3-substituted naphtho[2,3-b]isoxazole (thio)zole-4,9-dione derivatives for use in the treatment of psoriasis

Abstract

The application belongs to the field of dermatosis drugs and relates to application of 3-substituted naphtho[2,3-b]isoxazole(thiazole)-4,9-dione derivatives in anti-psoriasis, a structural formula of the 3-substituted naphtho[2,3-b]isoxazole(thiazole)-4,9-dione derivative is shown in general formula (I): wherein X is selected from oxygen or a sulfur atom; Y and Z are independently selected from carbon or nitrogen atoms; and substitution definitions of R1 and R2 are shown in the specification. The 3-substituted naphtho[2,3-b]isoxazole(thiazole)-4,9-dione derivative has significant HaCaT proliferation inhibition activity, cell activity of some compounds is more than 10 times higher than that of a positive control, and IC50 even reaches a nanomolar level. The 3-substituted naphtho[2,3-b]isoxazole(thiazole)-4,9-dione derivative can significantly down-regulate p-STAT3 level, dose-dependently inhibit the activation process of STAT3, and has better inhibition capacity than a control STA21. Animal experiments show that the anti-psoriasis effect is better than that of the drug anthralin.

Description

Technical Field

[0001] This invention belongs to the field of dermatological drugs and relates to the application of 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives in the treatment of psoriasis. Background Technology

[0002] Psoriasis is an immune-mediated chronic inflammatory skin disease, and its incidence has been increasing year by year in recent years. At the same time, psoriasis patients often develop various complications, such as metabolic syndrome, obesity, cardiovascular disease, diabetes, and psoriatic arthritis, which seriously endanger their health and even shorten their lifespan.

[0003] Clinically, small-molecule chemical drugs used to treat psoriasis mainly include glucocorticoids, vitamin D analogs, cyclosporine, and methotrexate. However, these drugs have serious drawbacks, such as toxic side effects or poor efficacy. Therefore, researching novel anti-psoriasis drugs is of great significance.

[0004] Targeted therapy is a key area of ​​research in psoriasis treatment. The etiology of psoriasis is extremely complex, and its pathogenesis remains unclear, primarily influenced by genetic, environmental, and immune factors. Psoriasis is characterized by persistent inflammation, leading to uncontrolled proliferation and impaired differentiation of keratinocytes (KCs). It is an inflammatory autoimmune disease primarily caused by CD4+ Th cells (Th17) produced by interleukin (IL)-17. Its formation is related to abnormal T cell activation and local infiltration, excessive proliferation and abnormal differentiation of keratinocytes (KCs), and abnormal proliferation of dermal microvessels. In this process, excessive activation of STAT3 (transcription and transcription activator 3) is a crucial factor in inducing psoriasis. Therefore, STAT3 plays a key role in the pathogenesis of psoriasis and is a potential new therapeutic target. In psoriasis patients, STAT3 is abnormally activated in keratinocytes (KCs). The possible mechanism of action is as follows: when the skin is stimulated by injury or infection, it releases antimicrobial peptides to activate dendritic cells, thereby producing cytokines such as interleukins IL-6 and IL-23. This, in turn, activates STAT3, promotes the production of IL-21, and forms an autocrine circuit of IL-21 / STAT3, promoting the differentiation of Th17 cells. Mature Th17 cells produce pro-inflammatory factors such as IL-17 and IL-22, which activate STAT3 in keratinocytes (KCs), causing them to proliferate excessively and differentiate abnormally, producing the autoantigen K17. This further activates T cells to form a positive feedback pathway, promoting the occurrence and development of psoriasis. Summary of the Invention

[0005] The purpose of this invention is to provide the use of 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives in the preparation of anti-psoriasis drugs.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] The application of 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives in the preparation of anti-psoriasis drugs, wherein the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives is shown in general formula (I):

[0008]

[0009] Where X is selected from oxygen or sulfur atoms; Y and Z are independently selected from carbon or nitrogen atoms;

[0010] R1 is selected from hydrogen, C1-C8 straight-chain or branched alkyl, C3-C8 cycloalkyl, aryl, substituted aryl, substituted C1-C8 straight-chain or branched alkyl, substituted C3-C8 cycloalkyl; the substituents on the substituted aryl, substituted C1-C8 straight-chain or branched alkyl and substituted C3-C8 cycloalkyl are selected from halogen, amino, nitro, carboxyl, phenyl, benzyl, =O, haloalkyl, alkyl, alkenyl, alkynyl, hydroxyl, alkoxy, carboxyl, ester, aminoethoxy, aminopropoxy, and aminobutoxy.

[0011] R2 is selected from hydrogen, hydroxyl, C1-C8 alkyl, halogen, C1-C8 alkyl, nitro, amino, and amino; the number of R2 is 1-4, and any substitution is at the 1-4 position.

[0012] In one preferred embodiment, R1 is selected from C1-C5 straight-chain or branched alkyl groups, or halogenated C1-C5 straight-chain or branched alkyl groups.

[0013] In one preferred embodiment, R1 is selected from methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, and halotert-pentyl.

[0014] In one preferred embodiment, R1 is selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0015] Aryl refers to: (1) an aromatic monocyclic or fused ring; preferably an aromatic carbocyclic ring with 5-12 carbon atoms (a cyclic structure in which all ring atoms are carbon). Examples of aryl include, but are not limited to: phenyl, naphthyl; (2) a ring structure that can be connected to partially saturated carbocyclic rings, for example: phenyl and C5-7 cycloalkyl or C5-7 cycloalkenyl groups are fused together to form a cyclic structure. Examples include, but are not limited to: tetrahydronaphthyl, indenyl or hydroindenyl, etc.

[0016] In one preferred embodiment, R1 is selected from substituted phenyl groups. The structure of the substituted phenyl group is as follows: The number of substituents R5 on the substituted phenyl group is 1-5, and R5 is selected from halogen, amino, nitro, carboxyl, ester, phenyl, benzyl, =O, haloalkyl, C1-C5 straight-chain or branched alkyl, amino C1-C5 straight-chain or branched alkoxy, alkenyl, alkynyl, hydroxyl, amide, C1-C5 straight-chain or branched alkoxy; R4 is selected from C1-C5 straight-chain or branched alkylene, carbonyl, amide, haloC1-C5 straight-chain or branched alkylene.

[0017] In one preferred embodiment, R5 is selected from hydroxyl, halogen, methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halo-n-propyl, halo-2-propyl, halo-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, carboxyl, ester. R4 is selected from methylene, ethylene, n-propylene, 2-propylene, n-butylene, isobutylene, tert-butylene, pentylene, isopentylene, tert-pentylene, halomethylene, haloethylene, halo-n-propylene, halo-2-propylene, halo-n-butylene, halo-isobutylene, halo-tert-butylene, halo-pentylene, halo-isopentylene, and halo-tert-pentylene.

