A klhl24 ubiquitin ligase inhibitor and use thereof

Abstract

This invention discloses a high-throughput screening method and system for KLHL24 ubiquitin ligase inhibitors. The method exhibits high stability and reproducibility, facilitating the efficient screening of KLHL24 ubiquitin ligase inhibitors and the development of related drugs. Based on the aforementioned method, this invention has screened several KLHL24 ubiquitin ligase inhibitors, which can be effectively used to treat KLHL24-related diseases, particularly those caused by KLHL24 mutations, such as hereditary epidermolysis bullosa, dilated cardiomyopathy, alopecia, and atrophic scarring, showing excellent application prospects. Given the function of KLHL24 in skin and hair regeneration, this inhibitor also shows good application potential in maintaining normal hair growth.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a KLHL24 ubiquitin ligase inhibitor and its application. Background Technology

[0002] KLHL24 belongs to the Kelch-like family of proteins (KLHLs). Proteins encoded by this family possess a highly conserved BTB and BACK domain, which interact with CUL3 and RBX1 to form the CUL3–RBX1–Kelch ubiquitin ligase complex. Kelch-like proteins also possess a Kelch domain for substrate recruitment, determining their E3 ligase specificity. KLHL24 is widely expressed in various human tissues, including the skin.

[0003] The research led by the first inventor of this invention is the first discovery that a truncated KLHL24-ΔN28 protein is produced due to a mutation in the start codon of the Klhl24 gene. This protein becomes more stable due to a decrease in its own ubiquitination, which leads to the ubiquitination and degradation of a large amount of keratin KRT14 in the skin by KLHL24. This makes the basal cells of the epidermis fragile and prone to shedding, 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 skinfragility). Nature genetics 48.12 (2016): 1508-1516.). Subsequent studies have reported that KLHL24 ubiquitination substrates include KRT15, ​​Vimentin, and Desmin, in addition to KRT14. Other studies have found that syndromic phenotypes caused by KLHL24 mutations are emerging. EBS-KLHL24 patients are born with extensive bare skin areas and skin fragility, which develop into atrophic scarring over time, accompanied by life-threatening cardiomyopathy (Yenamandra, VK, et al. Cardiomyopathy in patients with epidermolysis bullosa simplex with mutations in KLHL24). British Journal of Dermatology(2018) 179(5):1181-1183). Patients with EBS-KLHL24 may also experience hair loss, especially in areas of skin atrophy, and sometimes even affecting 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 is evident that developing drugs targeting KLHL24 is of great significance.

[0004] However, there are currently no specific drugs for treating diseases caused by KLHL24 mutations. Therefore, establishing a high-throughput screening system and successfully identifying drugs that specifically bind to and inhibit KLHL24 activity is of great significance for treating diseases caused by KLHL24 mutations. Given the function of KLHL24 in skin and hair regeneration, screening for its inhibitor is also of great significance in maintaining normal hair. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention 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 a first aspect of the invention, there is provided the use of a compound or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof as a KLHL24 ubiquitin ligase inhibitor, said compound having the following structure:

[0007] (I)

[0008] in, Represents a single bond or a double bond;

[0009] X is selected from: O, S, NH;

[0010] Ring A is a heterocyclic ring;

[0011] 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), ;

[0012] 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);

[0013] R COne 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 0-10 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-C6 alkylene) (4-10 membered heterocyclic groups), -(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, C1-C 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, C1-C 10 Haloalkyl, -(C0-C6 alkylene)-(C3-C6 alkylene) 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), -N(C 0-10 Alkyl)(C0-10 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);

[0014] 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 0-10Alkyl), -(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 0-10Alkyl), -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).

[0015] Specifically, the aforementioned nitric oxide (NO) donor residues can be selected from: -ONO2, , where R H and R K Independently selected from: H, C1-C 10 Alkyl, C1-C 10 alkenyl, -(C0-C6 alkylene)-(C6-C 10 Aryl), -(C0-C6 alkylene)-(4-6 membered heterocyclic) (e.g. ), or R H and R K Together with the nitrogen atom it is attached to, they form 4-6 membered heterocyclic groups (e.g. ).

[0016] In some embodiments of the present invention, X is 0.

[0017] Furthermore, ring A is a 5-10 membered heterocycle, selected from: Where V is selected from: -S-, -NH-, -CH2-, R1 and R2 have the above-mentioned R A Definition.

[0018] In some embodiments of the present invention, the compound has the following structure:

[0019] Preferred .

[0020] In some embodiments of the present invention, R1 is... .

[0021] In some embodiments of the present invention, R2 is... .

[0022] Further, L is 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)-N(C0-C 10 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).

[0023] 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).

[0024] 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)(C 0-10 alkyl), -CO(C) 0-10 Alkyl), -CO(C0-C6 alkylene) (4-10 membered heterocyclic groups), -(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, C1-C 10 Alkyl, C3-C 10 cycloalkyl, C6-C 10 The hydrogen atoms in aryl and 4-10 membered heterocyclic groups may optionally be substituted with one or more groups selected from the following: halogen, cyano, nitro, C1-C. 10 Alkyl, C1-C 10 Haloalkyl, -(C0-C6 alkylene)-(C3-C6 alkylene) 10 cycloalkyl), -(C0-C6 alkylene)-(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), -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).

[0025] Preferably, R C Selected from: H, halogens, C1-C 10 Alkyl, -O(C) 0-10 alkyl), -S(C 0-10alkyl), -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).

[0026] In some embodiments of the present invention, R1 is selected from: H, -F, -Cl, -Br, -I, .

