOD活性。
(2)皮肤黑色素细胞的病理形态学观察
所有豚鼠取备用皮肤组织1块，约2cm×1cm，10％多聚甲醛固定，病理组织检测，免疫组织化学染色，观察黑色素细胞染色和数目，并根据文献判断阳性细胞：无，0分；<15％，0.5分；<30％，1分；>30％，2分。每张切片分别观察5个视野，找到皮肤表皮和附件上皮细胞胞浆中呈现棕色反应的阳性目标后，用BX50F4北航病理图像分析系统定量分析，得到每只豚鼠5个视野黑色素阳性目标的平均面积、目标与统计场面积之比(面密度)、目标个数与统计场面积之比(数密度)、平均灰度、平均光密度及积分光密度。
1.4统计学方法
采用SPSS16.0软件统计，计量资料以均数±标准差(x±s)表示，多组间比较采用单因素方差分析，组间比较用t检验，P<0.05为差异有统计学意义。
2.实验结果
(1)各组豚鼠酪氨酸、MDA含量及SOD活性
各组豚鼠酪氨酸、MDA含量及SOD活性检测结果见表3。模型组豚鼠皮肤酪氨酸、MDA含量均高于空白组，SOD活性低于空白组，说明皮肤黄褐斑模型建立成功；与模型组比较，CLY系列治疗组和阳性治疗组皮肤中酪氨酸、MDA含量较低，而SOD活性则升高，差异均有统计学意义(P<0.05)。
表3各组豚鼠酪氨酸、MDA含量及SOD活性比较情况
*为与模型组比较P<0.05
组别 n 酪氨酸(ug) MDA(nmol/ml) SOD(NU/ml)
模型对照组 6 0.68±0.16 13.73±1.62 86.63±12.72
空白对照组 6 0.42±0.06* 10.16±0.95* 107.12±7.35*
CLY-1治疗组 6 0.45±0.07* 11.32±1.15* 103.65±9.63*
CLY-2治疗组 6 0.43±0.06* 10.86±1.19* 105.23±9.82*
CLY-3治疗组 6 0.51±0.07* 11.57±1.18* 97.45±9.23*
CLY-4治疗组 6 0.53±0.06* 11.62±1.15* 95.12±8.65*
CLY-5治疗组 6 0.43±0.04* 10.65±1.14* 105.89±9.86*
CLY-11治疗组 6 0.54±0.06* 11.62±1.21* 95.13±9.16*
CLY-19治疗组 6 0.56±0.07* 11.75±1.26* 96.53±9.21*
CLY-36治疗组 6 0.58±0.08* 11.83±1.27* 93.42±9.03*
阳性治疗组 6 0.49±0.09* 11.82±1.43* 101.26±10.36*
(2)各组豚鼠黑色素细胞的面积、数量及深浅度
各组豚鼠黑色素细胞的面积、数量及深浅度见表4和表5。与空白组比较，模型组豚鼠皮肤黑色素沉着面积增大、黑色素细胞个数增多，光密度变大，着色变深；与模型组比较，各CLY系列治疗组和阳性治疗组豚鼠皮肤的黑色素沉着面积缩小、黑色素细胞个数减少，光密度变小，着色变浅。
表4各组豚鼠黑色素细胞的面积、数量比较
*为与模型组比较P<0.05
组别 n 面积(um2) 面密度 个数 数密度
模型对照组 6 2335.12±436.45 0.013±0.005 3.24±1.23 5.53±1.69
空白对照组 6 316.97±55.12* 0.003±0.001* 1.14±0.38* 1.67±0.81*
CLY-1治疗组 6 372.31±58.32* 0.005±0.002* 1.93±0.55* 1.91±0.96*
CLY-2治疗组 6 366.26±62.15* 0.004±0.002* 1.75±0.53* 1.88±1.34*
CLY-3治疗组 6 436.32±79.12* 0.008±0.003* 2.13±0.61* 2.82±1.25*
CLY-4治疗组 6 496.56±86.23* 0.009±0.003* 2.45±0.67* 2.95±1.41*
CLY-5治疗组 6 345.31±56.21* 0.004±0.001* 1.43±0.32* 1.75±1.29*
CLY-11治疗组 6 417.53±73.16* 0.006±0.002* 1.98±0.56* 2.16±1.38*
CLY-19治疗组 6 405.62±73.45* 0.006±0.003* 1.95±0.52* 2.18±1.35*
CLY-36治疗组 6 495.32±79.43* 0.007±0.004* 1.98±0.59* 2.67±1.38*
阳性治疗组 6 489.13±95.67* 0.006±0.002* 2.39±0.72* 2.29±1.65*
表5各组豚鼠黑色素细胞深浅度比较
*为与模型组比较P<0.05
组别 n 平均灰度 平均光密度 积分光密度
模型对照组 6 0.68±0.16 13.59±1.45 87.65±11.32
空白对照组 6 0.39±0.06* 10.16±0.95* 108.63±8.12*
CLY-1治疗组 6 0.43±0.07* 11.23±1.21* 103.34±9.71*
CLY-2治疗组 6 0.41±0.06* 10.68±1.18* 106.96±9.84*
CLY-3治疗组 6 0.52±0.08* 11.42±1.32* 95.36±9.86*
CLY-4治疗组 6 0.58±0.09* 11.61±1.38* 92.45±9.36*
CLY-5治疗组 6 0.40±0.05* 10.59±1.13* 107.06±9.57*
CLY-11治疗组 6 0.46±0.06* 11.26±1.22* 101.38±9.92*
CLY-19治疗组 6 0.45±0.06* 11.28±1.24* 102.65±9.97*
CLY-36治疗组 6 0.47±0.07* 11.53±1.22* 97.62±9.91*
阳性治疗组 6 0.49±0.09* 11.91±1.36* 97.96±10.37*
3.实验结论
CLY-1、CLY-2、CLY-3、CLY-4、CLY-5、CLY-11、CLY-19和CLY-36能通过提高皮肤组织中SOD酶活性，降低酪氨酸、MDA的含量，抑制黑色素细胞和黑色素瘤细胞的酪氨酸酶活性，增强皮肤细胞的氧化还原反应，减少自由基产生，抑制黑色素的形成，从而治疗黄褐斑。
实施例8化合物CLY系列对大鼠疤痕模型的影响
1.实验方法
1.1治疗乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量CLY系列化合物混匀形成混合乳剂。本实施例所用的乳剂基质是指乳剂除去活性成分的基质成分。
1.2实验动物分组与造模：SPF级雄性大鼠，体重(210±28)g，来源于南医大动物中心。大鼠按体重编号，采用随机排列表法模型对照组(涂抹乳膏基质)、CLY-1治疗组(皮肤涂抹0.5％CLY-1乳膏)、CLY-2治疗组(皮肤涂抹0.5％CLY-2乳膏)、CLY-3治疗组(皮肤涂抹0.5％CLY-3乳膏)、CLY-4治疗组(皮肤涂抹0.5％CLY-4乳膏)、CLY-8治疗组(皮肤涂抹0.5％CLY-8乳膏)、CLY-11治疗组(皮肤涂抹0.5％CLY-11乳膏)、CLY-19治疗组(皮肤涂抹0.5％CLY-19乳膏)、CLY-36治疗组(皮肤涂抹0.5％CLY-36乳膏)，每组各6只。各组大鼠用2％戊巴比妥钠(120mg/kg)腹腔注射麻醉后固定于手术台上，然后在其背部中左侧选择一块4×5cm的完整皮肤，8％硫化钠脱毛，用组织剪在脱毛处各剪成一直径为2.4cm圆形深达肌筋膜的伤口，破坏部分肌肉表面筋膜。动物分笼饲养防止大鼠撕咬、舔蹭。创面每日涂2％碘酊常规消毒，观察大鼠创面愈合情况。