[0018] In one preferred embodiment, R1 is H, methyl, ethyl, haloethyl, 3,4,5-trimethoxyphenyl, benzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 3,4,5-trimethoxybenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 3-bromo-4-methoxybenzyl, 3-bromo-4-hydroxybenzyl, 2-bromo-3,4,5-trimethoxybenzyl, α-bromoethyl, α-bromobenzyl, α-hydroxyethyl, α-hydroxybenzyl, α-hydroxy-3,4-dimethoxybenzyl, acetyl, benzoyl, 4-methoxybenzoyl, 3,4-dimethoxybenzoyl.

[0019] In one preferred embodiment, R2 is selected from hydroxyl, C1-C5 alkoxy, halogen, and C1-C5 alkyl; the number of R2 is 1 to 4, and any substitution is made at the 1 to 4 positions.

[0020] In one preferred embodiment, R2 is selected from methoxy, ethoxy, propoxy, and butoxy; the number of R2 is 1 to 4, with any substitution at positions 1 to 4.

[0021] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0022]

[0023]

[0024] R1 is selected from hydrogen, C1-C8 straight-chain or branched alkyl, C3-C8 cycloalkyl, aryl, substituted aryl, substituted C1-C8 straight-chain or branched alkyl, substituted C3-C8 cycloalkyl; the substituents on the substituted aryl, substituted C1-C8 straight-chain or branched alkyl and substituted C3-C8 cycloalkyl are selected from halogen, amino, nitro, carboxyl, phenyl, benzyl, =O, haloalkyl, alkyl, alkenyl, alkynyl, hydroxyl, alkoxy;

[0025] R3 is selected from hydrogen, C1-C8 alkyloxy, and C1-C8 alkyl.

[0026] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0027]

[0028] R1 is selected from methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and substituted phenyl.

[0029] The number of substituents on the substituted phenyl group is 1-6, and the substituents are selected from halogen, amino, nitro, carboxyl, phenyl, benzyl, =O, haloalkyl, C1-C5 straight-chain or branched alkyl, haloC1-C5 straight-chain or branched alkyl, alkenyl, alkynyl, hydroxyl, C1-C5 straight-chain or branched alkoxy, C1-C5 straight-chain or branched alkylene, carbonyl, haloC1-C5 straight-chain or branched alkylene;

[0030] R3 is selected from hydrogen, C1-C8 alkyloxy, and C1-C8 alkyl.

[0031] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0032]

[0033] R1 is H, methyl, ethyl, haloethyl, 3,4,5-trimethoxyphenyl, benzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 3,4,5-trimethoxybenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 3-bromo-4-methoxybenzyl, 3-bromo-4-hydroxybenzyl, 2-bromo-3,4,5-trimethoxybenzyl, α-bromoethyl, α-bromobenzyl, α-hydroxyethyl, α-hydroxybenzyl, α-hydroxy-3,4-dimethoxybenzyl, acetyl, benzoyl, 4-methoxybenzoyl, 3,4-dimethoxybenzoyl.

[0034] R3 is selected from hydrogen, C1-C8 alkyloxy, and C1-C8 alkyl.

[0035] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0036]

[0037] R1 is H, methyl, ethyl, haloethyl, 3,4,5-trimethoxyphenyl, benzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 3,4,5-trimethoxybenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 3-bromo-4-methoxybenzyl, 3-bromo-4-hydroxybenzyl, 2-bromo-3,4,5-trimethoxybenzyl, α-bromoethyl, α-bromobenzyl, α-hydroxyethyl, α-hydroxybenzyl, α-hydroxy-3,4-dimethoxybenzyl, acetyl, benzoyl, 4-methoxybenzoyl, 3,4-dimethoxybenzoyl.

[0038] R3 is selected from H, C1-C8 alkyloxy, and C1-C5 alkyl.

[0039] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0040]

[0041] R1 is H, methyl, ethyl, haloethyl, 3,4,5-trimethoxyphenyl, benzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 3,4,5-trimethoxybenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 3-bromo-4-methoxybenzyl, 3-bromo-4-hydroxybenzyl, 2-bromo-3,4,5-trimethoxybenzyl, α-bromoethyl, α-bromobenzyl, α-hydroxyethyl, α-hydroxybenzyl, α-hydroxy-3,4-dimethoxybenzyl, acetyl, benzoyl, 4-methoxybenzoyl, 3,4-dimethoxybenzoyl.

[0042] R3 is selected from H, C1-C8 alkyloxy, and C1-C5 alkyl.

[0043] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0044]

[0045] R1 is selected from methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, substituted phenyl;

[0046] The number of substituents on the substituted phenyl group is 1-5, and the substituents are selected from methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, and halotert-pentyl.

[0047] R3 is selected from hydrogen, C1-C8 alkyloxy, and C1-C5 alkyl.

[0048] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0049]

[0050] R1 is selected from methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, substituted phenyl;

[0051] The substituted phenyl group has one substituent, which is located at the para or ortho position. The substituent is selected from methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, and halotert-pentyl.

[0052] R3 is selected from hydrogen, C1-C8 alkyloxy, and C1-C5 alkyl.

[0053] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0054]

[0055] R1 is selected from methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, substituted phenyl;

[0056] The substituted phenyl group has one substituent, which is located at the para or ortho position. The substituent is selected from methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, and halotert-pentyl.

[0057] R3 is selected from hydrogen, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, and tert-pentyl.

[0058] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0059]

[0060] R1 is H, methyl, ethyl, haloethyl, 3,4,5-trimethoxyphenyl, benzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 3,4,5-trimethoxybenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 3-bromo-4-methoxybenzyl, 3-bromo-4-hydroxybenzyl, 2-bromo-3,4,5-trimethoxybenzyl, α-bromoethyl, α-bromobenzyl, α-hydroxyethyl, α-hydroxybenzyl, α-hydroxy-3,4-dimethoxybenzyl, acetyl, benzoyl, 4-methoxybenzoyl, 3,4-dimethoxybenzoyl.

[0061] R3 is selected from hydrogen, C1-C8 alkyloxy, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, and tert-pentyl.

[0062] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0063]

[0064] R1 is selected from methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, substituted phenyl;

[0065] The number of substituents on the substituted phenyl group is 1-6, and the substituents are selected from methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, methylene, ethylene, n-propylene, 2-propylene, n-butylene, isobutylene, tert-butylene, pentylene, isopentylene, tert-pentylene, halomethylene, haloethylene, halon-n-propylene, halo2-propylene, halon-n-butylene, haloisobutylene, halotert-butylene, halopentylene, haloisopentylene, halotert-pentylene.