[0027] In some embodiments of the present invention, R2 is selected from: H, -F, -Cl, -Br, -I, .

[0028] 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 0-10Alkyl), -(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 group), -CO (C0-C6 alkylene group) (4-10 membered heterocyclic group), wherein the C0-C6 alkylene group, C1-C 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)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic), 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).

[0029] 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(C0-10 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).

[0030] In some embodiments of the present invention, R B Selected from: H, -F, -Cl, -Br, -I, -NO2, -NH2, -CN, -COOH, -OH, -CF3, -ONO2.

[0031] 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).

[0032] Preferably, two R B Together with the carbon atom it is attached to, it forms a 5-6 membered heterocycle.

[0033] In some embodiments of the present invention, two R BTogether with the carbon atom it is attached to, it forms a 5-6 membered heterocycle selected from: .

[0034] In some embodiments of the present invention, the compound has the following structure:

[0035]

[0036] Among them, R D Selected from: H, halogen, cyano, hydroxyl, carboxyl, C1-C6 alkyl, C1-C6 haloalkyl.

[0037] In some embodiments of the present invention, R D It is a C1-C6 alkyl group, such as methyl or ethyl.

[0038] In some embodiments of the present invention, R B Nitric oxide (NO) donor residues, such as -ONO2, .

[0039] In some embodiments of the present invention, the compound has the following structure:

[0040] .

[0041] In a second aspect of the invention, there is provided the use of a compound or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof in the preparation of a medicament for the prevention and / or treatment of KLHL24-related diseases, said compound having the definition of a compound as described in the first aspect of the invention.

[0042] Furthermore, the KLHL24-related diseases are those for which inhibition or degradation of KLHL24 can be beneficial for prevention and / or treatment, such as diseases caused by KLHL24 mutations (e.g., mutations in the start codon of the Klhl24 gene that produce truncated KLHL24-ΔN28 protein), keratin abnormalities, such as skin diseases, cardiovascular diseases, tumors, digestive system diseases, arthritis, and their complications.

[0043] Furthermore, the skin diseases mentioned are selected from: hair loss, bullous skin diseases, scars, keratosis, erythematous papulosquamous skin diseases, and connective tissue diseases.

[0044] Furthermore, the bullous skin diseases are selected from: bullous epidermolysis (such as hereditary bullous epidermolysis, acquired bullous epidermolysis), pemphigus, bullous pemphigoid, and cicatricial pemphigoid.

[0045] Furthermore, the scar is selected from: hypertrophic scars, keloids, and atrophic scars.

[0046] Furthermore, the keratosis is selected from: follicular keratosis, palmoplantar keratoderma, palmoplantar keratoderma, and its complications include dry skin, scaling, cracking, etc.

[0047] Furthermore, the erythematous papulosquamous skin disease is selected from: psoriasis, pityriasis rubra pilaris, and erythematous keratosis.

[0048] Furthermore, the connective tissue diseases mentioned are selected from: lupus erythematosus and scleroderma.

[0049] In some embodiments of the present invention, the disease is atrophic scarring.

[0050] In some embodiments of the present invention, the disease is hereditary epidermolysis bullosa and its complications (such as esophageal mucosal stricture, finger syndactyly, corneal scarring, and blindness).

[0051] In some embodiments of the present invention, the disease is hair loss.

[0052] Furthermore, the cardiovascular disease is cardiomyopathy (such as dilated cardiomyopathy or hypertrophic cardiomyopathy).

[0053] Furthermore, the 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, stomach cancer, adrenocortical carcinoma, pancreatic ductal adenocarcinoma, lung adenocarcinoma, pancreatic cancer, gonadal blastoma, multiple myeloma, mantle cell lymphoma, osteosarcoma, neuroblastoma, brain tumor, and melanoma.

[0054] Preferably, the tumor is selected from: colon cancer, prostate cancer, gonadal blastoma, and endometrial cancer.

[0055] More preferably, the tumor is selected from: colon cancer and prostate cancer.

[0056] Furthermore, the digestive system diseases mentioned are selected from: chronic hepatitis and drug-induced liver injury.

[0057] Furthermore, the arthritis mentioned is osteoarthritis.

[0058] Furthermore, the subjects of the disease are mammals, such as humans.

[0059] Furthermore, the compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound may be used alone or in combination with other types of active ingredients.

[0060] Furthermore, the drug is a veterinary drug or a human drug.

[0061] Furthermore, the drug also includes one or more pharmaceutically acceptable excipients.

[0062] Furthermore, the pharmaceutically acceptable excipients are selected from: binders, lubricants, disintegrants, antibacterial agents, suspending agents, solubilizers, thickeners, stabilizers, preservatives, fillers, antioxidants, and buffers.

[0063] Furthermore, the compound or its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates, or deuterated compounds can be administered via any suitable route of administration, such as gastrointestinal (e.g., oral, sublingual, rectal) or non-gastrointestinal (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, infusion, respiratory tract, etc.). The drug can be in any suitable dosage form, such as gastrointestinal dosage forms (e.g., oral dosage forms), including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, etc. Syrups, liquids, emulsions, suspensions, etc.; non-gastrointestinal dosage forms, such as injectable dosage forms (e.g., for subcutaneous, intravenous, intramuscular, and intraperitoneal injections), respiratory dosage forms (e.g., sprays, aerosols, powders), skin dosage forms (e.g., topical solutions, lotions, ointments, plasters, pastes, patches), mucosal dosage forms (e.g., eye drops, ophthalmic ointments, nasal drops, mouthwashes, sublingual tablets), and cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc.) for use in the rectum, vagina, urethra, nasal cavity, and ear canal.

