KLHL24 ubiquitin ligase inhibitor and use thereof

Abstract

Disclosed are a high-throughput screening method and system for KLHL24 ubiquitin ligase inhibitors. The present invention has high stability and good repeatability, which helps efficiently screen for KLHL24 ubiquitin ligase inhibitors and develop related drugs thereof. On the basis of the described method, some KLHL24 ubiquitin ligase inhibitors are obtained by means of screening in the present invention. These inhibitors can be effectively used for the treatment of KLHL24-related diseases, especially diseases caused by mutations in KLHL24, such as hereditary epidermolysis bullosa, dilated cardiomyopathy, alopecia, and atrophic scars, showing great promise. In view of the role of KLHL24 in skin and hair regeneration, these inhibitors also hold the promise of maintaining normal hair.

Description

A KLHL24 ubiquitin ligase inhibitor and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a KLHL24 ubiquitin ligase inhibitor and application thereof. BACKGROUND

[0002] KLHL24 belongs to the Kelch-like family proteins (KLHLs), which encode proteins with a highly conserved BTB domain and a BACK domain, which can interact with CUL3 and RBX1 to form a CUL3-RBX1-Kelch ubiquitin ligase complex. Kelch-like proteins also have a Kelch domain for recruiting substrates, which determines the specificity of E3 ligase. KLHL24 is widely expressed in various tissues of the human body, including the skin.

[0003] The research led by the first inventor of the present application first discovered that the truncated KLHL24-ΔN28 protein caused by the mutation of the initiation codon of the Klhl24 gene is more stable due to the decrease in its own ubiquitination degree, thereby leading to the ubiquitination and degradation of a large amount of keratin KRT14 in the skin by KLHL24, making the basal layer cells of the epidermis fragile and easy to fall off, and ultimately leading to the occurrence of hereditary epidermolysis bullosa (Lin, Zhimiao, et al. Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility. Nature genetics 48.12 (2016): 1508-1516.). Subsequent studies reported that the ubiquitination substrates of KLHL24, in addition to KRT14, also include KRT15, Vimentin and Desmin. Another study found that the syndrome phenotype caused by KLHL24 mutation is emerging. EBS-KLHL24 patients have extensive areas of bare skin and skin fragility at birth, and over time, they develop atrophic scars, accompanied by life-threatening cardiomyopathy (Yenamandra, V.K., et al. Cardiomyopathy in patients with epidermolysis bullosa simplex with mutations in KLHL24. British Journal of Dermatology (2018) 179(5): 1181-1183). EBS-KLHL24 patients can also experience hair loss, especially in areas of skin atrophy, and sometimes even terminal hair (Cui, Jun, et al. KLHL24-Mediated Hair Follicle Stem Cells Structural Disruption Causes Alopecia. Journal of Investigational Dermatology (2022) 142(8): 2079-2087). It can be seen that the development of drugs targeting KLHL24 has great significance.

[0004] However, there is currently no specific drug for treating diseases caused by KLHL24 mutations. Therefore, it is of great significance to establish a high-throughput screening system and successfully screen drugs that can specifically bind and inhibit the activity of KLHL24 for the treatment of diseases caused by KLHL24 mutations. Given the function of KLHL24 in skin and hair regeneration, screening such inhibitors also has great significance in maintaining normal hair. SUMMARY

[0005] To overcome the deficiencies of the prior art, the present application provides a KLHL24 ubiquitin ligase inhibitor and its application, and establishes a high-throughput screening method and system for KLHL24 ubiquitin ligase inhibitors.

[0006] In the first aspect of the present application, a compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound as a KLHL24 ubiquitin ligase inhibitor is provided, the compound having the following structure:

[0007] wherein, represents a single bond or a double bond;

[0008] X is selected from: O, S, NH;

[0009] A ring is a heterocyclic ring;

[0010] R A is one or more independent substituents on the A ring, selected from: H, halogen, hydroxyl, amino, cyano, nitro, azido, C1-C 10 alkyl, -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl),

[0011] L is a divalent group connected to the A ring, selected from: a single bond, H, C1-C6alkylene, -(C0-C6alkylene)-O-, -(C0-C6alkylene)-S-, -(C0-C6alkylene)-C(O)-, -C(O)-(C0-C6alkylene)-, -(C0-C6alkylene)-COO-, -(C0-C6alkylene)-C(S)-, -(C0-C6alkylene)-N(C0-C 10 alkylene)-, -(C0-C6alkylene)-CON(C0-C 10 alkylene)-, -(C0-C6alkylene)-N(C0-C 10 alkylene)CO-, -(C0-C6alkylene)-SO2-, -(C0-C6alkylene)-SO-, -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), wherein the C0-C6alkylene, C3-C 10 cycloalkyl, C6-C10 The hydrogen in the aryl or 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, azide, C1-C. 10 Alkyl, C1-C 10 Haloalkyl, -O(C) 0-10 alkyl), -S(C 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0- 10 Alkyl)(C 0-10 alkyl), -COO(C 0-10 Alkyl), -OCO(C) 0-10 Alkyl), -CON(C) 0-10 Alkyl)(C 0-10 Alkyl), -CO(C) 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)CO(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 alkyl), -N(C) 0-10 Alkyl)CON(C 0-10 alkyl), -N(C) 0-10 Alkyl)SO2(C 0-10 alkyl);

[0012] R C One or more independent substituents connected to L, selected from: H, halogen, cyano, nitro, azide, C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group, C1-C 10 Haloalkyl, -O(C) 0-10 alkyl), -S(C 0-10 alkyl), -SO(C) 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0- 10 alkyl), -N(C) 0-10 Alkyl)CO(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 alkyl), -N(C) 0-10 Alkyl)CON(C 0-10alkyl), -N(C 0-10 alkyl)SO2(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl), -CO(C0-C6alkylene)(4-10 membered heterocyclyl), -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), wherein the H of the C0-C6alkylene, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 10 cycloalkyl, C6-C 10 aryl, and 4-10 membered heterocyclyl is optionally substituted with one or more groups selected from halogen, cyano, nitro, azido, C1-C 10 alkyl, C1-C 10 haloalkyl, -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)COO(C 0-10 alkyl), -N(C 0-10 alkyl)CON(C 0- 10 alkyl), -N(C 0-10 alkyl)SO2(C 0-10 alkyl), -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO(C 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0-10 alkyl)(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10alkyl), -CO(C) 0-10 alkyl);

[0013] R B It is one or more independent substituents on the benzene ring, selected from: H, halogen, cyano, nitro, azido, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C6 alkylene) 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), -(C0-C6 alkylene)-(C1-C 10 (halogenated alkyl), -(C0-C6 alkylene)-(C1-C6) 10 Halogenated alkoxy), -(C0-C6 alkylene)-N(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)CO(C 0- 10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)CON(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -(C0-C6 alkylene)-O(C 0-10 Alkyl), -(C0-C6 alkylene)-S(C 0-10 Alkyl), -(C0-C6 alkylene)-SO(C 0-10 Alkyl), -(C0-C6 alkylene)-SO2(C 0-10 Alkyl), -(C0-C6 alkylene)-SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-COO(C 0-10 Alkyl), -(C0-C6 alkylene)-OCO(C 0-10 Alkyl), -(C0-C6 alkylene)-CON(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-CO(C 0- 10 Alkyl groups), -CO (C0-C6 alkylene groups) (4-10 membered heterocyclic groups), and nitric oxide (NO) donor residues, wherein the C0-C6 alkylene groups, C1-C6 alkylene groups, and C1-C6 alkylene groups are alkyl groups, C1-C6 alkylene ... 10 Alkyl, C2-C 10 alkenyl, C2-C10 alkynyl, C3-C 10 cycloalkyl, C6-C 10 aryl, 4-10 membered heterocyclyl, wherein H in aryl, 4-10 membered heterocyclyl is optionally substituted with one or more groups selected from halogen, cyano, nitro, azido, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), C1-C 10 haloalkyl, C1-C 10 haloalkoxy, -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)COO(C 0-10 alkyl), -N(C 0-10 alkyl)CON(C 0-10 alkyl), -N(C 0-10 alkyl)SO2(C 0-10 alkyl), -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO(C 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0-10 alkyl)(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl); or two R B together with the carbon atom to which they are attached form an aliphatic ring, an aromatic ring, or a heterocyclic ring, wherein H on said aliphatic ring, aromatic ring, or heterocyclic ring is optionally substituted with one or more groups selected from halogen, cyano, nitro, azido, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 haloalkyl, -N(C 0-10 alkyl)(C 0-10 alkyl), -O(C 0-10 alkyl), -COO(C 0- 10alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl).