2.实验结果
2.1大鼠创面观测结果
创面每天常规消毒，第1d、3d、5d、7d、12d、20d观察大鼠创面。自第5天开始各CLY系列治疗组伤口恢复速度明显比模型组快，创面面积逐渐变小。第12天，各治疗组创面已经基本恢复，而模型组仍有约0.5cm 2大小的创面。到第20天时，各组创面均已经恢复，模型对照组留下明显疤痕，而各治疗组只留下数量不等的色素沉着。
3.实验结论
CLY-1、CLY-2、CLY-3、CLY-4、CLY-8、CLY-11、CLY-19和CLY-36均能明显促进皮肤创面愈合，减少疤痕(瘢痕)形成。
实施例9化合物CLY系列对大鼠脱发模型的影响
1.实验方法
1.1材料
(1)化合物CLY-1、CLY-2、CLY-3、CLY-4、CLY-8、CLY-11、CLY-19、CLY-36酊制备方法：75％乙醇分别与适量以上化合物混匀配制成不同浓度的酊剂。
(2)阳性治疗药：5％米诺地尔酊(商品名：蔓迪，浙江万马药业有限公司)
(3)实验动物：SPF级Wistar大鼠，雄性，来源于上海斯莱克实验动物有限责任公司。
1.2动物分组与造模
Wistar大鼠按照体重随机分为阴性对照组(外用75％乙醇)、模型组(外用75％乙醇)、阳性对照组(外用5％米诺地尔酊)、CLY-1外用治疗组(外用5％CLY-1酊)、CLY-2外用治疗组(外用5％CLY-2酊)、CLY-3外用治疗组(外用5％CLY-3酊)、CLY-4外用治疗组(外用5％CLY-4酊)、CLY-5外用治疗组(外用5％CLY-5酊)、CLY-11外用治疗组(外用5％CLY-11酊)、CLY-19外用治疗组(外用5％CLY-19酊)、CLY-36外用治疗组(外用5％CLY-36酊)，CLY-1静脉注射组(2mg/kg.d)、CLY-2静脉注射组(2mg/kg.d)、CLY-3静脉注射组(2mg/kg.d)、CLY-4静脉注射组(2mg/kg.d)、CLY-5静脉注射组(2mg/kg.d)、CLY-11静脉注射组(2mg/kg.d)、CLY-19静脉注射组(2mg/kg.d)、CLY-36静脉注射组(2mg/kg.d)，每组各10只。实验前每只大鼠选取背部4cmx5cm面积的区域将毛脱去作为观察区。除阴性对照组外，大鼠颈后皮下注射丙酸睾酮注射液[5ml/(kg·d)]，每天1次，连续60d，建立SA模型。连续皮下注射丙酸睾丸酮4周后大鼠逐渐出现毛发脱落.残存毛发变得纤细、质脆，证明脱发模型成功建立。同时皮肤涂抹给药，均按2mL/(只·次)分别涂抹对应药物组大鼠背部观察区，每日2次，给药间隔8h。静脉给药每日一次。阴性对照组和模型对照组涂抹75％乙醇溶液，2mL/(只·次)，每日2次，连续60d。
1.3观察指标及测试方法
给药每15天于每只大鼠背部观察区拔取10根毛发，用游标卡尺测量毛发长度。给药60d后，取实验观察区皮肤，进行常规组织脱水、石蜡包埋、HE染色，光镜镜检，观察大鼠皮肤毛囊和皮脂腺组织病理学改变。对各组病变进行半定量分析。分级标准如下：皮肤真皮组织细胞和皮下毛囊、皮脂腺结构正常记为“一”：皮肤真皮未见有增生，毛囊、皮脂腺病变局限，皮下未见有炎症记为“±”：皮肤真皮组织未见有明显增生，毛囊明显囊性变.皮脂腺未见有明显增生，皮下未见有炎症记为“+”：皮肤真皮组织有节段性增生，不明显，少部分毛囊有囊性变，皮脂腺有轻度增生肥大.皮下未见有明显炎症记为“++”：皮肤真皮组织细胞有不同程度节段性增生，部分毛囊囊性变，表现毛囊大小不均匀，周边部无细胞。皮脂腺有增生，增生腺体内细胞核较少，个别大鼠皮下有轻度炎性增生记为“+++”。
2.实验结果
2.1CLY对大鼠毛发生长的影响
各治疗组大鼠在给药第15、30、45、60天的毛发长度均长于模型对照组，差异均有统计学意义(P<0.01)，与阳性治疗组比较，差异均有统计学意义(P<0.05)。见表6
表6各组对大鼠毛发生长长度的影响
Figure PCTCN2022099496-appb-000071
Figure PCTCN2022099496-appb-000072
*与模型对照组比较差异均有统计学意义(P<0.01)
2.2 CLY对大鼠观察区皮肤组织真皮浅层毛囊形态的影响
模型组大鼠部分皮肤真皮组织细胞有不同程度节段性增厚，大鼠皮下有轻度淋巴细胞增生；部分大鼠皮下毛囊有明显囊性变，毛囊大小不等，增大毛囊腔内有脱落角化物.周边有轻度纤维化，毛囊周边细胞消失或细胞层次明显减少，腔内似有钙化物染成蓝色，皮脂腺数目增多，部分腺体有肥大，肥大腺体细胞核明显减少，正常毛囊数减少。各治疗组大鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变与模型组比较有不同程度减轻。各治疗组大鼠皮肤损伤毛囊数与模型对照组比较均明显减少，差异有统计学意义(P<0.01)。与模型对照组比较，各治疗组大鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变明显减轻，差异有统计学意义(P<0.01)。与阳性治疗组比较，差异均有统计学意义(P<0.05)。见表7。
表7各组对大鼠皮肤毛囊和皮脂腺的影响(只)
组别 只数 — ± + ++ +++ P值*
阴性对照组 10 10 0 0 0 0 <0.01
模型对照组 10 0 0 1 4 5 —
阳性对照组 10 0 2 4 3 1 <0.01
CLY-1外用组 10 0 5 4 1 0 <0.01
CLY-2外用组 10 0 6 3 1 0 <0.01
CLY-3外用组 10 0 4 5 1 0 <0.01
CLY-4外用组 10 0 3 4 3 0 <0.01
CLY-5外用组 10 0 6 4 0 0 <0.01
CLY-11外用组 10 0 5 4 1 0 <0.01
CLY-19外用组 10 0 5 3 2 0 <0.01
CLY-36外用组 10 0 5 2 3 0 <0.01
CLY-1静脉组 10 0 6 4 0 0 <0.01
CLY-2静脉组 10 0 7 3 0 0 <0.01
CLY-3静脉组 10 0 5 4 1 0 <0.01
CLY-4静脉组 10 0 4 5 1 0 <0.01
CLY-5静脉组 10 0 8 2 0 0 <0.01
CLY-11静脉组 10 0 6 3 1 0 <0.01
CLY-19静脉组 10 0 6 2 2 0 <0.01
CLY-36静脉组 10 0 6 1 3 0 <0.01
注：*为与模型对照组比较差异有统计学意义(P<0.01)
3.实验结论
CLY-1、CLY-2、CLY-3、CLY-4、CLY-5、CLY-11、CLY-19和CLY-36无论外用还是系统应用均能明显促进大鼠脱发模型的毛发生长，减轻对皮下毛囊和皮脂腺的损伤，未见明显不良反应。
实施例10化合物CLY系列对小鼠脱发模型的影响
1.实验方法
1.1材料
(1)化合物CLY-1、CLY-2、CLY-3、CLY-4、CLY-8、CLY-11、CLY-19、CLY-36酊制备方法：60％乙醇分别与以上化合物混匀配制成2％浓度的酊剂。
(2)阳性治疗药：2％米诺地尔酊(中国医学科学院皮肤病研究所生产)
(3)实验动物：SPF级雄性健康C57BL/6小鼠，雌雄各半，6-8周龄，体重20-25g，由成都药康生物科技有限公司提供。
1.2动物分组与造模