[0064] In some embodiments of the present invention, the drug is a transdermal dosage form.

[0065] In some embodiments of the present invention, the drug is an oral dosage form.

[0066] In some embodiments of the present invention, the drug is an injectable dosage form.

[0067] In a third aspect of the invention, there is provided the use of a compound or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the preparation of a product for maintaining hair.

[0068] Furthermore, the subjects of the product are mammals, particularly humans, including healthy individuals and patients.

[0069] Furthermore, maintaining hair includes promoting hair growth, promoting hair regeneration and self-repair, prolonging the hair growth cycle, and preventing hair loss.

[0070] Furthermore, the product in question is a pharmaceutical or nursing product.

[0071] Furthermore, the product also includes one or more auxiliary materials.

[0072] In a fourth aspect of the present invention, a method for screening KLHL24 ubiquitin ligase inhibitors is provided, comprising the following steps: co-incubating the substance to be screened with cells expressing KLHL24 labeled with fluorescent protein, and detecting the fluorescence signal.

[0073] In some embodiments of the present invention, the fluorescent protein is GFP.

[0074] In some embodiments of the present invention, the cells are HEK293T cells.

[0075] Furthermore, the method also includes calculation and analysis steps, such as calculating the number or proportion of fluorescently positive cells.

[0076] In some embodiments of the present invention, the method further includes the step of transfecting cells with an expression vector containing KLHL24 labeled with a fluorescent protein.

[0077] In some embodiments of the present invention, the expression vector is a plasmid.

[0078] In some embodiments of the present invention, the method includes:

[0079] (a) Constructing the GFP-KLHL24 plasmid;

[0080] (b) Transfect HEK293T cells with the GFP-KLHL24 plasmid and incubate them;

[0081] (c) Mix the substance to be screened with the cells from step (b) and incubate together;

[0082] (d) Detect the fluorescence signal of the incubation system obtained in step (c).

[0083] In a fifth aspect of the invention, a screening system (such as a kit) for a KLHL24 ubiquitin ligase inhibitor is provided, comprising: cells expressing KLHL24 labeled with a fluorescent protein (as described in the fourth aspect).

[0084] Specifically, the screening system also includes reagents for incubating cells, such as culture medium.

[0085] Specifically, the screening system also includes containers for incubating cells, such as 96-well plates or 384-well plates.

[0086] Specifically, the screening system also includes reagents for detecting fluorescence signals, such as cell fixation reagents (e.g., paraformaldehyde), cell washing solutions, staining solutions, etc.

[0087] In a sixth aspect of the invention, the screening system described in the fifth aspect is provided for use in screening KLHL24 ubiquitin ligase inhibitors and medicaments for treating KLHL24-related diseases.

[0088] In a seventh aspect of the invention, a method for preventing and / or treating KLHL24-related diseases is provided, comprising administering to a subject in need of the compound described in the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof.

[0089] Furthermore, the KLHL24-related diseases have the definition of diseases described in the second aspect of this invention.

[0090] Furthermore, the subjects were mammals, particularly humans.

[0091] Furthermore, the administration can be carried out via 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, infusion, respiratory administration, etc.).

[0092] In an eighth aspect of the invention, a method for maintaining hair is provided, comprising administering to a subject in need of such a method the compound described in the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof.

[0093] Furthermore, the subjects are mammals, particularly humans, including healthy individuals and patients.

[0094] Furthermore, maintaining hair includes promoting hair growth, promoting hair regeneration and self-repair, prolonging the hair growth cycle, and preventing hair loss.

[0095] Furthermore, the administration can be carried out via 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, infusion, respiratory administration, etc.).

[0096] This invention designs a high-throughput screening method and system for KLHL24 ubiquitin ligase inhibitors. This method exhibits high stability and reproducibility, facilitating the efficient screening of KLHL24 ubiquitin ligase inhibitors and the development of related drugs. Based on the aforementioned method, this invention has screened several KLHL24 ubiquitin ligase inhibitors, which can be effectively used to treat KLHL24-related diseases (such as diseases caused by KLHL24 mutations, such as hereditary epidermolysis bullosa, dilated cardiomyopathy, alopecia, and atrophic scarring), showing excellent application prospects. Given the function of KLHL24 in skin and hair regeneration, this inhibitor also shows good application potential in maintaining normal hair growth. Attached Figure Description

[0097] Figure 1 The diagram shows a high-throughput screening method for KLHL24 inhibitors based on GFP-KLHL24 autoubiquitination.

[0098] Figure 2 The results show the GFP fluorescence detection of GFP-KLHL24 expression in 293T cells transiently transfected with GFP-KLHL24 24 h later. Figure 2 Image a shows a fluorescence image taken with a high-content fluorescence microscope. Figure 2 Figure b shows the fluorescence intensity statistics.

[0099] Figure 3 The experimental results show the stability and reproducibility evaluation of the high-throughput screening method.

[0100] Figure 4 The image shows candidate small molecule compounds obtained by high-throughput screening of Selleck's Express-Pick diversity library.

[0101] Figure 5 The image shows the effect of candidate small molecule compounds on GFP-KLHL24 expression, where ▲ indicates the small molecule compound STK731044.

