KLHL24 ubiquitin ligase inhibitor and application thereof

Abstract

The invention discloses a high-throughput screening method and a high-throughput screening system for a KLHL24 ubiquitin ligase inhibitor, which are high in stability and good in repeatability and are beneficial to efficient screening of the KLHL24 ubiquitin ligase inhibitor and development of related drugs. Based on the method, some KLHL24 ubiquitin ligase inhibitors are screened out, can be effectively used for treating diseases related to KLHL24, especially diseases caused by KLHL24 mutation, such as hereditary bullous epidermis, dilated cardiomyopathy, alopecia and atrophic scar, and have very good application prospects. In view of the function of KLHL24 in the aspect of skin and hair regeneration, the inhibitor also has a relatively good application prospect in the aspect of maintaining normal hair.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a KLHL24 ubiquitin ligase inhibitor and its application. Background Art

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

[0003] Research led by the first inventor of the present invention first discovered 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 level, resulting in a large amount of keratin KRT14 in the skin being ubiquitinated and degraded by KLHL24, making the basal epidermal cells fragile and prone to shedding, 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 research reports that in addition to KRT14, the ubiquitination substrates of KLHL24 also include KRT15, Vimentin, and Desmin. Another study found that the syndrome phenotypes caused by KLHL24 mutations are emerging. EBS-KLHL24 patients are born with extensive areas of bare skin and skin fragility, and over time, 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 may also experience hair loss, especially in areas of skin atrophy, and sometimes even affect 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 developing drugs targeting the KLHL24 target 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 screening out drugs that can specifically bind to and inhibit the activity of KLHL24 is of great significance for treating diseases caused by KLHL24 mutations. Given the function of KLHL24 in skin and hair regeneration, screening out this inhibitor is also of great significance in maintaining normal hair. Summary of the Invention

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

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

[0007]

[0008] Wherein, represents a single bond or a double bond;

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

[0010] Ring A is a heterocycle;

[0011] R A is one or more independent substituents on Ring A, and is selected from: H, halogen, hydroxyl, amino, cyano, nitro, azide, C1-C 10 alkyl, -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10-membered heterocyclic group),

[0012] L is a divalent group connected to Ring A, and 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)-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 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 10The H in cycloalkyl, C6-C 10 aryl, 4- to 10-membered heteroarylene 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 C is one or more independent substituents linked to L and is selected from: H, halogen, cyano, nitro, azide, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, 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 group), -(C0-C6 alkylene)-(C3-C 10 (cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 (aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the H in the C0-C6 alkylene, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 10 (cycloalkyl), C6-C 10 (aryl), 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from: halogen, cyano, nitro, azide, C1-C 10 alkyl, C1-C 10 haloalkyl, -(C0-C6 alkylene)-(C3-C 10 (cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 (aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), -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);

[0014] R B is one or more independent substituents on the benzene ring, and is selected from: H, halogen, cyano, nitro, azide, C1-C 10 alkyl, -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), -(C0-C6 alkylene)-(C1-C 10 haloalkyl), -(C0-C6 alkylene)-(C1-C 10 haloalkoxy), -(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), -CO(C0-C6 alkylene)(4-10 membered heterocyclic group), nitric oxide (NO) donor residue, wherein the C0-C6 alkylene, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 10 cycloalkyl, C6-C 10The H in aryl or 4- to 10-membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, azido, 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- to 10-membered heterocyclic group), 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 Rs B together with the carbon atom to which they are attached form an aliphatic ring, aromatic ring or heterocyclic ring, wherein the H on the aliphatic ring, aromatic ring or heterocyclic ring is optionally substituted by one or more groups selected from the following: 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 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10(alkyl), -CO(C 0-10 alkyl).

[0015] Specifically, the above-mentioned nitric oxide (NO) donor residue may be selected from: -ONO2, wherein, R H and R K are 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 group) (for example ), or R H and R K together with the nitrogen atom to which they are attached form a 4-6 membered heterocyclic group (for example

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

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

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

[0019]

[0020] Preferably,

[0021] In some embodiments of the present invention, R1 is

[0022] In some embodiments of the present invention, R2 is

[0023] Furthermore, 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 heteroalkylene), wherein the H in said C0-C6 alkylene, C3-C 10 cycloalkyl, C6-C 10 aryl, 4-10 membered heteroalkylene 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).

[0024] Preferably, L is selected from: a 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 membered heteroalkylene)-, wherein the H in said C0-C6 alkylene, C6-C 10 aryl, 4-10 membered heteroalkylene may optionally be substituted by one or more groups selected from the following: C1-C 10 alkyl, -COO(C 0-10 alkyl).

[0025] Further, R C is 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 group), -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the H in the C0-C6 alkylene, C1-C 10 alkyl, C3-C 10 cycloalkyl, C6-C 10 aryl, 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, C1-C 10 alkyl, C1-C 10 haloalkyl, -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), -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).

[0026] Preferably, R C is selected from: 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-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the H in the C0-C6 alkylene, C 0-10 alkyl, C3-C 10 cycloalkyl, C6-C 10 aryl, 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, nitro, C1-C10 alkyl, C1-C 10 haloalkyl, -O(C 0-10 alkyl), -N(C 0-10 alkyl)(C 0-10 alkyl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group).

[0027] In some embodiments of the present invention, R1 is selected from: H, -F, -Cl, -Br, -I,

[0028] In some embodiments of the present invention, R2 is selected from: H, -F, -Cl, -Br, -I,

[0029] Further, R B is selected from: H, halogen, cyano, nitro, azido, C1-C 10 alkyl, -(C0-C6 alkylene)-(C3-C 10 cycloalkyl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), -(C0-C6 alkylene)-(C1-C 10 haloalkyl), -(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(C 0-10 alkyl), -CO(C0-C6 alkylene)(4-10 membered heterocyclic group), wherein the C0-C6 alkylene, C1-C 10 alkyl, C2-C10 Alkenyl, C2-C 10 Alkynyl, C3-C 10 Cycloalkyl, C6-C 10 Aryl, 4-10 membered heterocyclic group, the H in which 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 group), C1-C 10 Halogenated 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), -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).