动物房12h-12h昼夜交替，保持动物自由饮水、进食，温度23-25℃，实验动物进入动物房适应性饲养一周后进入实验。实验小鼠分为11组，阴性对照组(外用60％乙醇)、模型组(外用60％乙醇)、阳性对照组(外用2％米诺地尔酊)、CLY-1外用治疗组(外用2％CLY-1酊)、CLY-2外用治疗组(外用2％CLY-2酊)、CLY-3外用治疗组(外用2％CLY-3酊)、CLY-4外用治疗组(外用2％CLY-4酊)、CLY-8外用治疗组(外用2％CLY-8酊)、CLY-11外用治疗组(外用2％CLY-11酊)、CLY-19外用治疗组(外用2％CLY-19酊)、CLY-36外用治疗组(外用2％CLY-36酊)。每组6只，雌雄各半。选取实验小鼠背部4cm×5cm面积的区域将毛脱去，用3％苦味酸溶液将脱毛区涂成黄色以确定脱毛实验观察区。除阴性对照组外，其他组小鼠颈后皮下注射丙酸睾酮注射液[8ml/(kg·d)]，每天1次，连续60d，建立SA模型。造模同时涂抹给药，均按1ml/(只·次)分别涂抹对应药物组大鼠背部观察区，每日2次，给药间隔2h。正常对照组和模型对照组涂抹赋型剂(60％乙醇溶液)，1ml/(只·次)，每日2次，连续60天。
1.3观察指标及测试方法
给药每15天于每只小鼠背部观察区拔取10根毛发，用游标卡尺测量毛发长度。给药60d后，取实验观察区皮肤，进行常规组织脱水、石蜡包埋、HE染色，光镜镜检，观察小鼠皮肤毛囊和皮脂腺组织病理学改变。对各组病变进行半定量分析。分级标准如下：皮肤真皮组织细胞和皮下毛囊、皮脂腺结构正常记为“一”：皮肤真皮未见有增生，毛囊、皮脂腺病变局限，皮下未见有炎症记为“±”：皮肤真皮组织未见有明显增生，毛囊明显囊性变.皮脂腺未见有明显增生，皮下未见有炎症记为“+”：皮肤真皮组织有节段性增生，不明显，少部分毛囊有囊性变，皮脂腺有轻度增生肥大.皮下未见有明显炎症记为“++”：皮肤真皮组织细胞有不同程度节段性增生，部分毛囊囊性变，表现毛囊大小不均匀，周边部无细胞。皮脂腺有增生，增生腺体内细胞核较少，个别皮下有轻度炎性增生记为“+++”。
2.实验结果
2.1 CLY系列对小鼠毛发生长的影响
各组小鼠在给药第15、30、45、60天的毛发长度均长于模型对照组，差异均有统计学意义(P<0.01)，与阳性治疗组比较，差异均有统计学意义(P<0.05)。见表8。各组小鼠在给药第30天的毛发生长情况见图1。
表8各组对小鼠毛发生长长度的影响
Figure PCTCN2022099496-appb-000073
Figure PCTCN2022099496-appb-000074
*与模型对照组比较差异均有统计学意义(P<0.01)
2.2 CLY对小鼠观察区皮肤组织真皮浅层毛囊形态的影响
模型组小鼠部分皮肤真皮组织细胞有不同程度节段性增厚，小鼠皮下有轻度淋巴细胞增生；部分小鼠皮下毛囊有明显囊性变，毛囊大小不等，增大毛囊腔内有脱落角化物.周边有轻度纤维化，毛囊周边细胞消失或细胞层次明显减少，腔内似有钙化物染成蓝色，皮脂腺数目增多，部分腺体有肥大，肥大腺体细胞核明显减少，正常毛囊数减少。各治疗组小鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变与模型组比较有不同程度减轻。各治疗组小鼠皮肤损伤毛囊数与模型对照组比较均明显减少，差异有统计学意义(P<0.01)。与模型对照组比较，各治疗组小鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变明显减轻，差异有统计学意义(P<0.01)。见表9。
表9各组对小鼠皮肤毛囊和皮脂腺的影响(只)
组别 只数 — ± + ++ +++ P值*
正常对照组 6 6 0 0 0 0 <0.01
模型对照组 6 0 0 0 1 5 —
阳性对照组 6 0 2 1 3 0 <0.01
CLY-1组 6 0 3 2 1 0 <0.01
CLY-2组 6 0 4 2 0 0 <0.01
CLY-3组 6 0 3 1 2 0 <0.01
CLY-4组 6 0 2 2 2 0 <0.01
CLY-8组 6 0 4 2 0 0 <0.01
CLY-11组 6 0 3 1 2 0 <0.01
CLY-19组 6 0 3 2 1 0 <0.01
CLY-36组 6 0 3 1 2 0 <0.01
注：*为与模型对照组比较差异有统计学意义(P<0.01)
3.实验结论
CLY-1、CLY-2、CLY-3、CLY-4、CLY-8、CLY-11、CLY-19、CLY-36能明显促进小鼠脱发模型的毛发生长，减轻对皮下毛囊和皮脂腺的损伤。
实施例11CLY系列对兔耳痤疮模型的影响
1.实验方法
1.1材料
(1)治疗乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量CLY系列化合物混匀形成混合乳剂。本实施例所用的乳剂基质是指乳剂除去活性成分的基质成分。
(2)阳性治疗药：0.1％阿达帕林凝胶(商品名：达芙文，法国高德美制药公司生产)
(3)实验动物：普通级新西兰家兔，1.8～2.1kg，雄性，来源于上海斯莱克实验动物有限责任公司。
1.2动物分组与造模
新西兰家兔按体重编号，采用随机排列表法分为模型对照组(涂抹乳膏基质)、空白对照组(涂抹乳膏基质)、阳性治疗组(皮肤涂抹达芙文)、CLY-1外用治疗组(皮肤涂抹0.25％CLY-1乳膏)、CLY-2外用治疗组(皮肤涂抹0.25％CLY-2乳膏)、CLY-3外用治疗组(皮肤涂抹0.25％CLY-3乳膏)、CLY-4外用治疗组(皮肤涂抹0.25％CLY-4乳膏)、CLY-8外用治疗组(皮肤涂抹0.25％CLY-8乳膏)、CLY-9外用治疗组(皮肤涂抹0.25％CLY-9乳膏)、CLY-11外用治疗组(皮肤涂抹0.25％CLY-11乳膏)、CLY-19外用治疗组(皮肤涂抹0.25％CLY-19乳膏)、CLY-36外用治疗组(皮肤涂抹0.25％CLY-36乳膏)；CLY-1静脉注射组(1mg/kg.d)，CLY-2静脉注射组(1mg/kg.d)、CLY-3静脉注射组(1mg/kg.d)、CLY-4静脉注射组(1mg/kg.d)、CLY-8静脉注射组(1mg/kg.d)、CLY-9静脉注射组(1mg/kg.d)、CLY-11静脉注射组(1mg/kg.d)、CLY-19静脉注射组(1mg/kg.d)、CLY-36静脉注射组(1mg/kg.d)，每组各10只。取兔右耳内侧脱毛处理作为观察区，所有家兔左耳内侧作为自身阴性对照，涂抹95％酒精，模型组和治疗组右耳内侧均涂2％煤焦油(Alfa Aesar中国公司，用95％酒精配制成2％的煤焦油溶液)，用无菌棉签均匀涂于家兔耳内侧面耳导管开口处约2cm×2cm范围，每天1次，每次0.5mL，并且用温水擦拭前次涂药部位，连续涂14d，建立痤疮微粉刺模型。肉眼观察局部皮肤的变化，包括耳厚薄、硬度、粗糙程度和毛囊口有无黑色角栓等。末次涂18h后处死取材，使用5mm打孔器在涂药部位打孔取皮肤组织，10％甲醛固定，石蜡包埋切片，HE染色后，进行病理组织学观察分析。
1.3观察指标
痤疮模型组织学判定分级标准：按组织学级别为3级。0级“一”为漏斗部仅有松散的角化的细胞，无粉刺生成；1级为兔耳表面皮肤发红，或毛囊漏斗部见少量致密角化物质，漏斗部不扩张“+”；2级为毛囊漏斗部见中等致密角化物质，并向皮脂腺延伸，伴随皮脂腺导管的增生，漏斗扩张“2+”；3级为毛囊内有广泛的角化物质，毛囊中紧密的角质栓塞引起毛囊重度扩张，皮脂腺导管上皮明显增生，皮肤凸起、瘢痕，部分皮脂腺发生退行变“3+”。
在显微镜下观察其组织病理改变情况，并用Biomias99图像分析系统测量一张切片上5处不同表皮的厚度，计算平均值；检测4张切片中位置相同而结构最完整的2个毛囊的面积和4个皮脂腺的直径，计算各自的平均值，然后将各组兔左、右外耳道数据相减，即得各兔的左、右耳表皮厚度差、毛囊面积差和皮脂腺直径差。
1.4统计学处理
用SPSS16软件进行统计分析。自身左右对照采用配对t检验，各组间比较用t检验，P<0.05为差异有统计学意义。