[0014] In particular, the above-mentioned nitric oxide (NO) donor residue can be selected from the group consisting of: -ONO2, wherein R H and R K are independently selected from the group consisting of: H, C1-C 10 alkyl, C1-C 10 alkenyl, -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-6 membered heterocyclyl) (e.g. ), or R H and R K together with the nitrogen atom to which they are attached form a 4-6 membered heterocyclyl (e.g. ).

[0015] In some embodiments of the present application, X is O.

[0016] Further, the A ring is a 5-10 membered heterocycle selected from the group consisting of: wherein V is selected from the group consisting of: -S-, -NH-, -CH2-, R1, R2have the definition of R A as described above.

[0017] In some embodiments of the present application, the compound has the following structure:

[0018] preferably,

[0019] In some embodiments of the present application, R1is

[0020] In some embodiments of the present application, R2is

[0021] Further, L is selected from the group consisting of: a single bond, H, C1-C6alkylene, -(C0-C6alkylene)-O-, -(C0-C6alkylene)-S-, -(C0-C6alkylene)-C(O)-, -C(O)-(C0-C6alkylene)-, -(C0-C6alkylene)-COO-, -(C0-C6alkylene)-N(C0-C10 alkylene)-, -(C0-C6 alkylene)-N(C0-C 10 alkylene)CO-, -(C0-C6 alkylene)-SO2-, -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the C0-C6 alkylene, C3-C 10 cycloalkyl, C6-C 10 The hydrogen in the aryl or 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, azide, C1-C. 10 Alkyl, -O(C) 0-10 Alkyl), -COO(C 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl).

[0022] Preferably, L is selected from: single bond, C1-C6 alkylene, -(C0-C6 alkylene)-C(O)-, -C(O)-(C0-C6 alkylene)-, -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 heterocyclic)-, wherein the C0-C6 alkylene, C6-C 10 The H in the aryl group, 4-10 membered heterocyclic group, may optionally be substituted by one or more groups selected from the following: C1-C 10 Alkyl, -COO(C 0-10 alkyl).

[0023] Furthermore, R C Selected from: H, halogen, cyano, nitro, C1-C 10 Alkyl, -O(C) 0-10 alkyl), -S(C 0-10 alkyl), -SO(C) 0-10 alkyl), -SO2(C 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)CO(C 0- 10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), CON(C) 0- 10 Alkyl)(C0-10 alkyl), -CO(C 0-10 alkyl), -CO(C0-C6alkylene)(4-10 membered heterocyclyl), -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), wherein the C0-C6alkylene, C 10 alkyl, C3-C 10 cycloalkyl, C6-C 10 aryl, 4-10 membered heterocyclyl, is optionally substituted by one or more substituents selected from the group consisting of halogen, cyano, nitro, C1-C 10 alkyl, C1-C 10 haloalkyl, -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -O(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl).

[0024] Preferably, R C is selected from the group consisting of H, halogen, C1-C 10 alkyl, -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO2(C 0- 10 alkyl), -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)COO(C 0-10 alkyl), -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), wherein the C0-C6alkylene, C 0-10 alkyl, C3-C 10 cycloalkyl, C6-C 10The hydrogen atoms in aryl and 4-10 membered heterocyclic groups may optionally be substituted by one or more groups selected from the following: halogen, nitro, C1-C. 10 Alkyl, C1-C 10 Haloalkyl, -O(C) 0- 10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 Alkyl group, -(C0-C6 alkylene group)-(4-10 membered heterocyclic group).

[0025] In some embodiments of the present invention, R1 is selected from: H, -F, -Cl, -Br, -I,

[0026] In some embodiments of the present invention, R2 is selected from: H, -F, -Cl, -Br, -I,

[0027] Furthermore, R B Selected from: H, halogen, cyano, nitro, azide, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C6 alkylene) 10 cycloalkyl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), -(C0-C6 alkylene)-(C1-C 10 Halogenated alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)CO(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -(C0-C6 alkylene)-O(C 0-10 Alkyl), -(C0-C6 alkylene)-SO2(C 0-10 Alkyl), -(C0-C6 alkylene)-SO2N(C 0-10 Alkyl)(C 0- 10 Alkyl), -(C0-C6 alkylene)-COO(C 0-10 Alkyl), -(C0-C6 alkylene)-OCO(C 0-10 Alkyl), -(C0-C6 alkylene)-CON(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-CO(C0-10 alkyl), -CO(Co-C6alkylene)(4-10 membered heterocyclyl), wherein the Co-C6alkylene, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 10 cycloalkyl, C6-C 10 aryl, 4-10 membered heterocyclyl, in which H is optionally replaced by one or more radicals selected from halogen, cyano, nitro, azido, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, -(Co-C6alkylene)-(C6-C 10 aryl), -(Co-C6alkylene)-(4-10 membered heterocyclyl), C1-C 10 haloalkyl, -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)COO(C 0-10 alkyl), -O(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl).

[0028] Preferably, R B is selected from the group consisting of H, halogen, cyano, nitro, C1-C 10 alkyl, C1-C 10 haloalkyl, -O(C 0-10 alkyl), -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -SO2(C 0-10 alkyl), -N(C 0- 10 alkyl)SO2(C 0-10 alkyl), -CO(C 0-10 alkyl), -COO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10Alkyl), -CO(C0-C6 alkylene) (4-10 membered heterocyclic group), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the C0-C6 alkylene, C1-C 10 The hydrogen atoms in alkyl or 4-10 membered heterocyclic groups may optionally be substituted with one or more groups selected from the following: halogens, C1-C... 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, -O(C 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -(C0-C6 alkylene)-(C6-C 10 Aryl).

[0029] In some embodiments of the present invention, R B Selected from: H, -F, -Cl, -Br, -I, -NO2, -NH2, -CN, -COOH, -OH, -CF3, -ONO2.

[0030] Furthermore, the two Rs B Together with the carbon atom to which it is attached, it forms a 5-6 membered aliphatic ring, aromatic ring, or heterocycle, wherein the H atom on the 5-6 membered aliphatic ring, aromatic ring, or heterocycle is optionally substituted with one or more groups selected from the following: halogen, cyano, nitro, C1-C 10 Alkyl, C1-C 10 Haloalkyl, -N(C) 0-10 Alkyl)(C 0-10 Alkyl), -O(C) 0-10 Alkyl), -COO(C 0-10 alkyl).

[0031] Preferably, two R B Together with the carbon atom it is attached to, it forms a 5-6 membered heterocycle.

[0032] In some embodiments of the present invention, two R B Together with the carbon atom it is attached to, it forms a 5-6 membered heterocycle selected from:

[0033] In some embodiments of the present invention, the compound has the following structure:

[0034] Among them, RD selected from: H, halogen, cyano, hydroxy, carboxy, C1-C6 alkyl, C1-C6 haloalkyl.

[0035] In some embodiments of the present application, R D is C1-C6 alkyl, such as methyl, ethyl.

[0036] In some embodiments of the present application, R B is a nitric oxide (NO) donor residue, such as -ONO2,

[0037] In some embodiments of the present application, the compound has the following structure:

[0038] In a second aspect of the present application, there is provided a use of a compound or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the manufacture of a medicament for preventing and / or treating a KLHL24-related disease, wherein the compound has the definition of the compound according to the first aspect of the present application.