[0030] Preferably, R B is selected from: H, halogen, cyano, nitro, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, -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 H in the C0-C6 alkylene, C1-C 10 Alkyl, 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, C1-C 10 Alkyl, C2-C10 Alkenyl, C2-C 10 Alkynyl, -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).

[0031] In some embodiments of the present invention, R B is selected from: H, -F, -Cl, -Br, -I, -NO2, -NH2, -CN, -COOH, -OH, -CF3,

[0032]

[0033] -ONO2.

[0034] Furthermore, two Rs B together with the carbon atom to which they are attached form a 5-6 membered aliphatic ring, aromatic ring or heterocyclic ring, wherein the H on the 5-6 membered aliphatic ring, aromatic ring or heterocyclic ring is optionally substituted by one or more groups selected from: 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).

[0035] Preferably, two Rs B together with the carbon atom to which they are attached form a 5-6 membered heterocyclic ring.

[0036] In some embodiments of the present invention, two Rs B together with the carbon atom to which they are attached form a 5-6 membered heterocyclic ring selected from:

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

[0038]

[0039] wherein, R D is selected from: H, halogen, cyano, hydroxy, carboxyl, C1-C6 alkyl, C1-C6 haloalkyl.

[0040] In some embodiments of the present invention, R D is a C1-C6 alkyl group, such as methyl, ethyl.

[0041] In some embodiments of the present invention, R B is a nitric oxide (NO) donor residue, such as -ONO2,

[0042] In some embodiments of the present invention, the compound has the following structure:

[0043]

[0044] In a second aspect of the present 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 preventing and / or treating KLHL24-related diseases, wherein the compound has the definition of the compound as described in the first aspect of the present invention.

[0045] Furthermore, the KLHL24-related diseases are diseases that can benefit from their prevention and / or treatment by inhibiting or degrading KLHL24, such as diseases caused by KLHL24 mutations (such as truncating KLHL24-ΔN28 protein generated by Klhl24 gene start codon mutation), keratin abnormalities, such as skin diseases, cardiovascular diseases, tumors, digestive system diseases, arthritis, etc., and their complications.

[0046] Furthermore, the skin diseases are selected from: alopecia, bullous skin diseases, scars, keratosis, erythematous papulosquamous skin diseases, connective tissue diseases.

[0047] Furthermore, the bullous skin diseases are selected from: epidermolysis bullosa (such as hereditary epidermolysis bullosa, acquired epidermolysis bullosa), pemphigus, bullous pemphigoid, cicatricial pemphigoid.

[0048] Furthermore, the scars are selected from: hypertrophic scars, keloids, atrophic scars.

[0049] Furthermore, the keratosis is selected from: keratosis follicularis, palmoplantar keratoderma, palmoplantar keratosis, and its complications include dry skin, scaling, chapping, etc.

[0050] Furthermore, the erythematous papulosquamous skin diseases are selected from: psoriasis, pityriasis rubra pilaris, erythrokeratodermia.

[0051] Furthermore, the connective tissue diseases are selected from: lupus erythematosus, scleroderma.

[0052] In some embodiments of the present invention, the disease is atrophic scar.

[0053] In some embodiments of the present invention, the disease is hereditary epidermolysis bullosa and its complications (such as esophageal mucosal stenosis, finger syndactyly contracture, corneal scarring, eye blindness).

[0054] In some embodiments of the present invention, the disease is alopecia.

[0055] Further, the cardiovascular disease is cardiomyopathy (such as dilated cardiomyopathy, hypertrophic cardiomyopathy).

[0056] Further, the tumors are 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, adrenocortical carcinoma, pancreatic ductal adenocarcinoma, lung adenocarcinoma, pancreatic cancer, gonadoblastoma, multiple myeloma, mantle cell lymphoma, osteosarcoma, neuroblastoma, brain tumor, melanoma.

[0057] Preferably, the tumors are selected from: colon cancer, prostate cancer, gonadoblastoma, endometrial cancer.

[0058] More preferably, the tumors are selected from: colon cancer, prostate cancer.

[0059] Further, the digestive system diseases are selected from: chronic hepatitis, drug-induced liver injury.

[0060] Further, the arthritis is osteoarthritis.

[0061] Further, the subjects of the disease are mammals, such as humans.

[0062] Further, the compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound can be used alone or in combination with other types of active ingredients.

[0063] Further, the drug is a veterinary drug or a human drug.

[0064] Further, the drug further comprises one or more pharmaceutically acceptable excipients.

[0065] Further, the pharmaceutically acceptable excipients are selected from: binders, lubricants, disintegrants, bacteriostatic agents, suspending agents, solubilizers, thickeners, stabilizers, preservatives, fillers, antioxidants, buffers.

[0066] Furthermore, the compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound can be administered by any suitable route, such as gastrointestinal administration (e.g., oral, sublingual, rectal administration) or parenteral administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, infusion, respiratory administration, etc.). The drug can be in any suitable dosage form, such as a gastrointestinal 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.; parenteral dosage forms, such as injection dosage forms: such as injections (e.g., for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), respiratory dosage forms: such as sprays, aerosols, powder aerosols, etc., skin dosage forms, such as topical solutions, lotions, ointments, plasters, pastes, patches, etc., mucosal dosage forms: such as eye drops, ophthalmic ointments, nasal drops, gargles, sublingual tablets, etc., cavity dosage forms: such as suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for the rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0067] In some embodiments of the present invention, the drug is a skin dosage form.