2.实验结果
肉眼观察：涂煤焦油14d后，所有组兔左耳皮肤柔软，其外耳道毛囊口排列整齐，未见粉刺、丘疹及脓疱等。模型对照组兔右耳涂煤焦油后耳厚度增加、皮肤变硬，表面粗燥，大部分毛囊口见黑头粉刺，毛囊口隆起呈丘疹，触之较硬，部分融合成片。各外用治疗组右耳表现为皮肤轻度粗燥肥厚，可见少量黑头粉刺。各静脉注射组均表现为兔右耳大部分毛囊性丘疹消退，皮肤变薄柔软，粉刺明显减少，毛孔明显缩小，无脱屑，基本接近正常兔耳。阳性治疗组兔右耳与左耳相比，皮肤轻度发红，有少许脱屑，可见少量粉刺。
组织切片观察：模型组左耳显示表皮较薄，可见毛囊，真皮与表皮交界清楚。模型组右耳造模后见表皮增厚，角化过度，颗粒层和棘层增厚，有角栓堵塞毛囊口，毛囊扩大，并向皮脂腺延伸，毛囊漏斗部充满角化物质，使毛囊漏斗部扩大呈壶状；真皮浅层毛细血管扩张，毛囊周围散在炎症细胞浸润，有少量中性粒细胞；皮脂腺数量增多，皮脂腺体积增大。
各组镜下实验性粉刺组织学分级(见表10)：模型组兔右耳(实验对照)与其左耳(空白对照)比较，差异有统计学意义(P<0.05)，说明兔耳痤疮模型造模成功；各治疗组兔右耳与模型组兔右耳进行比较，差异均有统计学意义(P均<0.05)，说明阳性对照组、各治疗组均能改善痤疮皮损。
表10各组痤疮粉刺的组织学分级
如表11所示，模型组兔右耳(实验对照)表皮厚度、毛囊画积和皮脂腺直径与其左耳(空
组别/组织学分级 n _ 1+ 2+ 3+
模型组左耳 10 10 0 0 0
模型组右耳 10 0 1 4 5
CLY-1外用组 10 5 3 2 0
CLY-2外用组 10 6 2 2 0
CLY-3外用组 10 4 4 2 0
CLY-4外用组 10 3 5 2 0
CLY-8外用组 10 8 1 1 0
CLY-9外用组 10 5 4 1 0
CLY-11外用组 10 5 3 2 0
CLY-19外用组 10 5 2 3 0
CLY-36外用组 10 5 1 4 0
CLY-1静脉组 10 7 2 1 0
CLY-2静脉组 10 8 1 1 0
CLY-3静脉组 10 6 2 2 0
CLY-4静脉组 10 5 3 2 0
CLY-8静脉组 10 9 1 0 0
CLY-9静脉组 10 7 3 0 0
CLY-11静脉组 10 7 2 1 0
CLY-19静脉组 10 8 1 1 0
CLY-36静脉组 10 7 1 2 0
阳性治疗组 10 5 2 3 0
白对照)比较，差异有统计学意义(P<0.05)，证明兔耳痤疮模型复制成功；各治疗组兔右耳表皮厚度、毛囊画积和皮脂腺直径与模型组兔右耳进行比较均有减少，差异有统计学意义(P<0.05)，提示阳性对照组、各治疗组均能改善痤疮皮肤病理损害。
表11各组耳表皮厚度、毛囊面积和皮脂腺直径
Figure PCTCN2022099496-appb-000075
Figure PCTCN2022099496-appb-000076
*为与模型组右耳比较P<0.05
3.实验结论
化合物CLY-1、CLY-2、CLY-3、CLY-4、CLY-8、CLY-9、CLY-11、CLY-19和CLY-36均能明显减轻兔耳痤疮模型症状，减少毛孔堵塞和粉刺形成，显示对痤疮具有明显的治疗作用。
实施例12化合物CLY系列抑制小鼠银屑病样炎症反应
1、材料：
阳性药(糖皮质激素药)：糠酸莫米松乳膏(艾洛松)，上海先灵葆雅制药有限公司产品。
动物：SPF级健康纯系小鼠(C57BL/6)；8周龄。
CLY乳膏制备方法：基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，与适量CLY系列化合物液形成混合乳剂。
本实施例所用的乳膏基质是指CLY乳膏除去活性成分的基质成分。
2、实验方法：
(1)购SPF级雌性C57BL/6小鼠8周龄，随机分为空白对照组、模型组、阳性对照组(外用艾洛松乳膏)、CLY-1治疗组(外用0.5％CLY-1乳膏)、CLY-2治疗组(外用0.5％CLY-2乳膏)、CLY-8治疗组(外用0.5％CLY-8乳膏)、CLY-9治疗组(外用0.5％CLY-9乳膏)、CLY-19治疗组(外用0.5％CLY-19乳膏)、CLY-36治疗组(外用0.5％CLY-36乳膏)，每组各5只。戊巴比妥钠80mg/kg腹腔注射麻醉后，背部剃毛，面积约为2cm×3cm，单笼饲养1天。
(2)空白对照组局部涂抹凡士林，模型组、阳性对照组和CLY治疗组背部每日定时涂抹5％咪喹莫特乳膏62.5mg，连续6天，每天拍照，进行PASI评分。
(3)在造模第1天，空白对照组和模型组外用乳膏基质，每天2次，治疗组外用0.5％CLY系列乳膏，每天2次。
3、实验结果：
(1)如图2所示，连续涂抹5％咪喹莫特乳膏6天后，模型组小鼠的背部涂药区域均出现明显红斑、鳞屑及浸润，而各CLY治疗组小鼠的背部涂药区域的红斑、鳞屑及浸润均明显比模型组轻微，治疗组的红斑、鳞屑及浸润接近阳性药治疗组。说明CLY系列化合物能够明显抑制银屑病样小鼠模型的炎症反应。
实施例13CLY系列化合物抑制小鼠湿疹模型的炎症反应
1、实验材料：
卵清蛋白(OVA)：PBS配成20g/L，保存于-20℃。
卡泊三醇搽剂(商品名：达力士搽剂)：丹麦利奥制药有限公司产品。
阳性药(糖皮质激素药)：糠酸莫米松乳膏(商品名：艾洛松)，上海先灵葆雅制药有限公司产品。
CLY乳膏制备方法：基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，与适量CLY系列化合物液形成混合乳剂。本实施例所用的乳膏基质是指CLY乳膏除去活性成分的基质成分。
动物：SPF级健康纯系小鼠(C57BL/6)；8周龄。
2、实验方法：
购SPF级雌性C57BL/6小鼠8周龄(0.02kg)，每组6只，随机分为空白对照组、模型组、阳性药组、CLY-1治疗组(外用0.1％CLY-1乳膏)、CLY-2治疗组(外用0.1％CLY-2乳膏)、CLY-3治疗组(外用0.1％CLY-3乳膏)、CLY-4治疗组(外用0.1％CLY-4乳膏)、CLY-8治疗组(外用0.1％CLY-8乳膏)、CLY-11治疗组(外用0.1％CLY-11乳膏)、CLY-19治疗组(外用0.1％CLY-19乳膏)和CLY-36治疗组(外用0.1％CLY-36乳膏)。
造模：正常对照组小鼠两侧耳部涂抹75％乙醇14.3ul，同时模型组、阳性药组和个治疗组每日定时先在两侧耳部涂抹1nmoI/L卡泊三醇搽剂14.3ul，风干后涂抹20g/L的OVA 25ul，每日1次，连续涂抹12d造模。
造模开始后4天起，在空白对照组和模型组小鼠耳部皮肤上涂抹乳膏基质，阳性药组小鼠耳部皮肤上涂抹艾洛松，各治疗组小鼠耳部皮肤上分别涂抹治疗乳膏，每天早晚2次，连续10天，每天拍照，进行评分。
分别在造模前和第14天用耳厚度测量仪测量记录小鼠耳廓厚度。于第14天测量完毕后脱颈处死小鼠，取血，分离血清。
用兔抗小鼠白介素(IL)-4抗体包被酶标板，4℃过夜，按照ELISA试剂盒说明书操作染色并终止反应，检测血清IL-4水平。ELISA试剂盒均购自美国Raybiotech公司。
3、实验结果：
(1)小鼠耳厚度比较：造模前，各组间耳厚度差异无统计学意义(P＞0.05)。造模后，各组小鼠耳厚度见表12。模型组显著高于各治疗组、阳性药组和空白对照组(P均＜0.01)，阳性药组与各治疗组间差异无统计学意义(P均＞0.05)。
表12造模后各组小鼠耳厚度
Figure PCTCN2022099496-appb-000077
(2)血清IL-4浓度：造模前，各组间血清IL-4浓度差异无统计学意义(P＞0.05)。造模后，各组小鼠外周血中血清IL-4水平见表13。模型组显著其他各组(P均＜0.01)，阳性药组与各治疗组间差异均无统计学意义(P＞0.05)。