[0039] Further, the KLHL24-related disease is a disease that can be beneficially prevented and / or treated by inhibiting or degrading KLHL24, such as a disease caused by a KLHL24 mutation (such as a Klhl24 gene start codon mutation resulting in a truncated KLHL24-ΔN28 protein), a keratin abnormality disease, such as a skin disease, a cardiovascular disease, a tumor, a digestive system disease, arthritis, and the like, and complications thereof.

[0040] Further, the skin disease is selected from: alopecia, a bullous skin disease, a scar, a keratosis, an erythematous papulovesicular scaly skin disease, a connective tissue disease.

[0041] Further, the bullous skin disease is selected from: epidermolysis bullosa (such as hereditary epidermolysis bullosa, acquired epidermolysis bullosa), pemphigus, bullous pemphigoid, cicatricial pemphigoid.

[0042] Further, the scar is selected from: a hypertrophic scar, a keloid, an atrophic scar.

[0043] Further, the keratosis is selected from: keratosis follicularis, keratosis palmaris, palmoplantar keratoderma, and complications thereof including dry skin, scaling, rhagades, and the like.

[0044] Further, the erythematous papulovesicular scaly skin disease is selected from: psoriasis, pityriasis rubra pilaris, erythrokeratodermia.

[0045] Further, the connective tissue disease is selected from: lupus erythematosus, scleroderma.

[0046] In some embodiments of the present application, the disease is atrophic scar.

[0047] In some embodiments of the present application, the disease is epidermolysis bullosa, and its complications (such as esophageal mucosal stenosis, finger syndactyly contracture, corneal scarring, eye blindness).

[0048] In some embodiments of the present application, the disease is alopecia.

[0049] Further, the cardiovascular disease is cardiomyopathy (such as dilated cardiomyopathy, hypertrophic cardiomyopathy).

[0050] Further, the tumor is selected from the group consisting of leukemia, colon cancer, colorectal cancer, prostate cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, liver cancer, gastric cancer, adrenal cortex cancer, pancreatic ductal adenocarcinoma, lung adenocarcinoma, pancreatic cancer, gonadoblastoma, multiple myeloma, mantle cell lymphoma, osteosarcoma, neuroblastoma, brain tumor, melanoma.

[0051] Preferably, the tumor is selected from the group consisting of colon cancer, prostate cancer, gonadoblastoma, endometrial cancer.

[0052] More preferably, the tumor is selected from the group consisting of colon cancer, prostate cancer.

[0053] Further, the digestive system disease is selected from the group consisting of chronic hepatitis, drug-induced liver injury.

[0054] Further, the arthritis is osteoarthritis.

[0055] Further, the subject of the disease is a mammal, for example, a human.

[0056] Further, the compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound can be used alone or in combination with other kinds of active ingredients.

[0057] Further, the drug is a veterinary drug or a human drug.

[0058] Further, the drug further comprises one or more pharmaceutically acceptable adjuvants.

[0059] Further, the pharmaceutically acceptable adjuvant is selected from the group consisting of binding agents, lubricants, disintegrants, bacteriostatic agents, suspending agents, solubilizing agents, thickening agents, stabilizing agents, preservatives, fillers, antioxidants, buffers.

[0060] Further, the compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof can be administered by any suitable route of administration, such as gastrointestinal administration (e.g., oral, sublingual, rectal administration) or non-gastrointestinal administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, drip, respiratory administration, etc.), and the drug can be in any suitable dosage form, such as a gastrointestinal administration dosage form (e.g., an oral dosage form), for example, including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.; a non-gastrointestinal administration dosage form, for example, an injection administration dosage form: such as an injection (e.g., for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), a respiratory administration dosage form: such as a spray, an aerosol, a powder spray, etc., a skin administration dosage form, such as a topical solution, a lotion, an ointment, a plaster, a paste, a patch, etc., a mucous membrane administration dosage form: such as eye drops, eye ointment, nose drops, gargle, sublingual tablets, etc., a cavity administration dosage form: such as suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0061] In some embodiments of the present application, the drug is a skin administration dosage form.

[0062] In some embodiments of the present application, the drug is an oral dosage form.

[0063] In some embodiments of the present application, the drug is an injection administration dosage form.

[0064] In a third aspect of the present application, a use of a compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the preparation of a product for maintaining hair is provided.

[0065] Further, the subject of the product is a mammal, in particular a human, including healthy people and patients.

[0066] Further, the maintenance of hair includes promoting hair growth, promoting hair regeneration and self-repair, prolonging the hair growth cycle, preventing hair loss, etc.

[0067] Further, the product is a drug, a food, a health product, a care product.

[0068] Further, the product further comprises one or more excipients.

[0069] In a fourth aspect of the present application, a screening method of a KLHL24 ubiquitin ligase inhibitor is provided, which comprises the following steps: co-incubating a substance to be screened with a cell expressing KLHL24 labeled with a fluorescent protein, and detecting the fluorescent signal.

[0070] In some embodiments of the present application, the fluorescent protein is GFP.

[0071] In some embodiments of the present application, the cell is HEK293T cell.

[0072] Further, the method further comprises the steps of calculation and analysis, such as calculating the number or proportion of fluorescent positive cells.

[0073] In some embodiments of the present application, the method further comprises the step of transfecting the cell with the expression vector comprising the KLHL24 labeled with fluorescent protein.

[0074] In some embodiments of the present application, the expression vector is plasmid.

[0075] In some embodiments of the present application, the method comprises:

[0076] (a) constructing GFP-KLHL24 plasmid;

[0077] (b) transfecting HEK293T cell with GFP-KLHL24 plasmid, and incubating;

[0078] (c) mixing the substance to be screened with the cell of step (b), and co-incubating;

[0079] (d) detecting the fluorescence signal of the incubation system of step (c).

[0080] In the fifth aspect of the present application, a screening system (such as a kit) for KLHL24 ubiquitin ligase inhibitor is provided, which comprises: cell expressing KLHL24 labeled with fluorescent protein (as described in the fourth aspect).

[0081] Specifically, the screening system further comprises reagents for incubating the cell, such as culture medium.

[0082] Specifically, the screening system further comprises containers for incubating the cell, such as 96-well plate or 384-well plate.

[0083] Specifically, the screening system further comprises reagents for detecting fluorescence signal, such as cell fixation reagent (such as paraformaldehyde), cell washing solution, staining solution, etc.

[0084] In the sixth aspect of the present application, the screening system of the fifth aspect is used for screening KLHL24 ubiquitin ligase inhibitor, and drug for treating KLHL24 related diseases.

[0085] In a seventh aspect of the present application, there is provided a method of preventing and / or treating a KLHL24-associated disease, comprising administering to a subject in need thereof a compound of the first aspect of the present application, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof.

[0086] Further, the KLHL24-associated disease has the definition of the disease of the second aspect of the present application.

[0087] Further, the subject is a mammal, particularly a human.

[0088] Further, the administration can employ any suitable route of administration, such as gastrointestinal administration (e.g. oral, sublingual, rectal administration) or non-gastrointestinal administration (e.g. intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, drip, respiratory tract administration, etc.).

[0089] In an eighth aspect of the present application, there is provided a method of maintaining hair, comprising administering to a subject in need thereof a compound of the first aspect of the present application, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof.

[0090] Further, the subject is a mammal, particularly a human, including healthy people and patients.

[0091] Further, the maintenance of hair includes promoting hair growth, promoting hair regeneration and self-repair, prolonging the hair growth cycle, preventing hair loss, etc.

[0092] Further, the administration can employ any suitable route of administration, such as gastrointestinal administration (e.g. oral, sublingual, rectal administration) or non-gastrointestinal administration (e.g. intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, drip, respiratory tract administration, etc.).