[0068] In some embodiments of the present invention, the drug is an oral dosage form.

[0069] In some embodiments of the present invention, the drug is an injection dosage form.

[0070] In the third aspect of the present invention, there is provided an application of a compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound in the preparation of a product for maintaining hair.

[0071] Furthermore, the subjects of the product are mammals, especially humans, including healthy people and patients.

[0072] Furthermore, the maintaining of hair includes promoting hair growth, promoting hair regeneration and self-repair, prolonging the hair growth cycle, preventing hair loss, etc.

[0073] Furthermore, the product is a drug, food, health product, or care product.

[0074] Furthermore, the product further includes one or more excipients.

[0075] In the fourth aspect of the present invention, there is provided a screening method for a KLHL24 ubiquitin ligase inhibitor, which includes the following steps: co-incubating a substance to be screened with cells expressing KLHL24 labeled with a fluorescent protein and detecting the fluorescence signal.

[0076] In some embodiments of the present invention, the fluorescent protein is GFP.

[0077] In some embodiments of the present invention, the cell is HEK293T cell.

[0078] Furthermore, the method further includes steps of calculation and analysis, such as calculating the number or proportion of fluorescent positive cells.

[0079] 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.

[0080] In some embodiments of the present invention, the expression vector is a plasmid.

[0081] In some embodiments of the present invention, the method includes:

[0082] (a) Constructing a GFP-KLHL24 plasmid;

[0083] (b) Transfecting HEK293T cells with the GFP-KLHL24 plasmid and incubating;

[0084] (c) Mixing the substance to be screened with the cells in step (b) and co-incubating;

[0085] (d) Detecting the fluorescence signal of the incubation system obtained in step (c).

[0086] In the fifth aspect of the present invention, there is provided a screening system (such as a kit) for KLHL24 ubiquitin ligase inhibitors, which comprises: cells expressing KLHL24 labeled with a fluorescent protein (as described in the fourth aspect).

[0087] Specifically, the screening system further comprises reagents for incubating cells, such as a culture medium.

[0088] Specifically, the screening system further comprises containers for incubating cells, such as a 96-well plate or a 384-well plate.

[0089] Specifically, the screening system further comprises reagents for detecting fluorescence signals, such as cell fixation reagents (such as paraformaldehyde), cell washing solutions, staining solutions, etc.

[0090] In the sixth aspect of the present invention, there is provided the use of the screening system described in the fifth aspect in screening KLHL24 ubiquitin ligase inhibitors and drugs for treating diseases related to KLHL24.

[0091] In a seventh aspect of the present invention, there is provided a method for preventing and / or treating KLHL24-related diseases, which comprises administering to a subject in need thereof a compound as described in the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof.

[0092] Further, the KLHL24-related diseases have the definitions of the diseases as described in the second aspect of the present invention.

[0093] Further, the subject is a mammal, particularly a human.

[0094] Further, the administration can be by any suitable route of administration, such as enteral administration (e.g., oral, sublingual, rectal administration) or parenteral administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, infusion, respiratory administration, etc.).

[0095] In an eighth aspect of the present invention, there is provided a method for maintaining hair, which comprises administering to a subject in need thereof a compound as described in the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof.

[0096] Further, the subject is a mammal, particularly a human, including healthy individuals and patients.

[0097] Further, the maintaining of hair includes promoting hair growth, promoting hair regeneration and self-repair, prolonging the hair growth cycle, preventing hair loss, etc.

[0098] Further, the administration can be by any suitable route of administration, such as enteral administration (e.g., oral, sublingual, rectal administration) or parenteral administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, infusion, respiratory administration, etc.).

[0099] The present invention designs a high-throughput screening method and system for KLHL24 ubiquitin ligase inhibitors, which have high stability and good reproducibility, and contribute to the efficient screening of KLHL24 ubiquitin ligase inhibitors and the development of related drugs. Based on the foregoing method, the present invention has screened 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 have 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 THE DRAWINGS

[0100] Figure 1 The figure shows a schematic diagram of a high-throughput screening for KLHL24 inhibitors based on the autoubiquitination of GFP-KLHL24.

[0101] Figure 2 The figure shows the experimental results of detecting the expression of GFP-KLHL24 by GFP fluorescence in 293T cells transiently transfected with GFP-KLHL24 for 24 h. Among them, Figure 2 a shows the fluorescence images taken by a high-content fluorescence microscope, Figure 2 b shows the statistical chart of fluorescence intensity.

[0102] Figure 3 The figure shows the experimental results of evaluating the stability and repeatability of the high-throughput screening method.

[0103] Figure 4 The figure shows the candidate small molecule compounds obtained by high-throughput screening of the Express-Pick diversity compound core library of Selleck.

[0104] Figure 5 The figure shows the effect of the candidate small molecule compounds on the expression of GFP-KLHL24, where the small molecule compound STK731044 is indicated by ▲.

[0105] Figure 6 The figure shows Klhl24 after treatment with the KLHL24 inhibitor STK731044 c.3G / T The experimental results of the tape stripping test on mice. Among them, Figure 6 a shows the intuitive display of the hair stripped from the mice. "-" represents the treatment with the control preparation (i.e., without treatment with the KLHL24 inhibitor), and "+" represents the treatment with the test preparation (i.e., treated with the KLHL24 inhibitor); Figure 6 b shows the statistical chart of the dry weight of the shed hair shafts.