表13造模后各组小鼠外周血中IL-4水平
Figure PCTCN2022099496-appb-000078
4、实验结论：
化合物CLY-1、CLY-2、CLY-3、CLY-4、CLY-8、CLY-11、CLY-19和CLY-36均能通 过降低血清IL-4水平抑制湿疹小鼠模型的炎症反应。
实施例14 CLY系列化合物对小鼠移植物抗宿主病小鼠模型的影响
1.实验方法
1.1实验动物：6-8周龄SPF级雄性C57BL/6小鼠(H-2b)和雌性BALB/c小鼠(H-2d)，体质量16-21g，所有实验小鼠均饲养于SPF级别隔离笼中，饲料及垫料均经灭菌处理，小鼠饲养1周后进行实验。
1.2动物分组与造模
骨髓细胞的制备：断颈处死供鼠，体积分数75％乙醇浸泡5min，在超净台内无菌条件下取出小鼠两股骨和胫骨，置于含体积分数2％胎牛血清的RPMI1640培养基中。用RPMI1640培养基冲洗骨髓腔，取出骨髓细胞，无菌洗液管吹打成骨髓悬液，200目滤网过滤制后，1 500r/min离心10min，收集细胞沉淀，加入生理盐水重悬。另外取少量细胞悬液，红细胞裂解液去除红细胞后，计数有核细胞的量。并调整骨髓悬液有核细胞浓度为5×1010L-1。
脾脏单个核细胞的制备：无菌剪剪开脾脏包膜，置于并200目滤网上，浸泡在含体积分数2％胎牛血清的RPMI1640培养基中，用无菌注射器柄端轻轻碾磨脾脏，用培养基冲洗滤网数次，收集过滤后的脾脏细胞悬液，1 500r/min离心10min，收集脾脏细胞沉淀。PBS重悬后吹打成单细胞悬液，轻轻置于小鼠脾脏淋巴细胞分离液上层，1 800r/min离心20min，收集白膜层细胞，PBS洗涤2次，细胞计数后，调整细胞密度为5×1010L-1，置于4℃冰箱待用。
以C57BL/6小鼠作为骨髓移植供鼠，BABL/c小鼠作为受体鼠。BABL/c小鼠按体重编号，采用随机排列表法分为对照组、模型组、CLY系列治疗组(每组静脉注射5mg/kg.d相应CLY化合物)，每组各6只。移植前1周喂食BALB/c小鼠含有200mg/L庆大霉素的无菌酸化水，移植前24h内给予小鼠8.5Gy全身照射(铯源)。模型组和各CLY系列治疗组尾静脉注射C57BL/6小鼠骨髓有核细胞(含细胞1×107)与脾脏单个核细胞(含细胞5×106)混合液0.5mL，建造小鼠急性GVHD模型，对照组BABL/c小鼠尾静脉输注0.5mL生理盐水。CLY系列治疗组同时分别静脉注射CLY系列化合物溶液各5mg/kg.d，其余组注射生理盐水5mg/kg.d。
1.3观察指标及测试方法
1.3.1小鼠GVHD症状评分：移植后每天观察小鼠的一般情况，包括体质量、皮毛、姿势、活动度、腹泻及精神状态，判断小鼠有无发生急性GVHD。根据临床GVHD症状评分标准，在移植后11d对各组小鼠的临床GVHD症状进行评分。
表14 GVHD症状进行评分标准
标准 0级 1级 2级
体质量减少 <10％ >10％且<25％ >25％
姿势 正常 休息时弓背可见 明显弓背影响活动
活动度 正常 轻度至中度减少 不刺激不活动
毛皮纹理 正常 皮毛轻度至中度卷曲 皮毛明显卷曲紊乱
皮肤完整度 正常 爪子或尾巴有鳞屑脱落 有明显裸露的皮肤
1.3.2小鼠生存时间分析：收集移植后3周各组小鼠的生存数据，通过kaplan-meier的方法绘制生存曲线，Log-rank分析方法比较各组小鼠生存率之间的差异。
1.3.3组织病理检查：移植后2周，每组取3只发生GVHD的小鼠，取背部皮肤组织、肝脏、小肠组织，40g/L多聚甲醛固定，石蜡固定包埋切片，苏木精-伊红染色，光学显微镜下观察各组织病理变化。
2.实验结果
2.1CLY系列化合物对急性GVHD小鼠临床症状及生存时间的影响
2.1.1临床症状：单纯照射对照组小鼠体质量减轻，活动减少，但未出现GVHD症状。急性GVHD组在移植后第6天小鼠开始出现弓背、体质量明显减轻、活动减少、颤抖、腹泻症状，而CLY系列治疗组也发生了GVHD症状，但症状明显减轻。移植后11d临床症状评分结果显示急性GVHD组为7.48±0.77，CLY-1组、CLY-2组、CLY-5组、CLY-8组、CLY-11组、CLY-36组分 别为5.28±0.53、4.68±0.42、4.91±0.46、4.56±0.39、5.06±0.41、4.93±0.39，CLY系列治疗组临床症状评分均低于急性GVHD组(P<0.01)。
2.1.2小鼠生存情况：通过kaplan-meier的方法绘制生存曲线，Log-rank分析方法比较各组小鼠生存率之间的差异，结果显示急性GVHD组、CLY-1组、CLY-2组、CLY-5组、CLY-8组、CLY-11组、CLY-36组小鼠存活率分别为12.13％、61.26％、68.36％、58.79％、63.13％、62.60％、67.75％。与急性GVHD组相比较，CLY系列组均可延长小鼠的生存时间(P<0.01)。
2.1.3皮肤、肝脏、小肠组织的病理变化：移植后2周，取发生GVHD症状小鼠皮肤、肝脏和小肠组织，进行苏木精-伊红染色，观察组织病理变化，结果显示各组发病小鼠皮肤组织变薄，均未见明显炎症细胞浸润；肝脏病理切片显示，急性GVHD组小鼠肝脏发生了多发性坏死病灶，大量炎症细胞浸润，而CLY系列化合物组小鼠肝脏仅有小的坏死病灶，可见少量炎症细胞浸润或在肝汇管区可见炎症因子浸润，较急性GVHD组明显减少。
3.实验结论
CLY系列化合物CLY-1、CLY-2、CLY-5、CLY-8、CLY-11、CLY-36均能明显提高急性GVHD小鼠生存时间，减轻临床症状，显示对急性GVHD具有治疗作用。
实施例15CLY系列化合物对大鼠肺纤维化模型的影响
1.实验方法
1.1材料：
(1)试剂：博来霉素(4mg/支，天津太和制药有限公司)，鼠抗鼠MMP单克隆抗体(NEO Mark-ers公司)，鼠抗鼠TIMP-1多克隆抗体(武汉博士德公司)，酶联免疫吸附试验(ELISA)试剂盒(美国R&D公司)，Quantscript RT Kit逆转录试剂盒(大连TaKaRa公司)。
(2)实验动物：SPF级Wistar大鼠，雌雄各半，鼠龄51～55d，体重(180±21)g，来源于南京医科大学动物中心。
1.2动物分组与造模
大鼠按体重编号，采用随机排列表法分为空白对照组、模型组、CLY系列治疗组，每组各12只，雌雄各半。各组大鼠用2％戊巴比妥钠(120mg/kg)腹腔注射麻醉后固定于手术台上，颈部气管切开注药。空白对照组注入生理盐水(1.25ml/kg)，模型组及CLY治疗组分别注入5U/mL博莱霉素溶液(5mg/kg)，每天1次，连续14天。于造模后一周开始，各治疗组分别尾静脉注射相应CLY化合物溶液(3mg/kg.d)，空白对照组及模型组尾静脉注射等量生理盐水，每天1次，连续14天。
1.3观察指标及测试方法
各组动物分别于造模后和治疗14d时经尾静脉取外周静脉血，检测外周血超氧化物歧化酶(SOD)、过氧化氢酶(CAT)水平。各组取血后分2次处死大鼠(造模后和治疗14d)，取动物右肺组织于-4℃冰箱储存用于检测VEGF，取左侧肺组织常规石蜡包埋、切片，用免疫组织化学染色法检测大鼠肺组织中MMP亚型及TIMP-1表达。取右肺组织研磨、组织匀浆，3000r/min高速离心后取上清液，采用ELISA法检测肺组织VEGF蛋白，应用反转录聚合酶链法测定VEGF-mRNA表达。
2.实验结果
2.1CLY系列化合物对大鼠肺组织MMP的影响