[0093] The present application designs a high-throughput screening method and system for KLHL24 ubiquitin ligase inhibitors, which has high stability, good repeatability, and helps to efficiently screen KLHL24 ubiquitin ligase inhibitors and the development of related drugs. Based on the foregoing method, the present application screens some KLHL24 ubiquitin ligase inhibitors, which can be effectively used for the treatment of KLHL24-related diseases (such as diseases caused by KLHL24 mutations, such as hereditary epidermolysis bullosa, dilated cardiomyopathy, hair loss, atrophic scarring, etc.), and has very good application prospects. In view of the function of KLHL24 in skin and hair regeneration, the inhibitor also has good application prospects in maintaining normal hair. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 shows a schematic diagram of high-throughput screening of KLHL24 inhibitors based on GFP-KLHL24 auto-ubiquitination.

[0095] Figure 2 shows the experimental results of GFP fluorescence detection of GFP-KLHL24 expression in 293T cells transiently transfected with GFP-KLHL24 after 24 hours. Figure 2a shows the fluorescence pictures taken by high-content fluorescence microscope, and Figure 2b shows the fluorescence intensity statistics.

[0096] Figure 3 shows the experimental results of stability and repeatability evaluation of the high-throughput screening method.

[0097] Figure 4 shows the candidate small molecule compounds obtained by high-throughput screening of the Express-Pick diversity compound mother nucleus library of Selleck Company.

[0098] Figure 5 shows the effect of candidate small molecule compounds on GFP-KLHL24 expression, in which the ▲ indicated small molecule compound is STK731044.

[0099] Figure 6 shows Klhl24 expression in the skin of mice treated with KLHL24 inhibitor STK731044. c.3G / T The experimental results of mouse tape-stripping hair test, in which Figure 6a shows a direct display of the hair stripped by the tape from the mice, and "-" represents control formulation treatment (i.e. without KLHL24 inhibitor treatment), and "+" represents test formulation treatment (i.e. with KLHL24 inhibitor treatment); Figure 6b shows the weight statistics of the shed hair shafts.

[0100] Figure 7 shows Klhl24 expression in the skin of mice treated with KLHL24 inhibitor STK731044. c.3G / T The experimental results of protein abundance of KRT15, Vimentin and GAPDH in mouse tissues, in which "-" represents control formulation treatment (i.e. without KLHL24 inhibitor treatment), and "+" represents test formulation treatment (i.e. with KLHL24 inhibitor treatment). DETAILED DESCRIPTION

[0101] Unless otherwise defined, all scientific and technical terms used in the present application have the same meanings as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0102] In the present application, the term "aliphatic" refers to straight chain or branched chain hydrocarbon chains that are completely saturated or that contain one or more units of unsaturation, or cyclic hydrocarbon groups (also referred to herein as "cycloalkyl", "alicyclic groups") that are completely saturated or that contain one or more units of unsaturation, that are attached to the remainder of the molecule by a single bond. Suitable aliphatic groups include, but are not limited to, linear or branched chain, substituted or unsubstituted alkyl groups, alkenyl groups, alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, (cycloalkyl)alkenyl, and the like. Typical aliphatic groups contain 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, preferably 1 to 6 carbon atoms.

[0103] The term "cycloalkyl" refers to alicyclic hydrocarbons, such as those containing from 1 to 4 rings and / or fused rings, containing 3 to 18 carbon atoms, preferably 3 to 10 (e.g. 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or adamantyl, and the like.

[0104] The term "alkyl" refers to straight chain or branched chain hydrocarbon radicals and is not intended to include unsaturated bonds. Typical alkyl groups contain 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, preferably 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, neopentyl, t-pentyl, n-hexyl, i-hexyl, and the like. If the alkyl group is substituted with an aryl group, it corresponds to an "aralkyl" group, such as benzyl, benzhydryl, or phenethyl. If the alkyl group is substituted with a heterocyclic group, it corresponds to a "heterocyclylalkyl" group. In the present application, a Coalkyl group refers to H, i.e. a C 0-10 alkyl (or C0-C 10 alkyl) includes H and C 1-10 alkyl (or C1-C 10 alkyl).

[0105] The term "alkylene" refers to a hydrocarbon radical (divalent alkyl) derived from an alkane molecule by loss of two hydrogen atoms, which can be straight chain or branched chain and which is attached to the remainder of the molecule by single bonds. Typical alkylene groups herein contain 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, preferably 1 to 6 carbon atoms, such as methylene (-CH2-), ethylene, propylene, butylene, and the like. In the present application, a Coalkylene group refers to a single bond, i.e. a C 0-10 alkylene (or C0-C 10 alkylene) includes a single bond and C 1-10 alkylene (or C1-C 10 alkylene).

[0106] The term "alkoxy" refers to a substituent formed by the replacement of the hydrogen of a hydroxyl group with an alkyl group, such as an alkoxy group containing 1 to 10 carbon atoms, for example methoxy, ethoxy, propoxy, butoxy, and the like.

[0107] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0108] The term "haloalkyl" refers to a group formed by the replacement of one or more hydrogens of an alkyl group with a halogen atom (e.g., fluorine, chlorine, bromine, or iodine), such as -CHF2, -CH2F, -CF3, -CH2-CF3, -CH2CH2-CF3, -CH2CH2CH2-CF3.

[0109] The term "aryl" refers to a monocyclic or polycyclic radical, including polycyclic radicals containing single aryl groups and / or fused aryl groups (also referred to herein as "aromatic rings"), such as C6-Ci8 (e.g., C6, C8, C10, C12, C14, C16, C18) carbon ring atoms comprising 1 to 3 single or fused rings, in the present invention, the aryl group is a C6-Ci2 (e.g., C6, C8, C10, C12) aryl group, which refers to an aryl group containing 6 to 12 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indenyl, and the like. 12 The term "aryl" refers to a monocyclic or polycyclic radical, including polycyclic radicals containing single aryl groups and / or fused aryl groups (also referred to herein as "aromatic rings"), such as C6-Ci8 (e.g., C6, C8, C10, C12, C14, C16, C18) carbon ring atoms comprising 1 to 3 single or fused rings, in the present invention, the aryl group is a C6-Ci2 (e.g., C6, C8, C10, C12) aryl group, which refers to an aryl group containing 6 to 12 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indenyl, and the like.

[0110] The term "heterocyclyl" refers to a 3- to 18-membered non-aromatic ring group comprising 2 to 17 carbon atoms and 1 to 10 heteroatoms. The heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused, spiro, or bridged ring systems. The heterocyclyl group (also referred to herein as "heterocycle") can be partially saturated (heteroaryl, also referred to herein as "heteroaromatic ring") or fully saturated (heterocycloalkyl). Suitable heteroaryl groups in the compounds of the present application contain 1, 2, or 3 heteroatoms selected from N, O, S, P atoms, and include, for example, coumarinyl, including 8-coumarinyl, quinolinyl, including 8-quinolinyl, isoquinolinyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phtalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furopyridinyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofuropyridinyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Suitable heterocycloalkyl groups in the compounds of the present application contain 1, 2, or 3 heteroatoms selected from N, O, or S atoms, and include, for example, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxasulfuranyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxiranyl, thiiranyl, azepinyl, oxazepinyl, diazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolizinyl, and quinolizinyl. In the present application, for an optionally substituted heterocyclyl group, the position of substitution can be on any suitable carbon atom or heteroatom, for example, for a 5- or 6-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 5-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 6-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 7-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 8-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 9-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 10-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 11-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 12-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 13-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 14-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 15-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 16-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 17-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom, for example, for a 18-membered ring, the position of substitution can be on any suitable carbon atom or nitrogen atom. wherein the position of substitution of R can be on any suitable carbon atom or nitrogen atom, which can be, for example,

[0111] The compounds of the present application also include isotopically-labelled forms, i.e. forms in which one or more atoms are enriched in an isotope. For example, compounds of the present application which have at least one hydrogen atom replaced by deuterium or tritium, or at least one carbon replaced by 13C or 14C enriched carbon, or at least one nitrogen replaced by 15N enriched nitrogen, are included within the scope of the present application.