[0102] Figure 6 The image shows Klhl24 after treatment with the Klhl24 inhibitor STK731044. c.3G / T Results of the mouse tape patching experiment, among which, Figure 6 Figure a shows a visual representation of the hair of a mouse that has been taped off. "-" represents the control treatment (i.e., no treatment with KLHL24 inhibitor) and "+" represents the test treatment (i.e., treatment with KLHL24 inhibitor). Figure 6 Figure b shows a statistical chart of the weight of the shed hair shafts.

[0103] Figure 7 The image shows Klhl24 after treatment with the Klhl24 inhibitor STK731044. c.3G / T Experimental results on the protein abundance of KRT15, ​​Vimentin, and GAPDH in mouse tissues. "-" indicates control treatment (i.e., no KLHL24 inhibitor treatment), and "+" indicates test treatment (i.e., treatment with KLHL24 inhibitor). Detailed Implementation

[0104] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0105] In this invention, the term "aliphatic ring" refers to a straight-chain or branched hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a cyclic hydrocarbon group (also referred to herein as "cycloalkyl" or "aliphatic group") that is fully saturated or contains one or more unsaturated units, connected to the rest of the molecule by a single bond. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, and mixtures thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, (cycloalkyl)alkenyl, etc. 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.

[0106] The term "cycloalkyl" refers to alicyclic hydrocarbons, such as those containing 1 to 4 monocyclic 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.

[0107] The term "alkyl" refers to a straight-chain or branched hydrocarbon radical that does not contain unsaturated bonds and is connected to the rest of the molecule by a single bond. 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, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. If the alkyl group is substituted with an aryl group, it is referred to as "aralkyl," such as benzyl, diphenylmethyl, or phenethyl. If the alkyl group is substituted with a heterocyclic group, it is referred to as "heterocyclic alkyl." In this invention, CO alkyl refers to H, i.e., C 0-10 Alkyl (or C0-C) 10 Alkyl groups include H and C. 1-10 Alkyl (or C1-C) 10 alkyl).

[0108] The term "alkylene" refers to a hydrocarbon group (divalent alkyl) formed by the loss of two hydrogen atoms from an alkane molecule. It can be straight-chain or branched and is connected to the rest of the molecule by a single bond. Typical alkylene groups described herein have 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, etc. In this invention, CO alkylene refers to a single bond, i.e., C... 0-10 Alkylene (or C0-C) 10 Alkylenes include single bonds and C 1-10 Alkylene (or C1-C) 10 Alkylene).

[0109] The term "alkoxy" refers to a substituent formed when the hydrogen in a hydroxyl group is replaced by an alkyl group, such as alkoxy groups containing 1-10 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy, etc.

[0110] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0111] The term "haloalkyl" refers to a group formed by replacing one or more hydrogen atoms in an alkyl group with a halogen atom (such as fluorine, chlorine, bromine or iodine), such as -CHF2, -CH2F, -CF3, -CH2-CF3, -CH2CH2-CF3, -CH2CH2CH2-CF3.

[0112] The term "aryl" refers to a monocyclic or polycyclic free radical, including polycyclic free radicals containing a monoaryl group and / or a fused aryl group (also referred to herein as "aromatic ring"), such as those containing 1-3 monocyclic or fused rings and 6-18 (e.g., 6, 8, 10, 12, 14, 16, 18) carbon ring atoms, C6-C as described in this invention. 12 The aryl group refers to an aryl group containing 6-12 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indene, etc.

[0113] The term "heterocyclic group" refers to a 3- to 18-membered non-aromatic ring group containing 2 to 17 carbon atoms and 1 to 10 heteroatoms. Heterocyclic groups can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, and can include fused, spirocyclic, or bridged ring systems. Heterocyclic groups (also referred to herein as "heterocycles") can be partially saturated (heteroaryl, also referred to herein as "heteroaromatic rings") or fully saturated (heterocyclic alkyl). Suitable heteroaryl groups in the compounds of the present invention contain one, two, or three heteroatoms selected from N, O, S, and P atoms. These heteroaryl groups include, for example, coumarin (including 8-coumarin), quinolinyl (including 8-quinolinyl, isoquinolinyl, pyridinyl, pyrazinyl, pyrazolyl, pyrimidinyl, furanyl, pyrroloyl, thiopheneyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazoleyl, indolyl, isoyndolyl, indazoleyl, inazinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazolidyl, pyridazinyl, triazinyl, cenolinyl, benzimidazolyl, benzofuranyl, benzofuranyl, benzothiopheneyl, benzothiazolyl, benzooxazolyl, quinazolinyl, quinoxolinyl, naphridinyl, and furanopyridinyl. Suitable heterocyclic alkyl groups in the compounds of the present invention contain one, two, or three heteroatoms selected from N, O, or S atoms. These heterocyclic alkyl groups include, for example, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydrothiophenyl, piperidinyl, morpholinyl, thiomorpholinyl, oxothiohexacyclohexyl, piperazine, aziridine, oxothiohexacyclohexyl, thiohexacyclohexyl, homopiperidinyl, oxopropane, thiopropane, acrylonitrile, oxo-aziridine, diaziridine, etc. Heptyl, triacetyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1,3-dioxapentyl, pyrazolinyl, dithiaalkyl, dithiopentyl, dihydropyranyl, dihydrothiophenyl, pyrazolinyl, imidazolinyl, imidazolinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinazinyl. In this invention, for optionally substituted heterocyclic groups, the substituted position can be any suitable carbon atom or heteroatom, for example, for The substitution position of R can be any suitable carbon or nitrogen atom, which can be, for example... .