[0066] In one preferred embodiment, the structural formula of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is shown below:

[0067]

[0068] R1 is H, methyl, ethyl, haloethyl, 3,4,5-trimethoxyphenyl, benzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 3,4,5-trimethoxybenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 3-bromo-4-methoxybenzyl, 3-bromo-4-hydroxybenzyl, 2-bromo-3,4,5-trimethoxybenzyl, α-bromoethyl, α-bromobenzyl, α-hydroxyethyl, α-hydroxybenzyl, α-hydroxy-3,4-dimethoxybenzyl, acetyl, benzoyl, 4-methoxybenzoyl, 3,4-dimethoxybenzoyl.

[0069] This invention also claims protection for a 3-substituted naphtho[2,3-b]isox(thia)azole-4,9-dione derivative, the structural formula of which is shown below:

[0070]

[0071] Wherein, X is selected from oxygen or sulfur atoms;

[0072] R10 and R11 are independently selected from H, C1-C8 alkyl, amino, and amine-substituted C1-C8 alkyl, respectively;

[0073] R12 is selected from hydrogen, C1-C8 straight-chain or branched alkyl, C3-C8 cycloalkyl, amino, aminoalkyl, aryl, substituted aryl, substituted C1-C8 straight-chain or branched alkyl, substituted C3-C8 cycloalkyl; the substituents on the substituted aryl, substituted C1-C8 straight-chain or branched alkyl and substituted C3-C8 cycloalkyl are selected from halogen, amino, amino, nitro, carboxyl, phenyl, benzyl, =O, halogenated C1-C8 straight-chain or branched alkyl, C1-C8 straight-chain or branched alkyl, alkenyl, alkynyl, hydroxyl, alkoxy; the number of substituents on the substituted aromatic heterocycle and substituted aryl is 1-5, and any substitution is at positions 1-5.

[0074] In one preferred embodiment, the substituents on the substituted aromatic heterocycle and the substituted aryl group are independently selected from methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, halomethyl, haloethyl, halon-n-propyl, halo2-propyl, halon-n-butyl, haloisobutyl, halotert-butyl, halopentyl, haloisopentyl, halotert-pentyl, methyl formate, ethyl formate, propyl formate, butyl formate, pentyl formate, and hexyl formate.

[0075] In one preferred embodiment, the C3-C8 cycloalkyl group is: furanyl, thiophenyl, pyrrolyl, pyrazolyl, triazolyl, thiazolyl, pyridinyl, piperidinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, pyridinyl, imidazoleyl, 3-phenylpyrrolyl, thiazolyl-oxazolyl, tetrazolyl, isoxazolyl, inzolyl, pyridazinyl, quinolinyl, purinyl, carbazoleyl, acridineyl, pyrimidinyl, 2,3'-bifuranyl, imidazole, oxazole, isoxazole, thiadiazole, oxadiazole, tetrazolyl, pyridazine, isoquinoline, or isoquinoline.

[0076] In one preferred embodiment, the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is a compound or its stereoisomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or cocrystal shown in the following structures.

[0077] In one preferred embodiment, the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is a compound or its stereoisomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt, or cocrystal shown in the following structures.

[0078]

[0079]

[0080] This invention also claims protection for the preparation method of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative.

[0081] A method, when the 3-substituted naphtho[2,3-b]isox(thia)azole-4,9-dione derivative is a compound represented by general formula (IV), includes the following steps:

[0082] S1. Raw material 4 undergoes a substitution reaction under the action of acetic anhydride and sodium hydroxide, and then undergoes an oxidation reaction under the action of hydrochloric acid and potassium dichromate to generate intermediate 5.

[0083] S2, raw material 3 and hippuric acid react in the presence of sodium acetate with acetic anhydride as solvent to obtain intermediate 6;

[0084] S3, intermediate 5 and intermediate 6 undergo a ring-closing reaction to obtain the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative;

[0085] The structural formulas of the raw materials and intermediates are as follows:

[0086]

[0087] The method for preparing the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative, when the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative is a compound represented by general formula (V), includes the following steps:

[0088] S1. Raw material 4 undergoes a substitution reaction under the action of acetic anhydride and sodium hydroxide, and then undergoes an oxidation reaction under the action of hydrochloric acid and potassium dichromate to generate intermediate 5.

[0089] S2, raw material 5 and chlorocarbonyl sulfinyl chloride were mixed with toluene as solvent to obtain intermediate 7;

[0090] S3, intermediate 5 and intermediate 7 undergo a ring-closing reaction to obtain the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative;

[0091] The structural formulas of the raw materials and intermediates are as follows:

[0092]

[0093] The present invention also claims the use of the described 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative in the preparation of anti-psoriasis drugs.

[0094] The present invention also claims the use of the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative in the preparation of antitumor drugs.

[0095] In one preferred embodiment, the tumor includes non-small cell lung cancer, breast cancer, liver cancer, and colorectal cancer.

[0096] The 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives of this invention exhibit significant HaCaT proliferation inhibitory activity, with some compounds showing cell activity more than 10 times higher than the positive control, and IC50 values ​​even reaching nanomolar levels. Furthermore, they significantly downregulate p-STAT3 levels, inhibiting STAT3 activation in a dose-dependent manner, with superior inhibitory ability compared to the control STA21. Evaluation using an imiquimod-induced mouse psoriasis model revealed that the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives of this invention were significantly more effective than Anthralin cream and reduced the production of IL-17A and IL-17F. Attached Figure Description

[0097] Figure 1 The effect of the target compound at different concentrations on the phosphorylation process of STAT3;

[0098] Figure 2 Results of experiments on the target compound's anti-psoriasis effects in mice;

[0099] Figure 3 Experimental results showing that the target compound inhibits the production of pro-inflammatory factors IL-17A and IL-17F. Detailed Implementation

[0100] The invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0101] Example 1

[0102] The chemical structural formulas of this invention are those described in Tables 1 and 2:

[0103] Table 1 Chemical Structures of A1-A20

[0104]

[0105]

[0106]

[0107] Table 2B1-B35 Chemical Structures

[0108]

[0109]

[0110] The preparation methods of the above compounds refer to existing technologies: Liu Suyou, Liu Lijun, Ma Dayou, et al. Naphtho[2,3-b]isoxazole-4,9-dione derivatives and their preparation methods and uses. CN201810985272.X [2024-07-01].

[0111] HaCaT cell activity was used as an in vitro model for evaluating anti-psoriasis treatment.

[0112] The inhibitory activity of the above-mentioned naphtho[2,3-b]isoxazol-4,9-dione derivatives on HaCaT cells was determined by the MTT assay. The specific experimental procedure was as follows: HaCaT cells in the logarithmic growth phase were collected, plated to a density of 3000 cells per well, and incubated at 37°C with 5% CO2 for 12 h. Then, a gradient of drug concentrations (five concentrations for each compound) was added, and incubation continued for 48 h. Next, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured for another 4 h. The culture medium was then removed, and 100 μL of dimethyl sulfoxide was added to each well. The absorbance (OD) of each well was measured at 570 nm using an ELISA reader, and the IC50 of the compound was calculated. 50 Values. Anthralin (an anti-psoriasis drug) was used as a control, and the results are shown in Tables 3 and 4.