[0106] Figure 7 The figure shows Klhl24 after treatment with the KLHL24 inhibitor STK731044 c.3G / T The experimental results of the protein abundances of KRT15, Vimentin, and GAPDH in the tissues of Klhl24 mice after treatment with the KLHL24 inhibitor STK731044. Among them, "-" represents the treatment with the control preparation (i.e., without treatment with the KLHL24 inhibitor), and "+" represents the treatment with the test preparation (i.e., treated with the KLHL24 inhibitor). Specific embodiments

[0107] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the technical field to which the present invention pertains.

[0108] In the present invention, the term "alicyclic" refers to a straight-chain or branched hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a cycloalkyl group (also referred to herein as a "cycloalkyl group", an "aliphatic group") that is fully saturated or contains one or more unsaturated units, which is connected to other parts 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 groups 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.

[0109] The term "cycloalkyl" refers to an alicyclic hydrocarbon, such as a monocyclic and / or fused-ring containing 1 to 4 rings and 3 - 18 carbon atoms, preferably 3 - 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or adamantyl, etc.

[0110] The term "alkyl" refers to a straight-chain or branched hydrocarbon chain radical that does not contain an unsaturated bond, and this hydrocarbon chain radical is connected to other parts 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 is substituted by an aryl group, then it is correspondingly an "arylalkyl", such as benzyl, diphenylmethyl or phenethyl. If the alkyl is substituted by a heterocyclic group, then it is correspondingly a "heterocyclic alkyl". In the present invention, C0 alkyl refers to H, that is, C 0-10 alkyl (or C0-C 10 alkyl) includes H and C 1-10 alkyl (or C1-C 10 alkyl).

[0111] The term "alkylene" refers to a hydrocarbon group (divalent alkyl) formed by removing two hydrogen atoms from an alkane molecule, which can be straight-chain or branched and is connected to other parts of the molecule by a single bond. In this context, typical alkylene 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 methylene (-CH2-), ethylene, propylene, butylene, etc. In the present invention, C0 alkylene refers to a single bond, that is, C 0-10 alkylene (or C0-C 10 alkylene) includes a single bond and C 1-10 alkylene (or C1-C 10 alkylene).

[0112] The term "alkoxy" refers to a substituent formed by replacing the hydrogen in a hydroxyl group with an alkyl group, such as an alkoxy group containing 1 to 10 carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, etc.

[0113] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0114] The term "haloalkyl" refers to a group formed by replacing one or more hydrogens in an alkyl group with halogen atoms (such as fluorine, chlorine, bromine, or iodine), for example, -CHF2, -CH2F, -CF3, -CH2-CF3, -CH2CH2-CF3, -CH2CH2CH2-CF3.

[0115] The term "aryl" refers to a monocyclic or polycyclic radical, including polycyclic radicals containing monoaryl groups and / or fused aryl groups (also referred to as "aromatic rings" herein), such as those containing 1 to 3 monocyclic or fused rings and 6 to 18 (for example, 6, 8, 10, 12, 14, 16, 18) carbon ring atoms. The C6-C 12 aryl in the present invention refers to an aryl group containing 6 to 12 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indanyl, etc.

[0116] The term "heterocyclic group" refers to a 3- to 18-membered non-aromatic ring group that contains 2 to 17 carbon atoms and 1 to 10 heteroatoms. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can contain a fused, spiro, or bridged ring system. The heterocyclic group (also referred to as "heterocycle" herein) can be partially saturated (heteroaryl, also referred to as "heteroaromatic ring" herein) or fully saturated (heterocycloalkyl). Suitable heteroaryls in the compounds of the present invention contain 1, 2, or 3 heteroatoms selected from N, O, S, and P atoms, and the heteroaryls include, for example, coumarin, including 8-coumarin, quinolinyl, including 8-quinolinyl, isoquinolinyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indazinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furanopyridyl. Suitable heterocycloalkyls in the compounds of the present invention contain 1, 2, or 3 heteroatoms selected from N, O, or S atoms, and the heterocycloalkyls include, for example, pyrrolidinyl, tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathianyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxiranyl, thiiranyl, azepinyl, oxazepinyl, diazepinyl, triazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 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-indolyl, and quinuclidinyl. In the present invention, for an optionally substituted heterocyclic group, the position of substitution can be any suitable carbon atom or heteroatom. For example, for where the position of substitution of R can be any suitable carbon atom or nitrogen atom, and it can be, for example

[0117] The compounds of the present invention also include isotopically labeled forms, i.e., compounds that differ only in the presence of one or more atoms enriched in isotopes. For example, compounds having the existing structure with at least one hydrogen atom replaced only by deuterium or tritium, or at least one carbon replaced by carbon enriched in 13C or 14C, or at least one nitrogen replaced by nitrogen enriched in 15N are all included within the scope of the present invention.

[0118] In the present invention, "D" refers to deuterium; "being deuterium-substituted" means substituting one or more hydrogen atoms with the corresponding number of deuterium atoms.

[0119] It should be recognized that depending on the source of the chemical materials used in the synthesis, there are some variations in the natural isotope abundances in the synthesized compounds. Thus, the compounds of the present invention will inherently contain small amounts of deuterated isotopologues. Despite this variation, the concentrations of the stable hydrogen and carbon isotopes of natural abundance are low and insignificant compared to the degree of stable isotope substitution of 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.

[0120] In the compounds of the present invention, any atom not specified as deuterium is present in its natural isotope abundance. Unless otherwise specified, when a position is specifically designated as "H" or "hydrogen", that position should be understood to have hydrogen with its isotopic composition according to natural abundance. Similarly, unless otherwise specified, when a position is specifically designated as "D" or "deuterium", that position should be understood to have deuterium with an abundance greater than the natural abundance of deuterium (which is 0.015%) by at least 3000-fold (i.e., at least 45% deuterium incorporation).