[0114] The compounds of the present invention also include isotopically labeled forms, that is, compounds distinguished only by the presence of one or more isotopically rich atoms. For example, compounds having existing structures in which at least one hydrogen atom is replaced only by deuterium or tritium, or at least one carbon atom is replaced by carbon rich in 13C or 14C, or at least one nitrogen atom is replaced by nitrogen rich in 15N are all included within the scope of the present invention.

[0115] In this invention, "D" refers to deuterium; "replaced by deuterium" means replacing one or more hydrogen atoms with a corresponding number of deuterium atoms.

[0116] It should be recognized that, depending on the source of the chemical materials used in the synthesis, there are variations in the natural isotopic abundance in the synthesized compounds. Therefore, the compounds of the present invention will inherently contain small amounts of deuterated isotopes. Despite this variation, the concentrations of these naturally abundant stable hydrogen and carbon isotopes are low and insignificant compared to the degree of stable isotopic substitution in the compounds of the present invention. See, for example, Wada, E et al., Seikagaku, 1994, 66: 15; Gannes, LZ et al., Comp Biochem Physiol Mol Integr Physiol, 1998, 119:725.

[0117] In the compounds of this invention, any atom not specifically designated as deuterium is present at its natural isotopic abundance. Unless otherwise stated, when a position is specifically designated as "H" or "hydrogen", that position should be understood as having hydrogen according to its natural abundance isotopic composition. Similarly, unless otherwise stated, when a position is specifically designated as "D" or "deuterium", that position should be understood as having deuterium at an abundance at least 3000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 45% deuterium doping).

[0118] As used herein, the term "isotope enrichment factor" refers to the ratio between the isotopic abundance of a particular isotope and its natural abundance. In other embodiments, the compounds of the present invention have isotope enrichment factors for each specified deuterium atom of at least 3500 (52.5% deuterium doping at each specified deuterium atom), at least 4000 (60% deuterium doping), at least 4500 (67.5% deuterium doping), at least 5000 (75% deuterium doping), at least 5500 (82.5% deuterium doping), at least 6000 (90% deuterium doping), at least 6333.3 (95% deuterium doping), at least 6466.7 (97% deuterium doping), at least 6600 (99% deuterium doping), or at least 6633.3 (99.5% deuterium doping).

[0119] The term "isotope" refers to a substance whose chemical structure differs from that of a specific compound of the present invention only in terms of its isotopic composition.

[0120] The term "pharmaceutically acceptable" means that when the molecular basis and the composition comprising it are properly administered to a subject, they do not produce adverse, allergic or other adverse reactions.

[0121] The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0122] The term "acid addition salt" includes, but is not limited to, salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphonic acid, as well as salts derived from organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanes, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Therefore, these salts include, but are not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromates, iodates, acetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, octanoates, sebacic acid salts, fumarates, maleates, amygdalinates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, and methanesulfonates, as well as salts of amino acids such as arginine salts, gluconates, and galacturonic acids. Acid addition salts can be prepared by contacting a sufficient amount of the desired acid in a conventional manner to form a salt. The free base can be regenerated by contacting the salt with a base, and the free base can be separated in a conventional manner.

[0123] The term "base addition salt" refers to a salt formed with a metal or amine, such as hydroxides of alkali metals and alkaline earth metals, or with an organic amine. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Suitable amines include, but are not limited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (1,2-diaminoethane), N-methylglucosamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid form can be regenerated by contacting the salt form with an acid, and the free acid can be separated in a conventional manner.

[0124] The term "stereoisomer" includes enantiomers, diastereomers, and geometric isomers. Some compounds of the present invention have cyclic hydrocarbon groups that can be substituted on more than one carbon atom; in this case, all their geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention.

[0125] The term "solvent" refers to the physical bond between the compound of this invention and one or more solvent molecules. This physical bond includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate can be isolated, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvents include solution phases and separable solvates. Representative solvates include ethanolides, methanolides, etc.

[0126] The term "prodrug" refers to a Formula I compound that is suitable for administration to patients without excessive toxicity, irritation, or allergic reactions, and is effective for its intended purpose. Prodrugs include acetals, esters, and zwitterionic forms. Prodrugs are converted in the body, such as through hydrolysis in the blood, to yield the parent compound.

[0127] The terms “patient” or “subject”, etc., may be used interchangeably herein to refer to any animal or its cells, whether in vitro or in situ, treated according to the methods described herein. Specifically, the aforementioned animals include mammals, such as rats, mice, guinea pigs, rabbits, dogs, monkeys, or humans, especially humans.

[0128] The term "treatment" refers to the prevention, cure, reversal, reduction, mitigation, minimization, suppression, cessation, and / or cessation of one or more clinical symptoms of a disease after its onset.

[0129] The term "prevention" refers to the treatment taken before a disease develops to avoid, minimize, or prevent the disease from developing or progressing.

[0130] The term "KLHL24 ubiquitin ligase inhibitor," also known as KLHL24 ubiquitin protease inhibitor or KLHL24 inhibitor, refers to a substance that has an inhibitory effect on the expression of KLHL24 protein. This inhibitory effect includes, but is not limited to: inhibiting the activity of KLHL24 protein, inhibiting the expression of the KLHL24 protein gene, or inhibiting the self-ubiquitination and degradation of KLHL24 protein.

[0131] The term "KLHL24-related diseases," also known as KLHL24-mediated diseases, mainly refers to a series of diseases caused by abnormal KLHL24 activity, such as diseases caused by KLHL24 mutations, diseases caused by other factors leading to KLHL24 dysfunction, keratin abnormalities, and especially diseases for which prevention and / or treatment may be beneficial by inhibiting / degrading KLHL24.