空白对照组大鼠肺组织TIMP-1和MMP亚型均有少量表达。模型组大鼠MMP-2、MMP-9表达在造模后升高，TIMP-1降低，与空白对照组比较差异有统计学意义(P<0.05)，说明造模成功。CLY系列化合物组的MMP-2、MMP-9表达降低，TIMP-1表达上调，与模型组比较差异均有统计学意义(P均<0.05)。见表15
表15各组大鼠肺组织TIMP-1和MMP表达的比较(n＝6,x±s)
组别 TIMP-1 MMP-2 MMP-9
模型对照组 5.71±0.63 3.86±0.29 5.17±0.39
空白对照组 8.95±0.56* 2.41±0.18* 3.29±0.22*
CLY-1组 7.82±0.45* 2.43±0.21* 3.32±0.23*
CLY-2组 8.06±0.49* 2.51±0.24* 3.36±0.25*
CLY-8组 8.82±0.53* 2.44±0.21* 3.31±0.21*
CLY-11组 8.13±0.38* 2.52±0.23* 3.53±0.26*
CLY-36组 7.63±0.52* 2.59±0.23* 3.46±0.25*
注：*与模型对照组比较，P<0.05。
2.2CLY系列化合物组对肺组织中VEGF的影响
各组大鼠肺组织VEGF蛋白及VEGF-mRNA表达见表16。模型组VEGF蛋白、VEGF-mRNA表达明显降低，与空白对照组比较差异有统计学意义(P<0.05)，说明肺纤维化模型造模成功。CLY系列化合物组VEGF蛋白及VEGF-mRNA表达与模型组比较差异均有统计学意义(P<0.05)。
表16各组肺组织中VEGF蛋白及VEGF-mRNA表达水平的比较(n＝6,x±s)
组别 VEGF蛋白(pg/ml) VEGF-mRNA
模型对照组 37.23±4.16 0.52±0.19
空白对照组 54.26±3.95* 0.83±0.13*
CLY-1组 52.16±3.61* 0.76±0.11*
CLY-2组 51.18±3.69* 0.71±0.12*
CLY-8组 53.16±3.62* 0.79±0.13*
CLY-11组 49.86±3.58* 0.76±0.13*
CLY-36组 53.87±3.73* 0.83±0.13*
注：*与模型对照组比较，P<0.05。
2.3CLY系列化合物对外周血中SOD、CAT酶活性的影响
各组大鼠外周血SOD、CAT酶水平见表17。模型组大鼠外周血SOD、CAT酶活性降低，与对空白对照组比较差异有统计学意义(P<0.05)；CLY系列化合物组大鼠外周血SOD、CAT酶活性均增强，与模型组比较差异均有统计学意义(P<0.05)。
表17各组外周血中SOD、CAT酶水平的比较(n＝6,x±s)
组别 SOD(U/ml) CAT(kU/g)
模型对照组 143.36±15.23 8.87±1.21
空白对照组 223.65±13.28* 15.16±1.29*
CLY-1组 214.58±13.53* 14.13±1.22*
CLY-2组 216.62±13.55* 14.28±1.39*
CLY-8组 220.86±13.78* 14.75±1.22*
CLY-11组 216.48±13.16* 13.96±1.36*
CLY-36组 221.76±13.57* 13.26±1.26*
注：*与模型对照组比较，P<0.05。
3.实验结论
化合物CLY-1、CLY-2、CLY-8、CLY-11、CLY-36均能明显降低肺纤维化小鼠模型肺组织中MMP-2、MMP-9水平，增加TIMP-1和VEGF水平，同时可提高外周血中SOD、CAT酶水平，显示对肺纤维化具有抑制作用。
实施例16、CLY系列化合物能改善类风湿关节炎(RA)小鼠的关节症状和炎症指标
1、实验材料与方法
(1)实验材料
弗氏完全佐剂(Freund＇s Adjuvant Complete，FCA，购于美国Sigma公司)；IL-10ELISA试剂盒(联科生物技术有限公司)；IL-17ELISA试剂盒(美国Raybiotech公司)。
(2)实验动物分组及处理
5周龄雌性Wistar大鼠18只，将动物随机分为对照组、RA模型组、CLY系列化合物治疗组，每组6只。对照组：用75％酒精对大鼠右足趾进行消毒，将0.15mL生理盐水注射于大鼠右足跖皮下。RA模型组：于实验开始第一天每只大鼠进行常规消毒，将FCA 0.15mL注射于大鼠右足跖皮下。CLY系列化合物治疗组：于实验开始后第一天，每只大鼠进行常规消毒，将FCA 0.15mL注射于大鼠右足跖皮下，7天后开始分别给予CLY系列化合物每天灌胃，剂量为10mg/kg，每日1次，RA模型组和对照组每天生理盐水灌胃(10mg/kg.d)，连续21天。CLY系列化合物干预后第21天对大鼠进行心脏采血3mL，分离血清，ELISA法检测大鼠血清中IL-10、IL-17的水平。
(3)观察指标
干预第21天后，对每组大鼠进行关节炎评分，测量每组大鼠右足趾的关节肿胀度。根据RA大鼠的炎症反应对踝关节的严重程度按0～4级进行评分0分，正常；1分，踝关节有微红及轻微肿胀；2分，踝关节至跖关节或掌关节有红斑及轻微肿胀；3分，踝关节至掌关节或跖趾关节有红斑及中度肿胀；4分，踝关节至趾关节严重红肿。将每只大鼠的四肢评分相加作为关节炎评分，最高分为16分。
2、实验结果
(1)大鼠一般表现
RA模型组大鼠与对照组相比，食欲减退、精神萎靡，活动受限，左右足趾逐渐肿胀。CLY系列化合物干预21天后较其他组均有所好转。
(2)大鼠的体质量、关节肿胀度和关节炎评分
药物干预21天后，对照组体质量高于其他各组，差异具有统计学意义(P均＜0.05)；RA模型组右足趾关节肿胀度及关节炎评分均高于对照组，差异具有统计学意义(P＜0.05)，CLY系列化合物治疗组关节肿胀度和关节炎评分低于RA模型组，差异具有统计学意义(见表18)。
表18各组大鼠关节炎指标比较
组别 N 体质量(g) 肿胀度(mm) 关节评分
对照组 6 367.1±18.62 * 8.5±0.32 * ——
RA模型组 6 295.4±9.63 # 10.4±1.45 # 6.63±0.39 #
CLY-1组 6 354.5±13.48 * 9.7±0.68 * 4.35±0.37 *
CLY-2组 6 358.2±13.92 * 9.4±0.53 * 4.21±0.29 *
CLY-8组 6 363.5±13.63 * 9.5±0.72 * 4.32±0.41 *
CLY-36组 6 352.6±12.96 * 9.3±0.51 * 4.46±0.42 *
注：*与RA模型组比较P＜0.05，#与治疗组比较P＜0.05
(3)各组大鼠血清中IL-17、IL-10水平比较
对照组和CLY系列化合物治疗组大鼠血清中IL-10含量高于RA模型组(P均＜0.05)；对照组和CLY系列化合物治疗组大鼠血清中IL-17水平低于RA模型组，差异具有统计学意义(P均＜0.05)(见表19)。
表19各组大鼠血清IL-10和IL-17水平比较
组别 N IL-17(pg/mL) IL-10(pg/mL)
对照组 6 36.83±3.28 * 72.57±8.42 *
RA模型组 6 60.16±6.05 # 45.76±5.63 #
CLY-1组 6 46.15±4.23 * 65.36±6.78 *
CLY-2组 6 46.23±3.96 * 67.03±7.02 *
CLY-8组 6 43.51±4.16 * 66.75±6.78 *
CLY-36组 6 44.18±3.97 * 58.96±5.89 *
注：*与RA模型组比较P＜0.05，#与治疗组比较P＜0.05
3、实验结论
CLY系列化合物能通过降低外周血中IL-17水平和升高IL-10等炎症指标来改善类风湿关节炎小鼠的关节炎症状。