[0112] In the present application, "D" refers to deuterium; "substituted with deuterium" means the replacement of one or more hydrogen atoms with the corresponding number of deuterium atoms.

[0113] It is recognized that depending on the source of the chemical materials used in the synthesis, there will be some variation in the natural isotopic abundance in the synthesized compounds. Thus, the compounds of the present application will inherently include small amounts of isotopologues that are deuterated. Despite this variation, the concentration of stable hydrogen and carbon isotopes of natural abundance is very low and insignificant compared to the degree of stable isotope substitution of the compounds of the present application. See, e.g., Wada, E, et al., Seikagaku, 1994, 66: 15; Gannes, LZ, et al., Comp Biochem Physiol Mol Integr Physiol, 1998, 119: 725.

[0114] In the compounds of the present application, any atom not designated as deuterium is present at its natural isotopic abundance. Unless otherwise indicated, when a position is designated specifically as "H" or "hydrogen," that position is to be understood to have hydrogen in its natural isotopic composition. Likewise, unless otherwise indicated, when a position is designated specifically as "D" or "deuterium," that position is to be understood to have deuterium in an abundance of at least 3000 times greater than the natural abundance of deuterium (i.e., at least 45% deuterium incorporation).

[0115] The term "isotopic enrichment factor" as used herein refers to the ratio between the isotopic abundance of a particular isotope and the natural abundance. In other embodiments, the compounds of the present application have an isotopic enrichment factor for each specified deuterium atom of at least 3500 (52.5% deuterium incorporation at each specified deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

[0116] The term "isotopologues" refers to substances in which the chemical structure differs from a particular compound of the present application only in its isotopic composition.

[0117] The term "pharmaceutically acceptable" means that the molecules per se, as well as the compositions containing them, are suitable for use in contact with the tissues of subjects without undue toxicity, allergic reaction, or other problem or complication commensurate with a rational benefit / risk ratio when administered as described.

[0118] The term "pharmaceutically acceptable salt" includes both acid and base addition salts.

[0119] The term "acid addition salt" includes but is not limited to salts from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, and phosphonic acids, as well as salts from organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Thus, these salts include but are not limited to sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartarate, and methanesulfonate, as well as salts of amino acids such as arginate, gluconate, galacturonate, and the like. Acid addition salts can be prepared by contacting the free base form with a sufficient amount of the desired acid, in a manner to form the salt. The free base form can be regenerated by contacting the salt form with a base, and the free base isolated in a conventional manner.

[0120] The term "base addition salt" refers to salts which are formed with metals or amines such as hydroxides of alkali and alkaline earth metals or with organic amines. Examples of metals useful as cations include but are not limited to sodium, potassium, magnesium, and calcium. Examples of appropriate amines include but are not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (1,2-diaminoethane), N-methylglucamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base, in a manner to form the salt. The free acid form can be regenerated by contacting the salt form with an acid, and the free acid isolated in a conventional manner.

[0121] The term "stereoisomers" includes enantiomers, diastereomers, and geometric isomers. Some of the compounds of the application have cyclic hydrocarbyl groups which can be substituted on more than one carbon atom, in which case all geometric isomers, including cis and trans, and mixtures thereof, are within the scope of the application.

[0122] The term "solvate" refers to a physical association between one or more solvent molecules and one or more of the compounds of the present application. The physical association can include ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate can be isolated, e.g., when one or more solvent molecules are incorporated in the crystal lattice of the solid state form. Solvates include solution-phase and isolatable solvates. Representative solvates include ethanolates, methanolates, and the like.

[0123] The term "prodrug" refers to forms of the compounds of Formula I which are suitable for administration to a patient without undue toxicity, irritation, allergic response, and the like, and which are effective for their intended use, including the ester, amide and zwitterionic forms. Prodrugs are converted to the parent compound in vivo, e.g., by hydrolysis in blood.

[0124] The terms "patient" or "subject" and the like are used interchangeably herein and refer to any animal or cell thereof, whether in vitro or in situ, to be treated according to the methods described herein. In particular, the aforementioned animal includes a mammal, for example, a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey, or a human, and particularly a human.

[0125] The term "treatment" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, arresting, and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0126] The term "prevention" refers to avoiding, minimizing, or making it difficult for a disease to occur or develop by treatment before the onset of the disease.

[0127] The term "KLHL24 ubiquitin ligase inhibitor", also known as KLHL24 ubiquitin protease inhibitor, KLHL24 inhibitor, refers to a substance that has an inhibitory effect on the expression of KLHL24 protein, including but not limited to: inhibiting the activity of KLHL24 protein, inhibiting the expression of KLHL24 protein gene or inhibiting the ubiquitination degradation of KLHL24 protein.

[0128] The term "KLHL24 related disease", also known as KLHL24 mediated disease, mainly refers to a series of diseases caused by abnormal activity of KLHL24, for example, diseases caused by KLHL24 mutation, diseases caused by abnormal function of KLHL24 due to other factors, keratin abnormality diseases, and diseases that can be beneficial to the prevention and / or treatment thereof by inhibiting / degrading KLHL24.

[0129] The term "keratin abnormality disease" refers to a class of diseases caused by keratin abnormalities, mainly affecting the structure and function of tissues composed of keratin, such as skin, hair, and nails.

[0130] The term "tumor" refers to an abnormal mass of tissue in which the growth of the mass exceeds and is not coordinated with the growth of normal tissue. Tumors can be "benign" or "malignant", depending on the following characteristics: degree of cellular differentiation (including morphology and function), rate of growth, local invasion, and metastasis. "Benign tumors" are generally well-differentiated, characterized by slower growth than malignant tumors, and remain confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. In some cases, certain "benign" tumors can later give rise to malignant tumors, possibly due to additional genetic changes in a subpopulation of the neoplastic cells of the tumor, and these tumors are referred to as "pre-malignant tumors". "Malignant tumors" are generally poorly differentiated (anaplastic), and are characterized by rapid growth, accompanied by progressive infiltration, invasion, and destruction of surrounding tissue. In addition, malignant tumors generally have the ability to metastasize to distant sites.

[0131] The term "cancer" refers to a malignant tumor (Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990).

[0132] The terms "compound", "agent", "therapeutic agent" are used interchangeably herein and include, but are not limited to, compounds and mixtures thereof, small molecule compounds, biological macromolecules, and the like, particularly small molecule compounds. The agents provided herein are capable of interfering with the activity of KLHL24, i.e., they have the ability to inhibit the expression and / or the biochemical or biological function of the KLHL24 protein.

[0133] The term "small molecule" refers to a non-biological agent or compound having a molecular weight of less than about 1000 g / mol.

[0134] The disclosures of various publications, patents and published patent specifications referenced herein are hereby incorporated by reference in their entireties.

[0135] STK731044 (chemical name: (5E)-2-[4-(3-methylphenyl)-1-piperazinyl]-5-[[5-(4- nitrophenyl)-2-furanyl]methylidene]-1,3-thiazol-4-one) used in the embodiments of the present application has the following structure:

[0136] MLN4924 (also known as Pevonedistat, purchased from Sellck company) used in the embodiments of the present application has the following structure:

[0137] The disclosures of various publications, patents and published patent specifications, referred to herein are hereby incorporated by reference in their entirety.

[0138] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0139] The experimental methods and materials involved in the following embodiments are as follows:

[0140] 1. Cell culture

[0141] 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, PAN), 1% penicillin-streptomycin (NEAA, Gibco). The culture environment was 37℃ and 5% CO2.

[0142] 2. Construction and transfection of plasmid

[0143] The KLHL24 gene fragment was cloned into the pEGFP-N1 vector by the Gateway cloning system (Invitrogen). Neofect DNA transfection reagent (TF201201) was selected as the transfection reagent, which was mixed with Opti-MEM (Gibco, 31985070) and the plasmid to be transfected, incubated at room temperature for 20 minutes, and then added to the cell culture medium. After 6 hours of transfection, the DMEM medium containing 10% FBS was replaced.