[0132] The term "keratin abnormality disease" refers to a group of diseases caused by abnormalities in keratin, which mainly affect the structure and function of tissues composed of keratin, such as skin, hair, and nails.

[0133] The term "tumor" refers to an abnormal mass of tissue that grows beyond and out of harmony with the growth of normal tissue. Tumors can be "benign" or "malignant," depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion, and metastasis. "Benign tumors" are typically well-differentiated, characterized by slower growth than malignant tumors, and remain confined to their site of origin. Furthermore, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. In some cases, certain "benign" tumors may later develop into malignant tumors, possibly due to additional genetic alterations in a subset of the tumor's proliferative cells, and these tumors are called "precancerous tumors." "Malignant tumors" are typically poorly differentiated (anaplastic) and characterized by rapid growth, accompanied by progressive infiltration, invasion, and destruction of surrounding tissues. Furthermore, malignant tumors often have the ability to metastasize to distant sites.

[0134] The term "cancer" refers to a malignant tumor ( Stedman's Medical Dictionary , 25th ed.; Hensyled.; Williams & Wilkins: Philadelphia, 1990).

[0135] The terms “compound,” “substance,” and “therapeutic agent” are used interchangeably herein, including but not limited to compounds and mixtures thereof, small molecule compounds, biomacromolecules, etc., especially small molecule compounds. The substances provided by this invention are capable of interfering with KLHL24 activity, i.e., they have the ability to inhibit KLHL24 protein expression and / or biochemical or biological functions.

[0136] The term "small molecule" refers to non-biological substances or compounds with a molecular weight of less than approximately 1000 g / mol.

[0137] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0138] The STK731044 used in the embodiments of this invention (chemical name: (5E)-2-[4-(3-methylphenyl)-1-piperazinyl]-5-[[5-(4-nitrophenyl)-2-furanyl]methylene]-1,3-thiazolyl-4-one; STK731044 used in high-throughput screening was obtained from Selleck's Pick compound library; STK731044 used in animal experiments was purchased from Vitasmlab) has the following structure:

[0139]

[0140] The MLN4924 (also known as Pevonedistat, purchased from Sellck) used in the embodiments of this invention has the following structure:

[0141]

[0142] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0143] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0144] The experimental methods and materials involved in the following embodiments are as follows:

[0145] 1. Cell Culture

[0146] 293T cells were cultured in DMEM medium (NEAA, Gibco) containing 10% fetal bovine serum (FBS, PAN) and 1% penicillin and streptomycin. The culture environment was 37°C and 5% CO2.

[0147] 2. Plasmid construction and transfection

[0148] The KLHL24 gene fragment was cloned into the pEGFP-N1 vector using the Gateway cloning system (Invitrogen). Neofect DNA transfection reagent (TF201201) was selected as the transfection reagent, and it was mixed with Opti-MEM (Gibco, 31985070) and the plasmid to be transfected. After incubation at room temperature for 20 minutes, the mixture was added to the cell culture medium. Six hours after transfection, the medium was replaced with DMEM containing 10% FBS.

[0149] 3. Laboratory animals

[0150] Laboratory animals and housing environment: 7-week-old SPF-grade C57BL / 6J mice weighing 16-20g were produced from the Beijing Vital River Laboratory Animal Center and were constructed using CRISPER / Cas9 technology with the assistance of Beijing Biocytogen. Klhl24 c.3G / T Mice.

[0151] 4. High-throughput screening

[0152] HEK293T cells transfected with GFP-KLHL24 plasmid were incubated in 10cm culture dishes for 8 hours, then re-seeded into 96-well or 384-well plates for 8 hours of adhesion. The compound was then added to the 96-well or 384-well plates using an Echo 550 nano-level ultrasonic pipetting system (Labcyte). After 24 hours of co-incubation, immunofluorescence was used to observe the fluorescence signal. The specific steps were as follows: Cells were fixed by adding an equal volume of 4% paraformaldehyde to the cell culture medium and fixing at room temperature for 15 minutes. After washing the cell surface three times with 1×PBS, 30 μL of DAPI staining solution (1 μg / mL) was added to each well of a 96-well plate, and staining was performed at room temperature for 5 minutes. The plates were then sealed with light-shielding aluminum foil before photography. This study used a laser confocal high-content imaging microscope (PerkinElmer, Opera Phenix) for imaging. When using the instrument, follow the operation manual. After creating a new working template, first select a field of view and focus. After aligning with the focal plane, take pictures using 488nm and 340nm excitation light (separate exposures). Analyze the data using the Opera data analysis station. First, count the cell nuclei N stained by DAPI, which is the total number of cells. GFP-positive cells emit green fluorescence under 488nm excitation light. Count the number of green fluorescent cells n, which represents the cells expressing GFP-KLHL24. When counting n, it is necessary to adjust the green fluorescence threshold parameter to select as many green fluorescent cells as possible. In addition, parameters such as cell area and cell shape can be set to reduce the influence of background.

[0153] 5. Protein blotting

[0154] After cell collection, cells were lysed on ice for 30 min using RIPA lysis buffer (Beyotime, P0013C). For animal tissue samples, centrifugation was performed at 12,000 rpm for 10 min at 4°C. The supernatant was collected, 5× loading buffer was added, and the mixture was incubated in a 95°C metal bath for 10 min. Proteins were then separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk (TBST preparation) at room temperature for 1 h and incubated with primary antibodies corresponding to the proteins: anti-Vimentin (Cell Signaling Technology, 5741S), anti-GAPDH (ZSGB-BIO, TA-08), and anti-K15 (ab52816, Abcam). The target bands were detected using an HRP-conjugated secondary antibody via an exposure analyzer.