[0121] As used herein, the term "isotope enrichment factor" refers to the ratio of the isotope abundance of a specific isotope to its natural abundance. In other embodiments, the compounds of the present invention have an isotope enrichment factor 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) for each specified deuterium atom.

[0122] The term "isotopologue" refers to a substance in which the chemical structure differs from a specific compound of the present invention only in its isotopic composition.

[0123] The term "pharmaceutically acceptable" means that when the molecular entity and the composition containing it are appropriately administered to a subject, they do not produce adverse, allergic, or other untoward reactions.

[0124] The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0125] 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, and salts derived from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Thus, these salts include, but are not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromides, iodides, acetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, and methanesulfonates, and also include salts of amino acids such as arginates, gluconates, galacturonates, etc. Acid addition salts can be prepared by contacting the free base form with a sufficient amount of the desired acid in a conventional manner to form the salt. The free base form can be regenerated by contacting the salt form with a base and the free base can be isolated in a conventional manner.

[0126] The term "base addition salt" refers to salts formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or formed with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of 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 isolated in a conventional manner.

[0127] The term "stereoisomers" includes enantiomers, diastereomers, and geometric isomers. Some compounds of the present invention have cycloalkyl groups which may be substituted on more than one carbon atom, in which case all geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention.

[0128] The term "solvate" refers to the physical association of a compound of the present invention with one or more solvent molecules. This physical association includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, solvates can be isolated, for example when one or more solvent molecules are incorporated into the lattice of a crystalline solid. Solvates include solution-phase and isolable solvates. Representative solvates include ethanolates, methanolates, etc.

[0129] The term "prodrug" refers to a form of a compound of formula I that is suitable for administration to a patient, has no excessive toxicity, irritation, allergic reaction, etc., and is effective for its intended purpose, including acetal, ester, and zwitterionic forms. The prodrug is converted in vivo, such as by hydrolysis in the blood, to give the parent compound.

[0130] The terms "patient" or "subject", etc., are used interchangeably herein and refer to any animal or its cells treated according to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animals include mammals, such as rats, mice, guinea pigs, rabbits, dogs, monkeys, or humans, especially humans.

[0131] 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.

[0132] The term "prevention" refers to, by treatment, avoiding, minimizing, or making it difficult for a disease to occur or develop before the onset of the disease.

[0133] 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, and the inhibitory effect includes, but is not limited to: inhibiting the activity of KLHL24 protein, inhibiting the gene expression of KLHL24 protein, or inhibiting the autoubiquitination degradation of KLHL24 protein.

[0134] The term "KLHL24-related disease", also known as KLHL24-mediated disease, mainly refers to a series of diseases caused by abnormal activity of KLHL24, such as diseases caused by KLHL24 mutation, diseases with abnormal KLHL24 function caused by other factors, keratin abnormality diseases, especially diseases that can benefit from the prevention and / or treatment by inhibiting / degrading KLHL24.

[0135] The term "keratin abnormality disease" refers to a class of diseases caused by keratin abnormality, mainly affecting the structure and function of tissues composed of keratin, such as skin, hair, nails, etc.

[0136] The term "tumor" refers to an abnormal mass of tissue, where the growth of the mass exceeds and is uncoordinated 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 usually well-differentiated, characterized by slower growth than malignant tumors and remaining 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 may later give rise to malignant tumors, which may be due to additional genetic alterations in subsets of the neoplastic cells of the tumor, and these tumors are called "precancerous tumors". "Malignant tumors" are usually poorly differentiated (anaplastic) and have characteristic rapid growth, accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. In addition, malignant tumors usually have the ability to metastasize to distant sites.

[0137] The term "cancer" refers to malignant tumors (Stedman’s Medical Dictionary, 25th ed.; Hensyled.; Williams & Wilkins: Philadelphia, 1990).

[0138] The terms "compound", "substance", "therapeutic agent" are used interchangeably herein and include, but are not limited to, compounds and their mixtures, small molecule compounds, biological macromolecules, etc., especially small molecule compounds. The substances provided by the present invention are capable of interfering with the activity of KLHL24, that is, they have the ability to inhibit the expression and / or biochemical or biological functions of the KLHL24 protein.

[0139] The term "small molecule" refers to a non-biological substance or compound having a molecular weight of less than about 1000 g / mol.

[0140] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0141] STK731044 (chemical name: (5E)-2-[4-(3-methylphenyl)-1-piperazinyl]-5-[[5-(4-nitrophenyl)-2-furyl]methylene]-1,3-thiazol-4-one) used in the examples of the present invention, STK731044 in high-throughput screening is from the Pick compound library of Selleck, and STK731044 used in animal experiments is purchased from Vitasmlab) has the following structure:

[0142]

[0143] MLN4924 (also known as Pevonedistat, purchased from Sellck) used in the embodiments of the present invention has the following structure:

[0144]

[0145] The disclosures of all various publications, patents and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0146] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0147] The experimental methods and materials involved in the following embodiments are as follows:

[0148] 1. Cell culture

[0149] 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, PAN) and 1% penicillin-streptomycin (NEAA, Gibco). The culture environment was under the conditions of 37 °C and 5% CO2.

[0150] 2. Construction and transfection of plasmids

[0151] The KLHL24 gene fragment was cloned into the pEGFP-N1 vector through 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, and added to the cell culture medium after incubation at room temperature for 20 minutes. After 6 hours of transfection, the DMEM medium containing 10% FBS was replaced.

[0152] 3. Experimental animals

[0153] Experimental animals and breeding environment: SPF-grade C57BL / 6J mice at 7 weeks old with a body weight of 16 - 20 g were produced by Beijing Vital River Laboratory Animal Technology Co., Ltd., and Klhl24 c.3G / T mice were constructed with the assistance of Beijing Bioocytogen Co., Ltd. using the CRISPER / Cas9 technology.