Claims (16)

  1. 具有式I结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，
    Figure PCTCN2022099496-appb-100001
    其中：
    R 1为取代或未取代的含有N、O、S中至少一种的5～6元的杂环、苯环并该杂环或者至少两个该杂环的并环；所述取代基为H、卤素、羟基、烷氧基或(C1-C4)烷基；
    R 2为H、卤素、羟基、甲氧基、乙氧基、氨基、甲基或乙基；
    R 3为H、卤素、羟基、甲氧基、乙氧基、氨基、(C1-C3)烷基、
    Figure PCTCN2022099496-appb-100002
    Figure PCTCN2022099496-appb-100003
    R 4为取代或未取代的5～6元的环烷基或具有选自N或O的1～3杂原子的4～7元杂环；所述取代基选自H、-NH 2、-OH、(C1-C4)烷基、(C1-C4)烷氧基、氨基、(C1-C4)烷氨基；
    其中：
    R 6和R 8各自独立地为H、卤素或(C1-C4)烷基，且R 6和R 8不同时是卤素；
    R 7为羟基、(C1-C4)烷氧基、(C1-C4)烷氧基羰基氧基(C1-C4)烷基或(C1-C4)烷基羰基氧基(Cl-C4)烷基；
    R l0和R 11各自独立地为H、(C1-C4)烷基或(C3-C6)环烷基；
    R 12选自H、卤素、-OH、-NH 2或(C 1-C 3)烷基；
    R 13为H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基；
    R 14为H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基；
    R 15为羟基、四唑基、(C1-C2)烷基磺酰基或三氟甲基磺酰基；
    R 16为H、(C1-C4)烷基、(C1-C4)烷基羰基氧基(C1-C4)烷基或(C1-C4)烷氧基羰基氧基(Cl-C4)烷基。
  2. 据权利要求1所述的具有式I结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，其特征在于，
    如果R 3为H、卤素、羟基、甲氧基、乙氧基、氨基、(C1-C3)烷基、
    Figure PCTCN2022099496-appb-100004
    Figure PCTCN2022099496-appb-100005
    时，则R 4为
    Figure PCTCN2022099496-appb-100006
    Figure PCTCN2022099496-appb-100007
    如果R 3为
    Figure PCTCN2022099496-appb-100008
    Figure PCTCN2022099496-appb-100009
    时，则R 4为
    Figure PCTCN2022099496-appb-100010
    Figure PCTCN2022099496-appb-100011
  3. 根据权利要求1所述的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，其特征在于，R 2是H、羟基或甲基。
  4. 根据权利要求1所述的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，其特征在于，R 1是取代或未取代的吡啶基、异喹啉基或吡咯并吡啶基；所述取代基为H、氯或甲基。
  5. 具有式I-a结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，
    Figure PCTCN2022099496-appb-100012
    其中，如果R 3选自H、卤素、羟基、甲氧基、氨基、甲基或者以下取代或未取代的基团：
    Figure PCTCN2022099496-appb-100013
    时，则R 4为
    Figure PCTCN2022099496-appb-100014
    Figure PCTCN2022099496-appb-100015
    如果R 3为
    Figure PCTCN2022099496-appb-100016
    Figure PCTCN2022099496-appb-100017
    时，则R 4为
    Figure PCTCN2022099496-appb-100018
    Figure PCTCN2022099496-appb-100019
    其中，R 6、R 7、R 8、R l0、R 11、R 12、R 13、R 14、R 15、R 16的限定与权利要求1一致。
  6. 具有式I-b结构的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，
    Figure PCTCN2022099496-appb-100020
    其中，如果R 3为H、卤素、羟基、甲氧基、氨基、甲基或以下取代或未取代的以下基团：
    Figure PCTCN2022099496-appb-100021
    则R 4为
    Figure PCTCN2022099496-appb-100022
    Figure PCTCN2022099496-appb-100023
    如果R 3为
    Figure PCTCN2022099496-appb-100024
    R 4为
    Figure PCTCN2022099496-appb-100025
    Figure PCTCN2022099496-appb-100026
    其中，R 6、R 8、R l0、R 11、R 12、R 13的限定与权利要求1一致。
  7. 根据权利要求1-6中任意一项所述的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，其特征在于，R 6是H或甲基，且R 8是H。
  8. 根据权利要求1-6中任意一项所述的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐，其特征在于，R 12选自H、卤素、-OH、-NH 2或甲基。
  9. 根据权利要求1-6中的任意一项所述的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐：其特征在于，其选自以下化合物：
    Figure PCTCN2022099496-appb-100027
    Figure PCTCN2022099496-appb-100028
    Figure PCTCN2022099496-appb-100029
  10. 一种如式(I)所示化合物的制备方法：
    ；
    Figure PCTCN2022099496-appb-100030
    由原料
    Figure PCTCN2022099496-appb-100031
    制备生成
    Figure PCTCN2022099496-appb-100032
    然后，
    Figure PCTCN2022099496-appb-100033
    与
    Figure PCTCN2022099496-appb-100034
    生产终产物I；
    其中：R 1、R 2、R 3、R 4与权利要求1-9中任意一项所限定的一致。
  11. 一种药物组合物，其特征在于，以权利要求1～9任一项所述化合物或通过权利要求10所制备得到的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐为活性成分或主要活性成分，辅以药学上可接受的载体组成。
  12. 权利要求1～9任一项所述化合物或权利要求10所制备的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐或权利要求11所述的组合物在制备治疗和/或预防疾病药物中的应用。
  13. 权利要求1～9任一项所述化合物或权利要求10所制备的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐或权利要求11所述的组合物在制备治疗和/或预防黄褐斑、疤痕、雄性激素性脱发、脂溢性脱发、斑秃、痤疮、鱼鳞病、汗孔角化症、毛周角化症、银屑病、湿疹、特应性皮炎、移植物抗宿主病、肺纤维化或类风湿性关节炎等疾病的药物中的应用。
  14. 权利要求1～9任一项所述化合物或权利要求10所制备的化合物，其互变异构体，其溶剂化物或其药学上可接受的盐或权利要求11所述的组合物制备的药学上允许的任何剂型。
  15. 根据权利要求14所述剂型，其特征在于，为适于口服、肠胃外、腹膜内、静脉内、动脉内、皮肤外用、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、眼内、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
  16. 根据权利要求14所述剂型，其特征在于，所述的剂型包括膏剂、凝胶、乳剂、搽剂、洗剂、溶液、喷雾剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂或注射剂。
PCT/CN2022/099496 2021-06-17 2022-06-17 Cly系列化合物及其制备方法和制备药物的用途 WO2022262854A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
KR1020247001632A KR20240022602A (ko) 2021-06-17 2022-06-17 Cly계열 화합물 및 그 제조방법과 약물의 제조용도
EP22824326.7A EP4357345A1 (en) 2021-06-17 2022-06-17 Cly series compound, preparation method therefor and use thereof in preparation of drugs
CN202310688735.7A CN116803391A (zh) 2021-06-17 2023-06-12 Wez系列化合物在制备预防或/和治疗脱发产品中的用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110668896.0 2021-06-17
CN202110668896.0A CN113248501B (zh) 2021-06-17 2021-06-17 Cly系列化合物及其制备方法和制备药物的用途