[0113] Table 3 shows the inhibitory effects of compounds A1-A20 on HaCaT cell activity.

[0114]

[0115]

[0116] Table 4 shows the inhibitory effects of compounds B1-B35 on HaCaT cell activity.

[0117]

[0118]

[0119]

[0120] As shown in the table, the naphtho[2,3-b]isoxazol-4,9-dione derivatives of this invention exhibit significant HaCaT proliferation inhibitory activity, with some compounds showing cell activity more than 10 times higher than the positive control, IC50... 50 It could even reach the level of Namor.

[0121] Example 2

[0122] The damage to cell membranes caused by target compounds, i.e., cytotoxic effects, is assessed by measuring LDH release activity.

[0123] HaCaT cells with good size and growth rate were selected and seeded into 96-well cell culture plates. After 12 hours, IC40 was added. 50 The culture medium dilution of the target compound with a concentration greater than 1 μM was incubated in an incubator (37℃, 5% CO2) for 4 h. One hour before the predetermined detection time point, LDH release reagent provided by the Lactate Dehydrogenase Cytotoxicity Kit (Shanghai Beyotime Biotechnology Co., Ltd., trade number C0017) was added to the "Sample Maximum Enzyme Activity Control Well". After the predetermined time, 120 μL of the supernatant from each well was taken and added to the corresponding well of a new 96-well plate. 60 μL of LDH detection working solution was added to each well, mixed well, and incubated at room temperature in the dark for 30 min. The absorbance was then measured at 490 nm. The results are shown in Table 5.

[0124] Table 5 shows the damaging effects of some compounds on HaCaT cells.

[0125]

[0126] The results showed that B20 caused the least damage to the cell membrane and had less toxicity.

[0127] Example 3

[0128] Western blot experiments were used to determine the effect of representative target compounds at different concentrations on the phosphorylation process of STAT3.

[0129] HaCaT cells in good growth condition were selected and diluted to a cell density of 250,000 cells / mL. The cell-containing culture medium was added to 6-well plates and incubated for 12 h. The drug was then diluted with culture medium to the required concentration gradient (B20 drug concentration was 0 / 0.25 / 0.5 / 1.0 μM). The culture medium in the 6-well plates was aspirated, and the drug culture medium dilution was added. After incubation for 24 h, sample proteins were extracted. SDS-PAGE gels were prepared, and 25 μg of protein was added to each well. Electrophoresis was performed at 100 V for 90 min. Then, the gel was transferred to an NC membrane at 100 V for 90 min. The target bands were excised. p-STAT3 was blocked with 5% BSA at room temperature for 1.5 h, and STAT3 and GAPDH were blocked with 5% skim milk powder at room temperature for 1.5 h. The corresponding antibodies were then added, and the plates were incubated overnight at 4 °C. The primary antibody was discarded overnight, and the plates were washed three times with PBST for 10 min each time. The plates were then incubated with secondary antibody for 1.5 h, washed three more times with PBST for 10 min each time, and then developed for detection. STA21 (a STAT3 inhibitor, 15.0 μM) and anthralin (anthralin, 3.0 μM) were selected as controls. The experimental results are as follows: Figure 1As shown (only compound B20 is displayed; other compounds also exhibit significant inhibitory effects), experimental results demonstrate that the naphtho[2,3-b]isoxazol-4,9-dione derivatives of this invention can significantly downregulate p-STAT3 levels, inhibiting STAT3 activation in a dose-dependent manner, and exhibiting superior inhibitory activity at high concentrations compared to the control STA21. Specifically, for compound B20, the inhibitory activity at 0.5 and 1.0 μM concentrations of compound B20 is superior to that of the control STA21.

[0130] Example 4

[0131] Animal experiments were conducted using a representative compound, B20, to verify its effectiveness.

[0132] Forty BALB / c mice (Hunan Slack Jingda Experimental Animal Co., Ltd.) were randomly divided into four groups: a blank control group (Vaseline), a model group (5% imiquimod cream), a positive control group (5% imiquimod cream + gel containing 2% anthralin), and a test drug group (5% imiquimod cream + gel containing 1% compound B20), with 10 mice in each group. The day before the experiment, the back hair of the mice was shaved with a razor, and then the downy hair on the back of the mice was removed with a depilatory agent, covering an area of ​​approximately 2cm × 3cm. After the experiment began, 5% imiquimod cream was applied to the shaved area on the back of the mice at 9:00 AM every day (the same amount of petroleum jelly was applied to the blank control group). At 9:00 PM every night, 2% anthralin gel (a drug gel made by adding 500 mg of glycerin, 40 mg of sodium hydroxide, 500 mg of ethanol, and distilled water to a final volume of 10,000 mg, etc., with 100 mg of carbomer as the base, was applied as the positive control, and a gel containing 1% B20 (a drug gel made by adding 500 mg of glycerin, 40 mg of sodium hydroxide, 500 mg of ethanol, and distilled water to a final volume of 10,000 mg, etc., with 1% B20, etc., was applied as the drug group or petroleum jelly (blank control group) for 7 consecutive days. During the experiment, skin lesion changes in each group of mice were recorded daily. Twenty-four hours after the last administration, the model establishment and skin lesion changes in each group were observed, and the skin lesion area and severity index (PASI) were used for scoring. Spleen changes were also observed, and mouse skin, heart, liver, spleen, lungs, and kidneys were stained with hematoxylin and eosin (HE). After inducing a mouse psoriasis model (model group) with imiquimod, the mice in the model group showed typical erythema and scaling, thickened lesions, and psoriasis-like lesions, indicating successful model establishment. The effects were then evaluated by treatment with anthralin (gel containing 2% anthralin, positive control) and B20 (gel containing 1% B20), respectively.

[0133] like Figure 2As shown in A-2B, compared with the control group mice, the model group mice exhibited typical erythema and scaling on their skin, with thickened lesions resembling psoriasis. The treatment group showed significantly reduced symptoms compared to the model group. Figure 2 In C-2G, PASI scores showed that B20 significantly improved skin lesions in mice. Figure 2 H showed that the spleen of mice treated with B20 was significantly reduced, indicating that B20 can improve IMQ-induced inflammation. In conclusion, compared with the positive control drug 2% anthralin, 1% B20 gel has a better therapeutic effect.

[0134] Example 5

[0135] Blood was drawn from the above-mentioned model animals to measure inflammatory factors associated with the formation of psoriasis.

[0136] Blood samples were collected from mice in four groups: a blank control group, a model group (5% imiquimod cream), a positive control group (5% imiquimod cream + gel containing 2% anthralin), and a test drug group (5% imiquimod cream + gel containing 1% compound B20). The samples were centrifuged at 800 rpm for 20 min at 4°C. The supernatant serum was collected and analyzed using the Solarbio mouse IL-17A ELISA kit. IL-17 is an inflammatory cytokine associated with STAT3. The ELISA results are shown below. Figure 3 , respectively Figure 3 A and Figure 3 As shown in Figure B, the experimental results show that the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives of the present invention can significantly reduce the production of IL-17A and IL-17F.