[0154] 4. High-throughput screening

[0155] After transfection of HEK293T cells with the GFP-KLHL24 plasmid, they were incubated in a 10-cm culture dish for 8 h, then re-seeded into a 96-well plate or a 384-well plate and allowed to adhere for 8 h. Then, compounds were added to the 96-well plate or 384-well plate using an Echo 550 nanoliter acoustic liquid handling system (Labcyte). After co-incubation for 24 h, immunofluorescence was used to observe the fluorescence signal. The specific steps were as follows: The cells were fixed by directly adding 4% paraformaldehyde equal in volume to the cell culture medium and fixing at room temperature for 15 min. After washing the cell surface 3 times with 1×PBS, taking the 96-well plate as an example, 30 μL of DAPI staining solution (1 μg / mL) was added to each well, stained at room temperature for 5 min, sealed with light-shielding aluminum foil, and then photography could be carried out. In this study, a laser confocal high-content imaging microscope (PerkinElmer, Opera Phenix) was used for photography. When using it, follow the instrument operation manual. After establishing a new working template, first select a field of view for focusing. After aligning the focal plane, select the 488-nm excitation light and 340-nm excitation light (separate exposures) for photography. The Opera data analysis station was used for analysis. First, count the number of nuclei N stained by DAPI, which is the total number of cells; GFP-positive cells emit green fluorescence under the excitation light of 488 nm. Count the number of green fluorescent cells n, which is the number of cells with positive expression of GFP-KLHL24. When counting n, parameters need to be set to adjust the threshold of green fluorescence to select as many green fluorescent cells as possible. In addition, parameters such as cell area and cell shape can also be set to reduce the influence of the background.

[0156] 5. Western Blot

[0157] After collecting the cells, they were lysed on ice for 30 min using RIPA lysis buffer (Beyotime, P0013C). For animal tissue samples, they were centrifuged at 12,000 rpm at 4 °C for 10 min, and the supernatant was taken. After adding 5×loading buffer, they were incubated in a 95 °C metal bath for 10 min. Subsequently, proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk (prepared with TBST) at room temperature for 1 h, and incubated with the corresponding primary antibodies, anti-Vimentin (Cell Signaling Technology, 5741S), anti-GAPDH (ZSGB-BIO, TA-08), and anti-K15 (ab52816, Abcam). HRP-conjugated secondary antibodies were used, and the target bands were detected using an exposure instrument.

[0158] 6. Tape Stripping Experiment

[0159] Drug Preparation Scheme:

[0160] Preparation of the test topical preparation (hereinafter referred to as the test preparation): 10 mM of STK731044 dissolved in DMSO was added to ethanol glycerol (ethanol and glycerol were mixed at a volume ratio of 1:1) to make the final concentration of STK731044 0.1 g / 100 ml;

[0161] Preparation of the control topical preparation (hereinafter referred to as the control preparation): An equal volume and equal concentration of DMSO solution (but without STK231044) used for the test preparation was added to ethanol glycerol (ethanol and glycerol were mixed at a volume ratio of 1:1).

[0162] Seven female or male mice at specific time points of Klhl24 c.3G / T were administered with the KLHL24 inhibitor. The administration time was the first resting period P15 - P30 for a total of 14 days. The administration method was to use 20 μL of the test preparation on the left side (test side) of the mouse's back and 20 μL of the control preparation on the right side (control side) every day. A tape stripping test was performed at the second resting period, i.e., P50, with self - control. When performing the tape stripping test, the mice were anesthetized with 0.25% tribromoethanol, and then the tape was pressed on the mouse's back with the same force for 30 s, after which the tape was removed, photographed, the pictures were collected and sorted out, and the weight of the shed hair shafts was statistically analyzed. When comparing the experimental group with the control group, the Dunnett - t test was used.

[0163] Example 1: Establishment of a high - throughput screening system for KLHL24 ubiquitin ligase inhibitors

[0164] KLHL24 belongs to a member of the KLHL E3 ubiquitin ligase family and has the characteristics of an E3 ubiquitin ligase, that is, it will ubiquitinate the substrate protein when there is a substrate protein, and it can ubiquitinate itself when there is no substrate protein. In the preliminary experimental exploration of the present invention, it was found that MLN4924 could 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 for the design of the screening method. According to the above ideas, a screening strategy as Figure 1 shown was designed. Specifically, it includes the following steps: Construct a plasmid of KLHL24 with a GFP tag and transfer the plasmid into 293T cells. Due to the autoubiquitination of KLHL24, it remains at a low level. Subsequently, a small - molecule inhibitor was added. If the small - molecule inhibitor could restore the content of KLHL24 protein, then an increase in KLHL24 protein could be observed through a fluorescence microscope.

[0165] According to the designed screening scheme, it is first necessary to determine whether the autoubiquitination of KLHL24 can be used as a screening index. The specific operation steps are as follows: Transfect 293T cells with GFP-KHL24, and then add the effective and selective NEDD8-activating enzyme (NAE) inhibitor MLN4924 (at concentrations of 0.156 μM, 0.313 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM respectively), and detect the expression of GFP-KLHL24 by GFP fluorescence. The results are as Figure 2 shown. The expression level of GFP-KLHL24 is very low ( Figure 2 a), and its content increases significantly after adding MLN4924, showing a dose-dependent relationship ( Figure 2 b). 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.