[0155] 6. Tape application test

[0156] Drug preparation plan:

[0157] Preparation of the test preparation (hereinafter referred to as the test preparation): 10 mM of STK731044 dissolved in DMSO was added to ethanol and glycerol (ethanol and glycerol were mixed in a volume ratio of 1:1) to make the final concentration of STK731044 0.1 g / 100 ml.

[0158] Preparation of the control preparation (hereinafter referred to as the control preparation): Add the same volume and concentration of DMSO solution (but without STK231044) used for the test preparation to ethanol and glycerol (ethanol and glycerol are mixed in a volume ratio of 1:1).

[0159] Take seven specific time points Klhl24 c.3G / T Female or male mice were administered KLHL24 inhibitors during the first resting phase (P15-P30), a total of 14 days. The administration method involved applying 20 μL of the experimental formulation to the left side of the mouse's back (experimental side) and 20 μL of the control formulation to the right side (control side), once daily. At the second resting phase (P50), a tape application test was performed as a self-control. For the tape application test, mice were anesthetized with 0.25% tribromoethanol, and the tape was applied to the mouse's back with equal force for 30 seconds. The tape was then removed, photographed, and the weight of shed hair shafts was recorded. The experimental and control groups were compared using Dunnett-900 micrograms. t test.

[0160] Example 1: Establishment of a high-throughput screening system for KLHL24 ubiquitin ligase inhibitors

[0161] KLHL24 belongs to the KLHL E3 ubiquitin ligase family and possesses the characteristics of E3 ubiquitin ligases: it ubiquitinates the substrate protein when present and ubiquitinates itself when the substrate protein is absent. Preliminary experiments in this invention showed that MLN4924 can significantly increase the content of KLHL24 protein. We can consider KLHL24 to be its own E3 ubiquitin ligase. Therefore, the stability of the KLHL24 protein itself is used as a screening indicator in the design of the screening method. Based on the above ideas, a method is designed as follows... Figure 1 The screening strategy shown includes the following steps: constructing a GFP-tagged KLHL24 plasmid, transforming the plasmid into 293T cells, where KLHL24 remains at a low level due to its own ubiquitination. Subsequently, a small molecule inhibitor is added. If the small molecule inhibitor can restore the KLHL24 protein content, then an increase in KLHL24 protein can be observed using a fluorescence microscope.

[0162] According to the designed screening protocol, the first step is to determine whether KLHL24 self-ubiquitination can be used as a screening indicator. The specific steps are as follows: GFP-KHL24 was transfected into 293T cells, and then the effective and selective NEDD8 activator (NAE) inhibitor MLN4924 (concentrations of 0.156 μM, 0.313 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM) was added. GFP-KLHL24 expression was detected by GFP fluorescence. The results are as follows... Figure 2 As shown, the expression level of GFP-KLHL24 is very low ( Figure 2 a) The content of MLN4924 increased significantly after its addition, and the increase was dose-dependent. Figure 2 (b) Based on the above experimental results, it can be determined that the stability of GFP-KLHL24 can be used as a screening indicator. This preliminarily confirms that the above screening scheme has high feasibility.

[0163] Secondly, before conducting high-throughput screening, it is necessary to evaluate the stability or reproducibility of the screening protocol. If the screening protocol has poor stability, the error between each group of experiments will be large, leading to the inability to accurately screen effective small molecule compounds and a high proportion of false negatives. To eliminate this error, the usual practice is to increase the number of replicate experiments; however, this will greatly increase the screening throughput and significantly increase the consumption of manpower and resources. To address this issue, a preliminary experiment was first conducted. The specific steps were as follows: 320 small molecule compounds (purchased from Ceramics) were added to different 96-well plates, and each small molecule compound was replicated twice. The results of the two replicates were then subjected to correlation analysis. The results are as follows: Figure 3 As shown, the correlation coefficient (R) between the two sets of results is 0.912, indicating a very strong correlation. Furthermore, regression analysis was performed on the two sets of repeated experiments, with the regression function being y = 0.8907x + 1.0135. Based on the coefficients of the regression function, the two sets of results are positively correlated, with a regression coefficient of 0.8907 and a constant of 1.0135, indicating that the two sets of data are very close. Figure 3 The experimental results above show that the screening method is very stable and has excellent reproducibility. Based on this result, we will only need one replicate in high-throughput screening, which will greatly improve screening efficiency.

[0164] Example 2: High-throughput screening of Selleck's Express-Pick diversity compound nucleus library

[0165] Based on the high-throughput screening method established in Example 1, small molecule compounds that inhibit KLHL24 activity were screened. The Express-Pick Library (catalog number L3600), containing 4208 small molecule compounds, was selected for screening. The reasons for choosing this library are as follows: 1) It is a unique collection of compounds with different core structures, suitable for high-throughput screening; 2) It originates from one of the world's largest pharmaceutical companies; 3) It can be linked to the target, allowing for the study of compound conformation and efficacy, thus accelerating new drug development; 4) The purity of the compounds was ensured through NMR and HPLC measurements.