[0144] 3. Experimental animals

[0145] Experimental animals and feeding environment: 7-week-old SPF C57BL / 6J mice weighing 16-20g were produced by Beijing Vital River Laboratory Animal Technology Co., Ltd. Klhl24 c.3G / T mice were constructed by Beijing Biosearch Technologies Co., Ltd. using CRISPER / Cas9 technology.

[0146] 4. High-throughput screening

[0147] HEK293T cells were transfected with GFP-KLHL24 plasmid, incubated in 10 cm dishes for 8 h, then reseeded in 96-well or 384-well plates for 8 h, and then compounds were added to the 96-well or 384-well plates by Echo 550 nanoliter acoustic liquid handling system (Labcyte). After co-incubation for 24 hours, immunofluorescence technology was used to observe the fluorescence signal. The specific steps are as follows: fix the cells, directly add and cell culture liquid of the same volume of 4% paraformaldehyde to fix the cells, and fix at room temperature for 15 min. After washing the cell surface with 1xPBS for 3 times, taking the 96-well plate as an example, 30 μL of DAPI staining solution (1 μg / mL) was added to each well, and after staining at room temperature for 5 min, the aluminum foil was sealed to take pictures. The laser confocal high-content imaging microscope (PerkinElmer, Opera Phenix) was used to take pictures. The instrument operation manual was followed, and after a new work template was established, a field of view was first selected for focusing, and after aligning the focal plane, 488 nm excitation light and 340 nm excitation light (separate exposure) were selected for taking pictures. The Opera data analysis station was used for analysis. First, count the DAPI-stained cell nucleus N, which is the total number of cells; GFP-positive cells emit green fluorescence under 488 nm excitation light. Count the number of green fluorescent cells n, which is the number of GFP-KLHL24-positive expression cells. When counting n, the threshold value of green fluorescence needs to be set to select as many green fluorescent cells as possible, and in addition, cell area, cell shape and other parameters can be set to reduce the influence of background.

[0148] 5. Western blot

[0149] After the cells were collected, RIPA lysis buffer (Beyotime, P0013C) was used for lysis on ice for 30 min. For animal tissue samples, centrifuge at 12000 rpm at 4°C for 10 min to take the supernatant, and then add 5xloading buffer and incubate at 95°C metal bath for 10 min. Then separate the proteins by 10% SDS-PAGE and transfer to PVDF membrane, block at room temperature for 1 h with 5% skim milk (TBST configuration), and incubate with the corresponding protein one antibody, anti-Vimentin (Cell Signaling Technology, 5741S), anti-GAPDH (ZSGB-BIO, TA-08), and anti-K15 (ab52816, Abeam). Use HRP-conjugated secondary antibody to detect the target band by exposure instrument.

[0150] 6. Tape hair sticking experiment

[0151] Drug preparation scheme:

[0152] Preparation of the test side external preparation (referred to as test preparation) : 10 mM STK731044 dissolved in DMSO was added to ethanol glycerol (ethanol and glycerol mixed at a volume ratio of 1:1) to make the final concentration of STK731044 0.1 g / 100 ml;

[0153] Preparation of the control side external preparation (referred to as control preparation) : the same volume and concentration of DMSO solution (without STK231044) as used in the test preparation was added to ethanol glycerol (ethanol and glycerol mixed at a volume ratio of 1:1).

[0154] Seven specific time point Klhl24 c.3G / T Female or male mice were administered with KLHL24 inhibitors, the administration time was the first resting period P15-P30, a total of 14 days, the administration method was to use 20 μL of test preparation on the left side (test side) of the mouse back and 20 μL of control preparation on the right side (control side), and to smear once a day. The tape hair sticking experiment was performed at the second resting period, i.e. P50, and the self-control was performed. When the tape hair sticking experiment was performed, the mouse was anesthetized with 0.25% tri bromoethanol, and then the tape was pressed on the back of the mouse with the same force for 30 s, and then the tape was removed, photographed, and the pictures were collected and sorted. The weight of the shed hair shaft was calculated, and Dunnett-t test was used for comparison between the experimental group and the control group.

[0155] Example 1: Establishment of KLHL24 ubiquitin ligase inhibitor high-throughput screening system

[0156] KLHL24 belongs to the KLHL E3 ubiquitin ligase family and has the characteristics of E3 ubiquitin ligase, i.e. it can ubiquitinate substrate proteins when there are substrate proteins, and it can ubiquitinate itself when there are no substrate proteins. The present application found in the previous experiment that MLN4924 can significantly increase the content of KLHL24 protein. We can consider that KLHL24 is the E3 ubiquitin ligase of KLHL24 itself. Therefore, the stability of KLHL24 protein itself was used as a screening index to design the screening method. According to the above idea, a screening strategy as shown in Figure 1 was designed. Specifically, the following steps are included: construction of GFP-tagged KLHL24 plasmid, transformation of the plasmid into 293T cells, and maintenance of low level due to KLHL24 ubiquitination. Subsequently, small molecule inhibitors are added, and if the small molecule inhibitors can restore the content of KLHL24 protein, then the increase of KLHL24 protein can be observed by fluorescence microscope.

[0157] According to the designed screening scheme, it is first necessary to determine whether KLHL24 self-ubiquitination can be used as an index for screening. The specific operation steps are as follows: GFP-KHL24 is transfected into 293T cells, and then an effective and selective NEDD8 activating enzyme (NAE) inhibitor MLN4924 (concentrations are 0.156 μM, 0.313 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM) is added, and the expression of GFP-KLHL24 is detected by GFP fluorescence. The results are shown in Figure 2, and the expression amount of GFP-KLHL24 is very low (Figure 2a), and after the addition of MLN4924, its content is significantly increased and shows a dose-dependent manner (Figure 2b). According to the above experimental results, it can be judged that the stability of GFP-KLHL24 can be used as a screening index. It is preliminarily confirmed that the above screening scheme has high feasibility.

[0158] Secondly, before carrying out high-throughput screening, it is also necessary to evaluate the stability or repeatability of the above screening scheme. If the stability of the screening scheme is poor, the error between each group of experiments will be large, which will lead to the inability to accurately screen effective small molecule compounds, and a large proportion of false negatives will occur. In order to eliminate such errors, the usual practice is to increase repeated experiments, however, this will greatly increase the throughput of screening, and greatly increase the consumption of manpower and material resources. In order to solve this problem, a preliminary experiment is first carried out, and the specific steps are as follows: 320 small molecule compounds (purchased from Tocris) are taken, and are added to different 96-well plates, and each small molecule compound is repeated twice, and the results of the two repeated experiments are analyzed for correlation. The results are shown in Figure 3, and the correlation coefficient R of the two groups of results is equal to 0.912, which indicates that they have very strong correlation. In addition, regression analysis is performed on the two repeated experiments, and the regression function is: y = 0.8907x + 1.0135. According to the coefficient of the regression function, the two results show a positive correlation, the regression coefficient is 0.8907, and the constant is 1.0135, indicating that the two groups of data are very close (Figure 3). According to the above experimental results, the above screening method is very stable and has very good repeatability. According to this result, we will only do one repetition in high-throughput screening, which will greatly improve the screening efficiency.

[0159] Example 2: High-throughput screening of the Express-Pick diversity compound mother nucleus library of Selleck company

[0160] Small molecule compounds that can inhibit the activity of KLHL24 were screened according to the high-throughput screening method established in Example 1. A diverse compound library (Express-Pick Library, Catalog No. L3600) was selected when the screening was performed, and the compound library contained 4208 small molecule compounds. The reasons for selecting this compound library include the following points: 1) the compound library is a unique collection of compounds with different core structures, suitable for high-throughput screening; 2) the compound library is derived from one of the world's largest pharmaceutical companies; 3) the compound library can be combined with the target, which can be used to study the conformation and efficacy of the compound and improve the speed of new drug development; 4) NMR and HPLC were used to ensure the purity of the compound.