[0166] Secondly, before conducting 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 very poor, the error between each group of experiments will be very large, which will lead to the inability to accurately screen out effective small molecule compounds and a large proportion of false negatives. To eliminate this error, the usual practice is to increase the number of repeated experiments. However, this will greatly increase the screening throughput and greatly increase the consumption of manpower and material resources. To solve this problem, a preliminary experiment was first carried out. The specific steps are as follows: Take 320 small molecule compounds (purchased from TargetMol), add them to different 96-well plates respectively, and perform two replicates for each small molecule compound, and perform a correlation analysis on the results of the two replicates. The results are as Figure 3 shown. The correlation coefficient between the two groups of results is R = 0.912, indicating that they have a very strong correlation. In addition, a regression analysis was performed on the two groups of repeated experiments, and the regression function is: y = 0.8907x + 1.0135. According to the coefficients of the regression function, the two groups of 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. Based on this result, we will only perform one replicate during high-throughput screening, which will greatly improve the screening efficiency.

[0167] Example 2: High-throughput screening of the Express-Pick diversity compound core library of Selleck

[0168] According to the high-throughput screening method established in Example 1, small molecule compounds that can inhibit the activity of KLHL24 were screened out. When conducting the screening, a diverse compound core library (Express-Pick Library, catalog number L3600) was selected, which contains 4208 small molecule compounds. The reasons for selecting this compound library include the following points: 1) This compound library is a unique collection of compounds with different core structures and is suitable for high-throughput screening; 2) This compound library is derived from one of the world's largest pharmaceutical companies; 3) This compound library can bind to the target, enabling the study of the conformation and pharmacodynamics of the compounds and accelerating the speed of new drug development; 4) After being determined by NMR and HPLC, the purity of the compounds is ensured.

[0169] After the compound library was selected, high-content based high-throughput screening was carried out. When conducting the screening, multiple negative controls (DMSO) were set in each group of experiments. After obtaining the original results, first, the number of nuclei N stained by DAPI was counted, which is the total number of cells; at the same time, the number of green fluorescent cells n was counted, which is the number of cells expressing GFP-KLHL24. When counting n, parameters need to be set to adjust the threshold of green fluorescence to select as many green fluorescent cells as possible. In addition, parameters such as cell area and cell shape can also be set to reduce the influence of the background. The calculation formula for the ratio of GFP-positive cell number is: n / N * 100%. The ratios of GFP-positive cells were all normalized according to the results of the negative control, and the critical value of Fold Change was set to 2, which will greatly reduce false positive results. At the same time, we also introduced the parameter of the number of cells per well. Small molecule compounds usually have a certain toxicity to cells, causing cell death and spontaneous fluorescence, which increases the possibility of false positives. Therefore, introducing the parameter of the number of cells per well can, on the one hand, reduce false positives, and on the other hand, exclude small molecule compounds with high toxicity, reducing the workload of subsequent verification. In addition, we also considered the factor of cell transfection efficiency. According to the transfection efficiency of the transfection reagent for the plasmid, we believe that a GFP-positive cell ratio higher than 80% is caused by spontaneous fluorescence of small molecules. Considering the three variables of FoldChange value, the number of cells per well, and transfection efficiency comprehensively, first, the cell nuclei were stained with DAPI, and whether the compound has obvious toxicity was judged by the number of cell nuclei. Those with obvious toxicity were excluded. In the case of non-toxic and effective, it was judged that the ratio of GFP-positive cells after compound treatment was normalized according to the results of the negative control, and the critical value of Fold Change was set to 2. The results are as Figure 4 shown, and finally 41 compounds were screened out.

[0170] Example 3: Verify the effect of the initially screened candidate small molecule compounds on the degradation of KLHL24

[0171] The above 41 small molecule compounds were preliminarily verified to see whether they could have a significant effect on the GFP-KLHL24 protein content. The specific steps were as follows: GFP-KLHL24 was transfected into 293T cells, and then treated with small molecule compounds, and the KLHL24 protein content was detected by Western blot. Since there were many compounds, we conducted multiple experiments to detect the effects on GFP-KLHL24 protein. In this experiment, DMSO treatment was set as a negative control and MLN4924 treatment was set as a positive control. The concentrations of the positive control and small molecule compounds were both 10 μM. The results are shown in Figure 2. 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.

[0172] Example 4: KLHL24 inhibitor restores Klhl24 c.3G / T Levels of KRT15 and Vimentin proteins in mice

[0173] Multiple cell experiments verified that among the 25 small molecules, STK731044 had the most significant effect in increasing the activity of GFP-KLHL24 and had the least cytotoxicity. Therefore, STK731044 was subsequently selected for animal experiments. The effect of KLHL24 ubiquitin ligase activity on mice after inhibitor treatment was evaluated. The specific operation steps were as described in the tape hair experiment above. The results are shown in Figure 6 As shown, compared with the negative control (i.e., control preparation treatment), the KLHL24 inhibitor STK731044 treated Klhl24 c.3G / T The anchoring ability of the back hair shafts of mice was enhanced, and the number of hair shafts that fell off the tape was reduced. According to the reported literature, compared with WT mice (wild-type mice), Klhl24 c.3G / T The levels of KRT15 (K15 for short) and Vimentin protein (vimentin) in mice were reduced. Further experiments found that giving Klhl24 c.3G / T After treatment with the mouse KLHL24 inhibitor STK731044, the expression levels of KRT15 and Vimentin proteins in the mouse skin increased ( Figure 7 ). Figure 7 In the experiment, GAPDH was used as a common housekeeping gene protein and was used as a reference protein for comparing various samples. The above results suggest that KLHL24 inhibitors can be used as drugs for the treatment of hair loss.

[0174] Summary: STK731044 (screening number: PickP4E22) is a potential KLHL24 ubiquitin ligase inhibitor, which has good application prospects in the treatment of diseases caused by KLHL24 mutations.

[0175] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

[0176] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0177] Listing the steps of the method in a certain order in the present invention does not constitute any limitation on the order of the method steps.