Publications (1)

Publication Number Publication Date
WO2022262854A1 true WO2022262854A1 (zh) 2022-12-22

Family

ID=77188283

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/099496 WO2022262854A1 (zh) 2021-06-17 2022-06-17 Cly系列化合物及其制备方法和制备药物的用途

Country Status (4)

Country Link
EP (1) EP4357345A1 (zh)
KR (1) KR20240022602A (zh)
CN (2) CN113248501B (zh)
WO (1) WO2022262854A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113248501B (zh) * 2021-06-17 2021-10-08 南京韦尔优众医药有限公司 Cly系列化合物及其制备方法和制备药物的用途

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105143203A (zh) * 2013-04-17 2015-12-09 辉瑞大药厂 用于治疗心血管疾病的n-哌啶-3-基苯甲酰胺衍生物
WO2016055901A1 (en) * 2014-10-08 2016-04-14 Pfizer Inc. Substituted amide compounds
CN107311996A (zh) * 2016-04-27 2017-11-03 成都贝斯凯瑞生物科技有限公司 一种酰胺化合物及其应用
WO2018192493A1 (zh) * 2017-04-21 2018-10-25 深圳信立泰药业股份有限公司 作为pcsk9抑制剂的哌啶类化合物
CN112094268A (zh) * 2020-11-09 2020-12-18 南京韦尔优众医药有限公司 化合物wez系列及其制备方法和制备药物的用途
CN113248501A (zh) * 2021-06-17 2021-08-13 南京韦尔优众医药有限公司 Cly系列化合物及其制备方法和制备药物的用途
CN113387945A (zh) * 2020-03-11 2021-09-14 陈敏 化合物ptm-3、ptm-4及其制备方法和制备药物的用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG11202008718UA (en) * 2018-03-09 2020-10-29 Brigham & Womens Hospital Inc Combination therapy for cardiovascular diseases
SG11202107615TA (en) * 2019-01-18 2021-08-30 Astrazeneca Ab Pcsk9 inhibitors and methods of use thereof
CR20210441A (es) * 2019-01-18 2022-03-11 Astrazeneca Ab Inhibidores de la pcsk9 y métodos de uso de los mismos

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105143203A (zh) * 2013-04-17 2015-12-09 辉瑞大药厂 用于治疗心血管疾病的n-哌啶-3-基苯甲酰胺衍生物
WO2016055901A1 (en) * 2014-10-08 2016-04-14 Pfizer Inc. Substituted amide compounds
CN107311996A (zh) * 2016-04-27 2017-11-03 成都贝斯凯瑞生物科技有限公司 一种酰胺化合物及其应用
WO2018192493A1 (zh) * 2017-04-21 2018-10-25 深圳信立泰药业股份有限公司 作为pcsk9抑制剂的哌啶类化合物
CN113387945A (zh) * 2020-03-11 2021-09-14 陈敏 化合物ptm-3、ptm-4及其制备方法和制备药物的用途
CN112094268A (zh) * 2020-11-09 2020-12-18 南京韦尔优众医药有限公司 化合物wez系列及其制备方法和制备药物的用途
CN113248501A (zh) * 2021-06-17 2021-08-13 南京韦尔优众医药有限公司 Cly系列化合物及其制备方法和制备药物的用途

Also Published As

Publication number Publication date
CN116803391A (zh) 2023-09-26
EP4357345A1 (en) 2024-04-24
CN113248501A (zh) 2021-08-13
CN113248501B (zh) 2021-10-08
KR20240022602A (ko) 2024-02-20

Similar Documents

Publication Publication Date Title
KR101398648B1 (ko) 리팍시민 직장 기능장애 치료 제제
JP2022116295A (ja) 炎症性皮膚疾患を処置するための局所組成物及び方法
JP2014503586A (ja) 油組成物
US10722493B2 (en) Methods for treating fibroproliferative disorders in a mammal
KR20210151816A (ko) 칸나비노이드 산 에스테르 조성물 및 이의 용도
JP2014503587A (ja) 界面活性剤組成物
JP2006524659A (ja) ケラチノサイトの過剰増殖、特に、アトピー性皮膚炎および乾癬を特徴とする疾患を治療するためのリルゾールの使用
KR20110131187A (ko) 여드름 및 기타 질환 치료용 비스파틴 치료제
JP2014507420A (ja) パラベン組成物
JP2015513359A (ja) 座瘡の排除又は予防用のピルフェニドンと改変ジアリルジスルフィド酸化物（ｍ−ｄｄｏ）とを含む半固形局所組成物
WO2022262854A1 (zh) Cly系列化合物及其制备方法和制备药物的用途
AU2013271836B2 (en) Method for treating skin inflammatory diseases
CN112979762B (zh) 环肽piz及其用途
JP2011524412A (ja) 脱毛治療組成物
KR20060011783A (ko) 데하이드로에피안드로스테론 스테로이드 및항-무스카린제와 더불어 호흡기 및 폐 질환을 치료하기위한 조성물, 제제 및 키트
JPWO2004024185A1 (ja) 医薬または化粧料
KR20090100416A (ko) 탈모증의 예방 또는 치료제
CA2967268A1 (en) 3-(piperidin-4-yl)-isoxazol-3(2h)-ones for treatment of dermatologic disorders
US9566262B2 (en) Itch suppression by fucoxanthin
JP6656890B2 (ja) フィラグリン産生促進剤
WO2020028745A1 (en) Methods for reducing abnormal scar formation
JP2016079152A (ja) エストロゲン受容体β活性化剤
WO2022002160A1 (zh) Pcsk9抑制剂在制备治疗多种疾病产品中的应用
JP2010215522A (ja) 皮膚疾患治療用又は皮膚疾患予防用医薬組成物、及び、皮膚疾患予防用化粧料
JP3568881B2 (ja) 皮膚疾患治療用外用剤

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22824326

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 20247001632

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1020247001632

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2022824326

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022824326

Country of ref document: EP

Effective date: 20240117