[0137] In summary, the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivatives of the present invention exhibit significant HaCaT proliferation inhibitory activity, with some compounds showing cell activity more than 10 times higher than the positive control, and IC50... 50 It even reaches nanomolar levels. Furthermore, it significantly downregulates p-STAT3 levels, inhibiting STAT3 activation in a dose-dependent manner, with superior inhibitory ability compared to the control STA21. Evaluation using an imiquimod-induced mouse psoriasis model revealed that the 3-substituted naphtho[2,3-b]isoxazol-4,9-dione derivative of this invention was significantly more effective than Anthralin cream and could reduce the production of IL-17A and IL-17F.

[0138] Example 6

[0139] Preparation of quinoline-2,5,8(1H)-trione (intermediate 5)

[0140]

[0141] 1000 mg (6.9 mmol) of 8-hydroxyquinoline was dissolved in 20 mL of acetonitrile, and 1725 mg (10 mmol) of mCPBA was added. The reaction was carried out at room temperature for 12 h, and the reaction was monitored by TLC until it was complete. 20 mL of water was added, and 10 mL of 1 M NaOH solution was added to adjust the pH. A solid precipitated, which was filtered and dried to give 1096 mg of bright yellow solid intermediate 5-1, with a yield of 98.7%. mp 137.6-138.1℃.

[0142] 1000 mg (6.21 mmol) of intermediate 5-1 was dissolved in 10 mL of acetic anhydride and reacted at 60 °C for 8 h. The reaction was monitored by TLC until complete. 100 mL of 1 M NaOH solution was added, and the mixture was allowed to stand at 0 °C for a period of time to obtain a solid precipitate. The precipitate was filtered and dried to give 1225 mg of a light yellow solid, intermediate 5-2, with a yield of 97.1%. mp 252.6–253.7 °C.

[0143] 1000 mg (4.93 mmol) of intermediate 5-2 was added to 10 mL of 8 M hydrochloric acid solution and reacted at 90 °C for 1 h. The reaction was monitored by TLC until complete. The precipitated solid was filtered and dried to give 785 mg of gray solid intermediate 5-3, with a yield of 98.9%. mp 298.3-299.2 °C.

[0144] 1000 mg (6.21 mmol) of intermediate 5-3 was dissolved in 10 mL of DCM, 20 mL of 2 M sulfuric acid solution was added, and 1883 mg (6.4 mmol) of potassium dichromate was added. The reaction was carried out at 0 °C for 1 h. The organic layer was monitored by TLC. The reaction was complete. 100 mL of water was added, and the mixture was extracted three times with DCM (30 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was removed by vacuum distillation using a rotary evaporator to obtain 423 mg of crude solid. The crude product was recrystallized from 10 mL of EA to obtain 315 mg of orange-yellow solid intermediate 5, with a yield of 30.0%. 1 HNMR (500MHz, CDCl3) δ10.03 (s, 1H), 7.98 (d, J = 9.5Hz, 1H), 7.01-6.87 (m, 3H).

[0145] Example 7

[0146] Preparation of 3-methylisothiazolium[5,4g]quinoline-4,6,9[5H]-trione (compound 24) and 3-methylisothiazolium[4,5-g]quinoline-4,7,9[8H]-trione (compound 30)

[0147]

[0148] 177 mg (3 mmol) of acetamide feedstock 5 was dissolved in 8 mL of toluene, and 252 μL (2 mmol) of chlorocarbonyl sulfoxide chloride was added. The reaction was carried out at 100 °C for 3 h, monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. 20 mL of water was added, and the mixture was extracted three times with EA (15 mL × 3). The combined organic layers were dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation under reduced pressure to obtain 200 mg of colorless oily intermediate 6-1, with a yield of 57.0%. The crude product was used directly in the next reaction without further purification.

[0149] 175 mg (1 mmol) of intermediate 5 was dissolved in 5 mL of xylene, and 117 mg (1 mmol) of compound intermediate 6-1 was added. The mixture was refluxed under nitrogen protection for 12 h. TLC monitoring showed the reaction was complete. The mixture was extracted three times with EA (15 mL × 3). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by 200-300 mesh silica gel column chromatography using a DCM / EA mixed solution [V(DCM):V(EA) = 30:1]. This yielded 26 mg of yellow solid 24 (10.6% yield) and 19 mg of orange solid 30 (7.7% yield). Compound 24: 1 H NMR (500MHz, DMSO-d6) δ12.41(s,1H),7.97(d,J=9.2Hz,1H),6.75(d,J=9.3Hz,1H),2.72(s,3H). 13 C NMR(126MHz,DMSO-d6)δ177.62(s),176.95(s),173.05(s),168.83(s),168.35(s) ,150.86(s),134.02(s),130.60(s),129.70(s),128.50(s),22.18(s).ESI-HR-MS m / z[M+H] + calcd for C 11 H7N2O3S, 246.0099; found, 246.0101. Compound 30: 1 H NMR (500MHz, DMSO-d6) δ12.28 (s, 1H), 7.73 (d, J = 7.0Hz, 2H), 7.57-7.47 (m, 3H), 6.69 (s, 1H), 2.51 (s, 3H). 13C NMR(126MHz,DMSO-d6)δ177.80(s),173.00(s),168.39(s),165.75(s),163.10(s) ,143.21(s),136.50(s),132.45(s),125.41(s),117.22(s),19.77(s).ESI-HR-MS m / z[M+H] + calcd forC 11 H7N2O3S,246.0099; found,246.0099.

[0150] Example 8

[0151] Preparation of 3-ethylisothiazolium[5,4g]quinoline-4,6,9[5H]-trione (compound 25) and 3-ethylisothiazolium[4,5-g]quinoline-4,7,9[8H]-trione (compound 31)

[0152]

[0153] Following the synthesis method of Example 7, a yellow solid (compound 25) was obtained in 10.8% yield. An orange solid 31 was also obtained in 8.1% yield. Compound 25: 1 H NMR (500MHz, DMSO-d6) δ12.42(s,1H),7.98(d,J=9.4Hz,1H),6.75(d,J=9.4Hz,1H),3.14(q,J=7.3Hz,2H),1.29(t,J=7.3Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ175.24(s),174.16(s),173.19(s),165.46(s),162.72(s),143 .14(s),135.86(s),131.78(s),125.33(s),115.55(s),26.59(s),12.20(s).ESI-HR-MS m / z[M+H] + calcd forC 12 H9N2O3S, 260.0256; found, 260.0259. Compound 31: 1 H NMR (500MHz, DMSO-d6) δ12.49(s,1H),8.05(d,J=8.6Hz,1H),6.83(d,J=8.9Hz,1H),3.18(q,J=7.4Hz,2H),1.29(t,J=7.4Hz,3H). 13C NMR(126MHz,DMSO-d6)δ180.57(s),173.24(s),168.78(s),164.23(s),159.11(s),141.86( s),138.43(s),132.00(s),128.11(s),117.27(s),26.62(s),12.39(s).ESI-HR-MSm / z[M+H] + calcd for C 12 H9N2O3S,260.0256; found,260.0259.