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof as a KLHL24 ubiquitin ligase inhibitor, or in the preparation of a drug for preventing and / or treating a KLHL24-related disease, or in the preparation of a product for maintaining hair, wherein the compound has the following structure: in, represents a single bond or a double bond; X is selected from: O, S, NH; Ring A is a heterocyclic ring; 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-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), L is a divalent group connected to the A ring, which is selected from: a 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 heterocyclylene), wherein the C0-C6 alkylene, C3-C 10 Cycloalkyl, C6-C 10 The H in the aryl group and the 4-10 membered heterocyclyl group may be optionally substituted by one or more groups selected from the following: halogen, cyano, nitro, azido, 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 is one or more independent substituents connected to L, which are selected from: H, halogen, cyano, nitro, azido, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, 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), -(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, C3-C 10 Cycloalkyl, C6-C 10 The H in the aryl group and the 4-10 membered heterocyclic group may be optionally substituted by one or more groups selected from the following: halogen, cyano, nitro, azido, C1-C 10 Alkyl, C1-C 10 Haloalkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), -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 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-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), -(C0-C6 alkylene)-(C1-C 10 haloalkyl), -(C0-C6 alkylene)-(C1-C 10 Haloalkoxy), -(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), -CO(C0-C6 alkylene)(4-10 membered heterocyclyl), nitric oxide (NO) donor residue, wherein the C0-C6 alkylene, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C3-C 10 Cycloalkyl, C6-C 10 The H in the aryl group and the 4-10 membered heterocyclic group may be optionally substituted by one or more groups selected from the following: halogen, cyano, nitro, azido, 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 group), C1-C 10 Halogenated alkyl, 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 it is attached, it forms an aliphatic ring, an aromatic ring or a heterocyclic ring, wherein the H on the aliphatic ring, the aromatic ring or the heterocyclic ring is optionally substituted by one or more groups selected from the following: 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 0-10 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: -ONO2, Among them, 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 group), or R H and R K Together with the nitrogen atom to which it is attached, it forms a 4-6 membered heterocyclic group.

2. The use according to claim 1, characterized in that: The compound has the following structure: Wherein, V is selected from: -S-, -NH-, -CH2-, R1 and R2 have the above R A Definition of .

3. The use according to claim 2, characterized in that: R1 and R2 are independently Preferably, L is selected from: a 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 membered heterocyclylene)-, wherein the C0-C6 alkylene, C6-C 10 The H in the aryl group, the 4-10 membered heterocyclyl group, or the 4-10 membered heterocyclyl group may be optionally substituted by one or more groups selected from the following: 10 Alkyl, -COO(C 0-10 alkyl); Preferably, R C Selected from: 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-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 H in the aryl group and the 4-10 membered heterocyclic group may be optionally 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 -(C0-C6 alkylene)-(4-10 membered heterocyclyl); 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 heterocyclyl), -(C0-C6 alkylene)-(4-10 membered heterocyclyl), wherein the C0-C6 alkylene, C1-C 10 The H in the alkyl group and the 4-10 membered heterocyclic group may be optionally substituted by one or more groups selected from the following: halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, -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 to which it is attached, it forms a 5-6 membered heterocyclic ring.

4. The use according to claim 3, characterized in that: R1 is selected from: H, -F, -Cl, -Br, -I, Preferably, R2 is selected from: H, -F, -Cl, -Br, -I, Preferably, R B Selected from: H, -F, -Cl, -Br, -I, -NO2, -NH2, -CN, -COOH, -OH, -CF3, -ONO2; Preferably, the two R B Together with the carbon atom to which it is attached, it forms 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 following structure: Among them, R D Selected from: H, halogen, cyano, hydroxyl, carboxyl, C1-C6 alkyl, C1-C6 haloalkyl.

6. The use according to claim 5, characterized in that: R D C1-C6 alkyl, especially methyl, ethyl; Preferably, R B A nitric oxide (NO) donor residue, especially -ONO2, 7. The use according to claim 1, characterized in that: More preferably, the compound has the following structure:

8. The use according to claim 1, characterized in that: The KLHL24-related disease is selected from: skin diseases, cardiovascular diseases, tumors, digestive system diseases, arthritis, and complications thereof; Preferably, the skin disease is selected from the group consisting of alopecia, bullous skin diseases, scars, keratosis, erythematous papular scaly skin diseases, and connective tissue diseases; The cardiovascular disease is cardiomyopathy; 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 cortical carcinoma, pancreatic ductal adenocarcinoma, lung adenocarcinoma, pancreatic cancer, gonadoblastoma, multiple myeloma, mantle cell lymphoma, osteosarcoma, neuroblastoma, brain tumor, and melanoma; The digestive system disease is selected from: chronic hepatitis, drug-induced liver injury; The arthritis is osteoarthritis; More preferably, the disease is selected from: hereditary epidermolysis bullosa and its complications, atrophic scars, alopecia, dilated cardiomyopathy, hypertrophic cardiomyopathy, colon cancer, prostate cancer, gonadoblastoma, endometrial cancer, and osteoarthritis.

9. The use according to claim 1, characterized in that: The compound or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound is used alone or in combination with other types of active ingredients; Preferably, the drug further comprises one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipient is selected from the group consisting of: binders, lubricants, disintegrants, antibacterial agents, suspending agents, solubilizers, thickeners, stabilizers, preservatives, fillers, antioxidants, and buffers; Preferably, the dosage form of the drug is selected from: gastrointestinal dosage form, injection dosage form, respiratory tract dosage form, skin dosage form, cavity administration dosage form, more preferably oral dosage form, skin dosage form, injection dosage form.

10. The use according to claim 1, characterized in that: The products are medicines, foods, health products, and nursing products; Preferably, the hair maintenance includes promoting hair growth, promoting hair regeneration and self-repair, extending the hair growth cycle, and preventing hair loss.

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