[0161] After selecting the compound library, high-throughput screening based on high content was performed. When the screening was performed, multiple negative controls (DMSO) were set in each experiment. After obtaining the original results, the number of DAPI-stained cell nuclei N was first counted, which was the total number of cells; At the same time, the number of green fluorescent cells n was counted, which was the number of GFP-KLHL24-expressing cells. When counting n, the threshold value of green fluorescence was set to adjust the green fluorescent cells as much as possible, and in addition, parameters such as cell area and cell shape can be set to reduce the influence of background. The calculation formula of the ratio of GFP-positive cells is: n / N*100%. The ratio of GFP-positive cells was standardized according to the results of the negative control, and the critical value of Fold Change was set to 2, which would greatly reduce the false positive results. At the same time, we also introduced the number of cells per well. Small molecule compounds usually have certain toxicity to cells, which can cause cell death and spontaneous fluorescence, which increases the possibility of false positives, therefore, the introduction of the number of cells per well can reduce false positives on the one hand, and can exclude small molecule compounds with high toxicity, reducing the workload of subsequent verification. In addition, we also considered the factor of cell transfection rate. According to the efficiency of the transfection reagent transfecting the plasmid, we believe that more than 80% of the proportion of GFP-positive cells is caused by small molecule spontaneous fluorescence. Considering the three variables of Fold Change value, number of cells per well, and transfection efficiency, first, the number of DAPI-stained cell nuclei was counted, and whether the compound had obvious toxicity was determined by the number of cell nuclei. In the case of no toxicity, the ratio of GFP-positive cells after compound treatment was standardized according to the results of the negative control, and the critical value of Fold Change was set to 2, and the results are shown in Figure 4. Finally, 41 compounds were screened out.

[0162] Example 3: Verification of the effect of the candidate small molecule compound screened in the preliminary screening on the degradation of KLHL24

[0163] To preliminarily verify whether the above 41 small molecule compounds can significantly affect the content of GFP-KLHL24 protein, the specific operation steps are as follows: transfect 293T cells with GFP-KLHL24, then treat with small molecule compounds, and detect the content of KLHL24 protein by Western blot. Because there are many compounds, we detect the effect on GFP-KLHL24 protein through multiple experiments. In this experiment, DMSO treatment is set as negative control and MLN4924 treatment is set as positive control, and the concentration of positive control and small molecule compounds is 10 μM. The results are shown in Figure 5, and a total of 25 small molecule compounds can increase the content of GFP-KLHL24 protein at a concentration of 10 micromole / liter.

[0164] Example 4: KLHL24 inhibitor restores Klhl24 c.3G / T The levels of KRT15 and Vimentin proteins in mice

[0165] Multiple cell experiments verified that STK731044 in 25 small molecules has the most significant activity in promoting GFP-KLHL24, and has the smallest cytotoxicity. Therefore, STK731044 was selected for subsequent animal experiments. The effect of KLHL24 ubiquitin ligase activity on mice after inhibitor treatment was evaluated, and the specific operation steps are as described in the previous adhesive tape hair experiment. The results are shown in Figure 6, compared with the negative control (i.e. control preparation treatment), KLHL24 inhibitor STK731044 treatment Klhl24 c.3G / T The anchoring ability of the back hair shaft of the mouse is enhanced, and the number of hair shafts falling off on the adhesive tape is reduced. According to the reported literature, compared with WT mice (wild type mice), Klhl24 c.3G / T The levels of KRT15 (K15) and Vimentin proteins in mice are reduced. Through further experiments, it was found that Klhl24 c.3G / T After the mouse KLHL24 inhibitor STK731044 treatment, the expression levels of KRT15 and Vimentin proteins in the skin of the mouse are increased (Figure 7). In Figure 7, GAPDH is a commonly used housekeeping gene protein, which is used as a reference protein for comparison of each sample. The above results suggest that KLHL24 inhibitors can be used as drugs for treating alopecia.

[0166] Summary: STK731044 (screening number PickP4E22) is a potential KLHL24 ubiquitin ligase inhibitor, which has good application prospect in treating diseases caused by KLHL24 mutation.

[0167] The above merely describes preferred embodiments of the present application, but is not used to limit the present application, and any modification, equivalent replacement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0168] The foregoing embodiments and methods described in the present application can vary based on the ability, experience and preference of the person skilled in the art.

[0169] The fact that the steps of the method are listed in a certain order in the present application does not constitute any limitation on the order of the steps of the method.

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof as a KLHL24 ubiquitin ligase inhibitor, or for use in the preparation of a medicament for the prevention and / or treatment of KLHL24-related diseases, or in the preparation of a product for maintaining hair, said compound having the following structure: wherein represents a single or double bond; X is selected from: O, S, NH; A ring is a heterocycle; R A It is one or more independent substituents on ring A, selected from: H, halogen, hydroxyl, amino, cyano, nitro, azide, C1-C. 10 Alkyl, -(C0-C6 alkylene)-(C3-C6 alkylene) 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), L is a divalent group attached to ring A, selected from: single bond, H, C1-C6 alkylene, -(C0-C6 alkylene)-O-, -(C0-C6 alkylene)-S-, -(C0-C6 alkylene)-C(O)-, -C(O)-(C0-C6 alkylene)-, -(C0-C6 alkylene)-COO-, -(C0-C6 alkylene)-C(S)-, -(C0-C6 alkylene)-N(C0-C 10 alkylene)-, -(C0-C6 alkylene)-CON(C0-C 10 alkylene)-, -(C0-C6 alkylene)-N(C0-C 10 -(C0-C6 alkylene)CO-, -(C0-C6 alkylene)-SO2-, -(C0-C6 alkylene)-SO-, -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the C0-C6 alkylene, C3-C 10 cycloalkyl, C6-C 10 The hydrogen in the aryl or 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, azide, C1-C. 10 Alkyl, C1-C 10 Haloalkyl, -O(C) 0-10 alkyl), -S(C 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0- 10 Alkyl)(C 0-10 alkyl), -COO(C 0-10 Alkyl), -OCO(C) 0-10 Alkyl), -CON(C) 0-10 Alkyl)(C 0-10 Alkyl), -CO(C) 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)CO(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 alkyl), -N(C) 0-10 Alkyl)CON(C 0-10 alkyl), -N(C) 0-10 Alkyl)SO2(C 0-10 alkyl); R C one or more independent substituents for L selected from H, halogen, cyano, nitro, azido, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 10 haloalkyl, -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO(C 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)(C 0- 10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)COO(C 0-10 alkyl), -N(C 0-10 alkyl)CON(C 0-10 alkyl), -N(C 0-10 alkyl)SO2(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl), -CO(C0-C6alkylene)(4-10 membered heterocyclyl), -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-10 membered heterocyclyl), wherein the C0-C6alkylene, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 10 cycloalkyl, C6-C 10 aryl, 4-10 membered heterocyclyl, can be optionally substituted with one or more groups selected from halogen, cyano, nitro, azido, C1-C 10 alkyl, C1-C 10 haloalkyl, -(C0-C6alkylene)-(C3-C 10 cycloalkyl), -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), -N(C 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)CO(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 alkyl), -N(C) 0-10 Alkyl)CON(C 0- 10 alkyl), -N(C) 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C) 0-10 alkyl), -S(C 0-10 alkyl), -SO(C) 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 alkyl), -COO(C 0-10 Alkyl), -OCO(C) 0-10 Alkyl), -CON(C) 0-10 Alkyl)(C 0-10 Alkyl), -CO(C) 0-10 alkyl); R B It is one or more independent substituents on the benzene ring, selected from: H, halogen, cyano, nitro, azido, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C6 alkylene) 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), -(C0-C6 alkylene)-(C1-C 10 (halogenated alkyl), -(C0-C6 alkylene)-(C1-C6) 10 Halogenated alkoxy), -(C0-C6 alkylene)-N(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)CO(C 0- 10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)CON(C 0-10 Alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -(C0-C6 alkylene)-O(C 0-10 Alkyl), -(C0-C6 alkylene)-S(C 0-10 Alkyl), -(C0-C6 alkylene)-SO(C 0-10 Alkyl), -(C0-C6 alkylene)-SO2(C 0-10 Alkyl), -(C0-C6 alkylene)-SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-COO(C 0-10 Alkyl), -(C0-C6 alkylene)-OCO(C 0-10 Alkyl), -(C0-C6 alkylene)-CON(C 0-10 Alkyl)(C 0-10 Alkyl), -(C0-C6 alkylene)-CO(C 0- 10 Alkyl groups), -CO (C0-C6 alkylene groups) (4-10 membered heterocyclic groups), and nitric oxide (NO) donor residues, wherein the C0-C6 alkylene groups, C1-C6 alkylene groups, and C1-C6 alkylene groups are alkyl groups, C1-C6 alkylene ... 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, C3-C 10 cycloalkyl, C6-C 10 The hydrogen atoms in aryl and 4-10 membered heterocyclic groups may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, azide, C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), C1-C 10 Haloalkyl, C1-C 10 Halogenated alkoxy groups, -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)CO(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 alkyl), -N(C) 0-10 Alkyl)CON(C 0-10 alkyl), -N(C) 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C) 0-10 alkyl), -S(C 0-10 alkyl), -SO(C) 0-10 alkyl), -SO2(C 0-10 alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 alkyl), -COO(C 0-10 Alkyl), -OCO(C) 0-10 Alkyl), -CON(C) 0-10 Alkyl)(C 0-10 Alkyl), -CO(C) 0-10 Alkyl); or two R B Together with the carbon atom to which it is attached, it forms an aliphatic ring, aromatic ring, or heterocycle, wherein the hydrogen atom on the aliphatic ring, aromatic ring, or heterocycle is optionally substituted with one or more groups selected from: halogen, cyano, nitro, azide, C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group, C1-C 10 Haloalkyl, -N(C) 0-10 Alkyl)(C 0-10 Alkyl), -O(C) 0-10 alkyl), -COO(C 0- 10 Alkyl), -OCO(C) 0-10 Alkyl), -CON(C) 0-10 Alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl); the nitric oxide (NO) donor residue is selected from the group consisting of: -ON02, wherein R H and R K are independently selected from H, C1-C 10 alkyl, C1-C 10 alkenyl, -(C0-C6alkylene)-(C6-C 10 aryl), -(C0-C6alkylene)-(4-6 membered heterocyclyl), or R H and R K together with the nitrogen atom to which they are attached form a 4-6 membered heterocyclyl.