[0154] Example 9

[0155] Preparation of 3-phenylisothiazolium[5,4g]quinoline-4,6,9[5H]-trione (compound 26) and 3-phenylisothiazolium[4,5-g]quinoline-4,7,9[8H]-trione (compound 32)

[0156]

[0157] Following the synthesis method of Example 7, yellow solid 26 was obtained with a yield of 20.1%; orange solid 32 was obtained with a yield of 11.4%. Compound 26: 1 H NMR (500MHz, DMSO-d6) δ12.18(s,1H),8.01(d,J=9.5Hz,1H),7.80(d,J=7.2Hz,2H),7.60-7.47(m,3H),6.77(d,J=9.5Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ175.15(s),173.25(s),168.72(s),166.89(s),162.51(s),143.56(s),135. 61(s),134.11(s),131.74(s),130.51(s),129.71(s),128.41(s),125.50(s),115.13(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H8N2O3S, 308.0256; found, 308.0260. Compound 32: 1 HNMR (500MHz, DMSO-d6) δ12.52(s,1H),8.03(s,1H),7.76(d,J=7.4Hz,2H),7.59-7.47(m,3H),6.86(s,1H). 13CNMR(126MHz,DMSO-d6)δ174.58(s),172.56(s),167.92(s),162.76(s),134.31(s),130.40(s),129.85(s),128.37(s),126.60(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H8N2O3S,308.0256; found,308.0260.

[0158] Example 10

[0159] Synthesis and preparation of 3-(1-morpholinyl)isothiazol[5,4g]quinoline-4,6,9[5H]-trione (compound 27)

[0160]

[0161] Following the synthesis method of Example 7, purple solid 27 was obtained in a yield of 30.0%. Compound 27: 1 H NMR (500MHz, DMSO-d6) δ12.29(s,1H),7.97(d,J=9.6Hz,1H),6.72(d,J=9.1Hz,1H),3.77(s,4H),3.42(s,4H). 13 C NMR (126MHz, DMSO-d6) δ175.29(s), 172.30(s), 167.34(d, J=10.3Hz), 162.51(s), 143.70(s) ),135.55(s),125.24(s),123.80(s),114.51(s),66.22(s),55.41(s),49.97(s).ESI-HR-MS m / z[M+H] + calcd for C 14 H 11 N3O4S,317.0470; found,317.0474.

[0162] Example 11

[0163] Synthesis and preparation of 3-(1-piperidinyl)isothiazol [5,4g]quinoline-4,6,9[5H]-trione (compound 28)

[0164]

[0165] Following the synthesis method of Example 7, purple solid 28 was obtained in a yield of 27.9%. Compound 28: 1H NMR (500MHz, DMSO-d6) δ12.24(s,1H),7.96(d,J=9.3Hz,1H),6.71(d,J=9.4Hz,1H),3.38(s,4H),1.68(s,4H)),1.62(s,2H). 13 C NMR(126MHz,DMSO-d6)δ175.35(s),172.19(s),171.73(s),167.95(s),167.16(s),135.54(s) ),125.08(s),123.67(s),121.01(s),117.98(s),50.72(s),25.66(s),24.17(s).ESI-HR-MS m / z[M+H] + calcd for C 15 H 13 N3O3S,315.0678; found,315.0682.

[0166] Example 12

[0167] Synthesis and preparation of 3-(1-pyrrolo)isothiazol[5,4g]quinoline-4,6,9[5H]-trione (compound 29)

[0168]

[0169] Following the synthesis method of Example 7, purple solid 29 was obtained in a yield of 29.6%. Compound 29: 1 H NMR (500MHz, DMSO-d6) δ12.24(s,1H),7.96(d,J=9.5Hz,1H),6.71(d,J=9.5Hz,1H),3.59(s,4H),1.92(s,4H). 13 C NMR(126MHz,DMSO-d6)δ175.18(s),164.60(s),137.50(s),135.59(s),129.15(s),121.74(s),51.01(s),25.68(s).ESI-HR-MS m / z[M+H] + calcd forC 14 H 11 N3O3S,301.0521; found,301.0521.

[0170] Example 13

[0171] Preparation of the synthesis of 8-methyl-3-phenylisothiazolium[5,4g]quinoline-4,6,9[5H]-trione (compound 33) and 8-methyl-3-phenylisothiazolium[4,5-g]quinoline-4,7,9[8H]-trione (compound 34).

[0172]

[0173] Following the synthesis method of Example 7, yellow solid 33 was obtained in 13.4% yield; orange solid 34 was obtained in 8.1% yield. Compound 33: 1 H NMR (500MHz, DMSO-d6) δ12.11 (s, 1H), 7.82 (d, J = 7.2Hz, 2H), 7.61-7.44 (m, 3H), 6.59 (s, 1H), 2.56 (s, 3H). 13 C NMR(126MHz,DMSO-d6)δ176.95(s),173.05(s),168.83(s),168.35(s),150.88(s) ,134.02(s),130.79(s),130.60(s),129.70(s),128.50(s),22.18(s).ESI-HR-MS m / z[M+H] + calcd for C 17 H 12 N2O3S, 322.0412; found, 322.0415. Compound 34: 1 H NMR (500MHz, DMSO-d6) δ12.27(s,1H),7.73(d,J=7.0Hz,2H),7.52(q,J=6.4Hz,3H),6.69(s,1H),2.51(s,3H). 13 C NMR(126MHz, DMSO-d6)δ178.73(d,J=4.2Hz),169.02(s),134.63(s),133.45(s),130.21(s),129.79(s),128.37(s),22.64(s).ESI-HR-MS m / z[M+H] + calcd for C 17 H 10 N2O3S,322.0412; found,322.0417.

[0174] Example 14

[0175] Preparation of 3-phenylisoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 35) and 3-phenylisoxazole[5,4g]quinoline-4,6,9[5H]-trione (compound 42)

[0176]

[0177] 1000 mg (5.59 mmol) hippuric acid was dissolved in 5 mL of acetic anhydride, and 815 μL (8 mmol) of benzaldehyde starting material 3 was added. 328 mg (4 mmol) of sodium acetate was added, and the reaction was carried out at 140 °C for 4 h. The organic layer was monitored by TLC, and the reaction was complete. After cooling to room temperature, a yellow solid precipitated. The solid was filtered, washed with a small amount of EA, and dried at 50 °C. The crude product was recrystallized from 10 mL of EA to give 837 mg of a yellow solid, with a yield of 60.1%. Intermediate 6-1: 1 H NMR (400MHz, CDCl3) δ8.23(td,J=8.5,1.7Hz,4H),7.65(t,J=7.4Hz,1H),7.57(t,J=7.5Hz,2H),7.54-7.47(m,3H),7.29(s,1H).