[0166] After selecting the compound library, high-throughput screening based on high content was performed. During screening, multiple negative controls (DMSO) were set up in each experiment. After obtaining the raw results, the number of DAPI-stained cell nuclei (N) was counted first, representing the total number of cells; simultaneously, the number of green fluorescent cells (n) was counted, representing GFP-KLHL24-expressing cells. When counting n, parameters were set to adjust the green fluorescence threshold to select as many green fluorescent cells as possible. Additionally, parameters such as cell area and cell shape could be set to reduce background interference. The formula for calculating the GFP-positive cell ratio was: n / N*100%. The GFP-positive cell ratio was standardized based on the negative control results, and the Fold Change threshold was set to 2, which significantly reduced false positives. We also introduced the cell count per well parameter. Small molecule compounds often have some toxicity to cells, causing cell death and autofluorescence, which increases the possibility of false positives. Therefore, introducing the cell count per well parameter can reduce false positives and exclude highly toxic small molecule compounds, reducing the workload of subsequent validation. Furthermore, we considered the cell transfection rate. Based on the transfection efficiency of the plasmid using the transfection reagent, we believe that a proportion of GFP-positive cells exceeding 80% is due to the autofluorescence of small molecules. Considering three variables—FoldChange value, number of cells per well, and transfection efficiency—we first stained cell nuclei with DAPI. The number of cell nuclei was used to determine the toxicity of the compound; cells with significant toxicity were removed. In cases where the compound was ineffective, the ratio of GFP-positive cells after treatment was normalized based on the negative control results, and the FoldChange threshold was set to 2. The results are as follows: Figure 4 As shown, 41 compounds were ultimately selected.

[0167] Example 3: Verification of the effect of preliminary screening candidate small molecule compounds on the degradation of KLHL24

[0168] To preliminarily verify whether the aforementioned 41 small molecule compounds could significantly affect the content of GFP-KLHL24 protein, the specific procedures were as follows: GFP-KLHL24 was transfected into 293T cells, followed by treatment with the small molecule compounds, and the KLHL24 protein content was detected by Western blot. Due to the large number of compounds, we conducted multiple experiments to detect their effects on GFP-KLHL24 protein. In this experiment, DMSO treatment was set as a negative control and MLN4924 treatment as a positive control, with both the positive control and the small molecule compounds at a concentration of 10 μM. The results are as follows: Figure 5 As shown, a total of 25 small molecule compounds can increase the content of GFP-KLHL24 protein at a concentration of 10 μmol / L.

[0169] Example 4: KLHL24 inhibitor restores Klhl24 c.3G / T Levels of KRT15 and Vimentin proteins in mice

[0170] Multiple cell experiments verified that among the 25 small molecules, STK731044 significantly enhanced GFP-KLHL24 activity with the least cytotoxicity. Therefore, STK731044 was subsequently selected for animal experiments. The effect of KLHL24 ubiquitin ligase activity after inhibitor treatment on mice was evaluated, and the specific procedures were as described in the tape-on-fur experiment above. The results are as follows: Figure 6 As shown, compared to the negative control (i.e., control treatment), the KLHL24 inhibitor STK731044 treatment of Klhl24 c.3G / T After treatment, the anchoring ability of the hair shafts on the back of the mice was enhanced, and the number of hair shafts detached from the tape was reduced. According to previously reported literature, compared with WT mice (wild-type mice), Klhl24... c.3G / T The levels of KRT15 (K15) and Vimentin protein were reduced in mice. Further experiments revealed that administration of Klhl24... c.3G / T Treatment with the KLHL24 inhibitor STK731044 in mice increased the expression levels of KRT15 and Vimentin proteins in the mouse skin. Figure 7 ). Figure 7 In this study, GAPDH, a commonly used housekeeping gene protein, was used as a reference protein for comparing various samples. These results suggest that KLHL24 inhibitors could be used as treatments for hair loss.

[0171] In summary, STK731044 (selection number PickP4E22) is a potential KLHL24 ubiquitin ligase inhibitor with promising potential for treating diseases caused by KLHL24 mutations.

[0172] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0173] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0174] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. The use of a compound or a pharmaceutically acceptable salt or deuterated compound thereof in the preparation of a medicament for the prevention and / or treatment of KLHL24-related diseases, said compound having the following structure: in, R B It is -NO2; R D Selected from: H, C1-C6 alkyl, C1-C6 haloalkyl; The condition associated with KLHL24 is hair loss.

2. The use of a compound or a pharmaceutically acceptable salt or deuterated compound thereof in the preparation of a medicament for maintaining hair, said compound having the following structure: in, R B It is -NO2; R D Selected from: H, C1-C6 alkyl, C1-C6 haloalkyl; Maintaining hair includes promoting hair growth, promoting hair regeneration and self-repair, extending the hair growth cycle, and preventing hair loss.

3. The application according to claim 1 or 2, characterized in that, R D It is a C1-C6 alkyl group.

4. The application according to claim 3, characterized in that, R D It can be methyl or ethyl.

5. The application according to claim 1 or 2, characterized in that, The compound has the following structure: 。 6. The application according to claim 1 or 2, characterized in that, The compound or its pharmaceutically acceptable salt or deuterated compound may be used alone or in combination with other types of active ingredients.

7. The application according to claim 6, characterized in that, The drug also includes one or more pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable excipients are selected from: binders, lubricants, disintegrants, suspending agents, solubilizers, thickeners, stabilizers, preservatives, fillers, antioxidants, and buffers.

9. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is selected from: gastrointestinal dosage form, injection dosage form, respiratory dosage form, skin dosage form, and cavity dosage form.

10. The application according to claim 9, characterized in that, The dosage form of the drug is selected from: oral dosage form, transdermal dosage form, and injectable dosage form.

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