2. Use according to claim 1, characterized in that, The compound has the structure: wherein V is selected from: -S-, -NH-, -CH2-, R1, R2have the above definitions of R A R1, R2have the above definitions of R 3. Use according to claim 2, characterized in that, R1and R2are independently Preferably, L is selected from: single bond, C1-C6 alkylene, -(C0-C6 alkylene)-C(O)-, -C(O)-(C0-C6 alkylene)-, -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 heterocyclic)-, wherein the C0-C6 alkylene, C6-C 10 The H in the aryl group, 4-10 membered heterocyclic group, may optionally be substituted by one or more groups selected from the following: C1-C 10 Alkyl group, -COO(C 0-10 alkyl); Preferably, R C Selected from: H, halogens, C1-C 10 Alkyl, -O(C) 0-10 alkyl), -S(C 0-10 alkyl), -SO2(C 0- 10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 Alkyl), -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the C0-C6 alkylene, C 0-10 Alkyl, C3-C 10 cycloalkyl, C6-C 10 The hydrogen atoms in aryl and 4-10 membered heterocyclic groups may optionally be substituted by one or more groups selected from the following: halogen, nitro, C1-C. 10 Alkyl, C1-C 10 Haloalkyl, -O(C) 0- 10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 Alkyl group, -(C0-C6 alkylene group)-(4-10 membered heterocyclic group); Preferably, R B Selected from: H, halogen, cyano, nitro, C1-C 10 Alkyl, C1-C 10 Haloalkyl, -O(C) 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)CO(C 0-10 alkyl), -SO2(C 0-10 alkyl), -N(C) 0- 10 Alkyl)SO2(C 0-10 Alkyl), -CO(C) 0-10 alkyl), -COO(C 0-10 Alkyl), -CON(C) 0-10 Alkyl)(C 0-10 Alkyl), -CO(C0-C6 alkylene) (4-10 membered heterocyclic group), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the C0-C6 alkylene, C1-C 10 The hydrogen atoms in alkyl or 4-10 membered heterocyclic groups may optionally be substituted with one or more groups selected from the following: halogens, C1-C... 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, -O(C 0-10 alkyl), -N(C) 0-10 Alkyl)(C 0-10 alkyl), -N(C) 0-10 Alkyl)COO(C 0-10 alkyl), -COO(C 0-10 Alkyl), -(C0-C6 alkylene)-(C6-C 10 Aryl); or, two R B Together with the carbon atom it is attached to, it forms a 5-6 membered heterocycle.

4. Use according to claim 3, characterized in that, R1is selected from: H, -F, -CI, -Br, -I, Preferably, R2is selected from: H, -F, -Cl, -Br, -I, Preferably, R B selected from the group consisting of: H, -F, -CI, -Br, -I, -N02, -NH2, -CN, -COOH, -OH, -CF3, - ONO2; Preferably, two R B together with the carbon atom to which they are attached form a 5-6 membered heterocyclic ring selected from:

5. The use according to any one of claims 1 to 4, characterized in that, The compound has the structure: wherein R D is selected from: H, halogen, cyano, hydroxy, carboxyl, Ci-C6alkyl, Ci-C6haloalkyl.

6. Use according to claim 5, characterized in that, R D is C1-C6 alkyl, in particular methyl, ethyl; Preferably, R B is a nitric oxide (NO) donor residue, in particular -ON02, 7. Use according to claim 1, characterized in that, More preferably, the compound has the structure:

8. Use according to claim 1, characterized in that, said KLHL24-related disease is selected from: a skin disease, a cardiovascular disease, a tumor, a digestive system disease, arthritis, and complications thereof; preferably, said skin disease is selected from: alopecia, a bullous skin disease, a scar, a keratosis, an erythematous papulosa squamous skin disease, a connective tissue disease; said cardiovascular disease is a cardiomyopathy; said tumor is selected from: leukemia, colon cancer, colorectal cancer, prostate cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, liver cancer, gastric cancer, adrenal cortex cancer, pancreatic ductal adenocarcinoma, lung adenocarcinoma, pancreatic cancer, gonadoblastoma, multiple myeloma, mantle cell lymphoma, osteosarcoma, neuroblastoma, brain tumor, melanoma; said digestive system disease is selected from: chronic hepatitis, drug-induced liver injury; said arthritis is osteoarthritis; more preferably, said disease is selected from: epidermolysis bullosa and complications thereof, atrophic scar, alopecia, dilated cardiomyopathy, hypertrophic cardiomyopathy, colon cancer, prostate cancer, gonadoblastoma, endometrial cancer, osteoarthritis.

9. The use according to claim 1, characterized in that, said compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound is used alone or in combination with other kinds of active ingredients; preferably, said medicament further comprises one or more pharmaceutically acceptable adjuvants; preferably, said pharmaceutically acceptable adjuvant is selected from: a binder, a lubricant, a disintegrant, a bacteriostatic agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a preservative, a filler, an antioxidant, a buffer; preferably, the dosage form of said medicament is selected from: a gastrointestinal administration dosage form, an injection administration dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a cavity administration dosage form, more preferably an oral dosage form, a skin administration dosage form, an injection administration dosage form.

10. The use according to claim 1, characterized in that, said product is a medicine, a food, a health product, a care product; preferably, said maintaining hair includes promoting hair growth, promoting hair regeneration and self-repair, prolonging the hair growth cycle, preventing hair loss.

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