[0178] 263 mg (1.5 mmol) of intermediate 5 was dissolved in 5 mL of 1,4-dioxane, followed by 249 mg (1 mmol) of intermediate 6-1, 484 mg (2 mmol) of copper nitrate trihydrate, and 166 mg (1 mmol) of potassium iodide. The mixture was refluxed at 80 °C for 3 h. TLC monitoring showed the reaction was complete. The solvent was evaporated, and 40 mL of water was added. The mixture was extracted three times with EA (15 mL × 3). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by 200-300 mesh silica gel column chromatography using a DCM / EA mixed solution [V(DCM):V(EA) = 30:1]. This yielded 29 mg of yellow solid 35 (9.9%) and 48 mg of orange solid 42 (16.4%). Compound 35: 1 H NMR (500MHz, DMSO-d6) δ12.67(s,1H),8.07(t,J=11.1Hz,3H),7.66-7.57(m,3H),6.88(d,J=7.2Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ177.15(s),169.22(s),166.41(s),160.52(s),136.76(s),131.84(s),129.62-129.16(m),126.34(s),117.60(s).ESI-HR-MS m / z[M+H] + calcd for C 16H8N2O4, 292.0484; found, 292.0490. Compound 42: 1 H NMR (500MHz, DMSO-d6) δ12.52(s,1H),8.04(t,J=8.7Hz,3H),7.64(dt,J=14.2,7.1Hz,3H),6.80(d,J=9.4Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ174.09(s),171.35(s),167.96(s),166.36(s),160.36(s),143.54(s),140. 35(s),135.67(s),134.34(s),131.97(s),129.39(s),126.21(s),122.30(s),117.59(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H8N2O4,292.0484; found,292.0489.

[0179] Example 15

[0180] Preparation of 3-(4-fluorophenyl)isoxazole[5,4g]quinoline-4,6,9[5H]-trione (compound 36) and 3-(4-fluorophenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 43)

[0181]

[0182] Following the synthesis method of Example 14, a pale yellow solid 36 was obtained with a yield of 6.8%; a deep yellow solid 43 was obtained with a yield of 11.3%. Compound 36: 1 H NMR (500MHz, DMSO-d6) δ12.60(s,1H),8.14(s,3H),7.43(s,2H),6.89(s,1H). 13 C NMR(126MHz,DMSO-d6)δ177.29(s),166.43(s),165.33(s),163.36(s),159.57(s) ,136.79(s),132.11(s),122.87(s),117.50(s),116.53(s),116.35(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7FN2O4,310.0390; found,310.0392. Compound 43:

[0183] 1 H NMR (500MHz, DMSO-d6) δ12.53(s,1H),8.12(dd,J=8.5,5.5Hz,2H),8.06(d,J=9.4Hz,1H),7.49(t,J=8.8Hz,2H),6.81(d,J=9.4Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ171.31(s),166.32(s),165.40(s),163.42(s),159.47(s),135.69(s) ,131.96(d,J=9.0Hz),124.36-122.88(m),122.72(d,J=3.2Hz),124.36-116.81(m).ESI-HR-MS m / z[M+H] + calcd for C 16 H7FN2O4,310.0390; found,310.0393.

[0184] Example 16

[0185] Preparation of the synthesis of 3-(4-bromophenyl)isoxazole[5,4g]quinoline-4,6,9[5H]-trione (compound 37) and 3-(4-bromophenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 45).

[0186]

[0187] Following the synthesis method of Example 14, yellow solid 37 was obtained in 10.0% yield; orange solid 45 was obtained in 14.9% yield. Compound 37: 1 H NMR (500MHz, DMSO-d6) δ12.53(s,1H),8.05(d,J=9.4Hz,1H),8.00(d,J=8.4Hz,2H),7.86(d,J=8.4Hz,2H),6.81(d,J=9.4Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ174.13(s),171.25(s),166.37(s),162.77(s),159.53(s),143.47(s),135. 69(s),132.50(s),131.33(s),125.77(s),125.43(s),120.64(s),117.62(s),114.74(s).ESI-HR-MS m / z[M+H] + calcd for C16 H7BrN2O4, 369.9589; found, 369.9592. Compound 45: 1 HNMR (500MHz, DMSO-d6) δ12.68(s,1H),8.08(d,J=7.4Hz,1H),8.03(d,J=8.3Hz,2H),7.82(d,J=8.3Hz,2H),6.87(d,J=9.1Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ177.14(s),166.49(s),159.68(s),136.77(s),132.90(s) ,132.40(s),131.49(s),130.64(s),125.60(d,J=10.2Hz),117.57(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7BrN2O4,369.9596; found,369.9589.

[0188] Example 17

[0189] Preparation of the synthesis of 3-(3-bromophenyl)isoxazole[5,4g]quinoline-4,6,9[5H]-trione (compound 38) and 3-(3-bromophenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 46).

[0190]

[0191] Following the synthesis method of Example 14, yellow solid 38 was obtained with a yield of 6.2%; orange solid 46 was obtained with a yield of 9.5%. Compound 38: 1 H NMR (500MHz, DMSO-d6) δ12.55(s,1H),8.27(s,1H),8.04(t,J=7.8Hz,2H),7.87(d,J=8.1Hz,1H),7.59(t,J=7.9Hz,1H),6.81(d,J=9.5Hz,1H). 13C NMR(126MHz,DMSO-d6)δ174.12(s),171.20(s),166.29(s),162.70(s),159.10(s),143.62(s),135.63(s),134. 77(s),131.99(s),131.61(s),128.35(s),128.19(s),125.15(s),122.39(s),117.68(s),114.59(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7BrN2O4, 369.9589; found, 369.9591. Compound 46: 1 H NMR (500MHz, DMSO-d6) δ12.70(s,1H),8.32(s,1H),8.12(s,1H),8.07(d,J=7.8Hz,1H),7.86(d,J=8.7Hz,1H),7.58(t,J=7.9Hz,1H),6.89(s,1H). 13 CNMR(126MHz,DMSO-d6)δ177.17(s),171.54(s),166.44(s),163.64(s),159.25(s),136.80(s),134. 65(s),132.09(s),131.53(s),128.52(s),128.39(s),127.62(s),122.32(s),117.66(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7BrN2O4,369.9589; found,369.9591.

[0192] Example 18

[0193] Preparation of 3-(2-bromophenyl)isoxazole[5,4g]quinoline-4,6,9[5H]-trione (compound 39) and 3-(2-bromophenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 47)

[0194]

[0195] Following the synthesis method of Example 14, yellow solid 39 was obtained with a yield of 5.7%; orange solid 47 was obtained with a yield of 10.3%. Compound 39: 1H NMR (500MHz, DMSO-d6) δ12.65(s,1H),7.99(s,1H),7.91-7.84(m,1H),7.62-7.53(m,3H),6.84(s,1H). 13 C NMR(101MHz,DMSO-d6)δ176.67(s),165.68(s),159.21(s),136.75(s),131. 32(s),130.39(s),129.80(s),129.46(s),125.22(s),117.05(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7BrN2O4, 369.9589; found, 369.9594. Compound 47: 1 HNMR (500MHz, DMSO-d6) δ12.51(s,1H),8.06(d,J=5.6Hz,1H),7.89(d,J=6.8Hz,1H),7.65-7.53(m,3H),6.81(d,J=7.7Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ173.72(s),171.26(s),165.56(s),159.64(s),155.98(s),135.78(s),133. 52(s),133.13(s),132.63(s),132.31(s),128.40(s),127.64(s),122.86(s),118.60(s).ESI-HR-MS m / z[M+H] + calcd forC 16 H7BrN2O4,369.9589; found,369.9593.

[0196] Example 19

[0197] Preparation of the synthesis of 3-(4-acetoxyphenyl)isoxazole[5,4g]quinoline-4,6,9[5H]-trione (compound 40) and 3-(4-acetoxyphenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 48).

[0198]

[0199] Following the synthesis method of Example 14, 40 mg of yellow solid was obtained, with a yield of 7.1%; 48 mg of orange solid (53 mg) was obtained, with a yield of...

[0200] The rate was 15.1%. Compound 40:1 H NMR (400MHz, DMSO-d6) δ12.51(s,1H),8.08(t,J=11.7Hz,3H),7.41(d,J=8.4Hz,2H),6.81(d,J=9.4Hz,1H),2.34(s,3H). 13 C NMR(101MHz,DMSO-d6)δ171.32(s),169.52(s),166.35(s),159.69(s),153.29(s),140 .03(s),130.83(s),123.71(s),122.95(d,J=9.5Hz),117.57(s),21.37(s).ESI-HR-MS m / z[M+H] + calcdfor C 16 H7BrN2O4, 350.0539; found, 350.0542. Compound 48: 1 H NMR (400MHz, DMSO-d6) δ12.69(s,1H),8.13(d,J=8.6Hz,3H),7.39(d,J=8.6Hz,2H),6.87(s,1H),2.34(s,3H). 13 C NMR(101MHz,DMSO-d6)δ173.29(s),169.52(s),166.49(s),158.78(s),153.22(s),130. 99(s),130.06(s),125.85(s),123.87(s),122.93(s),120.42(s),21.39(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7BrN2O4,350.0539; found,350.0542.

[0201] Example 20

[0202] Preparation of 3-(4-nitrophenyl)isoxazole[5,4g]quinoline-4,6,9[5H]-trione (compound 41) and 3-(4-nitrophenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 51)

[0203]

[0204] Following the synthesis method of Example 14, yellow solid 41 was obtained with a yield of 9.5%; orange solid 51 was obtained with a yield of 16.9%. Compound 41: 1H NMR (500MHz, DMSO-d6) δ12.62(s,1H),8.48(d,J=7.9Hz,2H),8.31(d,J=8.0Hz,2H),8.08(d,J=9.1Hz,1H),6.83(d,J=9.0Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ171.18(s),166.50(s),163.02(s),158.95(s),149.66(s),135.74(s) ,134.49(s),133.92(s),132.22(d,J=19.9Hz),130.89(s),129.15(s),124.50(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7N3O6, 337.0335; found, 337.0338. Compound 51: 1 H NMR (500MHz, DMSO-d6) δ12.71(s,1H),8.46(d,J=8.3Hz,2H),8.36(d,J=8.2Hz,2H),8.09(s,1H),6.90(s,1H). 13 CNMR(126MHz,DMSO-d6)δ151.10(s),149.60(s),132.45(s),131.05(s),124.41(s),119.31(s),116.75(s),114.25(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H7N3O6,337.0335; found,337.0339.

[0205] Example 21

[0206] Synthesis and preparation of 3-(4-chlorophenyl)isoxazole[4,5g]quinoline-4,6,9[5H]-trione (compound 44)

[0207]

[0208] Following the synthesis method of Example 14, an orange solid 44 was obtained in 9.8% yield. Compound 44: 1 H NMR (400MHz, DMSO-d6) δ12.73(s,1H),8.11(d,J=8.5Hz,3H),7.70(d,J=8.6Hz,2H),6.87(d,J=9.5Hz,1H). 13C NMR(101MHz,DMSO-d6)δ176.67(s),165.68(s),159.21(s),136.75(s),131. 32(s),130.39(s),129.80(s),129.46(s),125.22(s),117.05(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H9ClN2O4,328.0254; found,328.0251.

[0209] Example 22

[0210] Synthesis and preparation of 3-(4-hydroxyphenyl)-3-phenylisothiazol [4,5g]quinoline-4,6,9[5H]-trione (compound 49)

[0211]

[0212] 35 mg (0.1 mmol) of compound 48 was added to 2 mL of 2 M hydrochloric acid solution and reacted at 90 °C for 1 h. The reaction was monitored by TLC until complete. The mixture was filtered, washed with EA, and dried to give 29 mg of a red solid, with a yield of 94.2%. Compound 49: 1 HNMR(500MHz,DMSO-d6)δ12.61(s,1H),10.23(s,1H),8.08(d,J=6.6Hz,1H),7.96(d,J=8.6Hz,2H),6.96(d,J=8.6Hz,2H),6.87(s,1H).13C NMR(126MHz,DMSO-d6)δ177.35(s),169.21(s),166.29(s),160.82(s),160.31(s),136.90(s),131 .18(d,J=16.0Hz),124.94(s),117.38(s),116.81(s),116.64(s),116.06(d,J=8.9Hz).ESI-HR-MS m / z[M+H] + calcd for C 16 H9N2O5, 308.0433; found, 308.0434.

[0213] Example 23

[0214] Synthesis and preparation of 3-(4-acetoxyphenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 50)

[0215]

[0216] Following the synthesis method of Example 31, a yellow solid was obtained in 4.9% yield. Compound 50: 1 H NMR (500MHz, DMSO-d6) δ8.42(d,J=8.6Hz,1H),8.14(d,J=8.6Hz,2H),7.39(d,J=8.5Hz,2H),7.30(d,J=8.6Hz,1H),4.06(s,3H),2.35(s,3H). 13 C NMR(126MHz,DMSO-d6)δ176.88(s),172.45(s),169.44(s),167.20(s),166.64(s),160.06(s),153.22(s),149.27(s) ),137.91(s),130.97(s),124.33(s),123.90(s),122.86(s),119.22(s),116.30(s),54.96(s),21.37(s).ESI-HR-MS m / z[M+H] + calcd for C 18 H 10 N2O6,364.0695; found,364.0699.

[0217] Example 24

[0218] Synthesis and preparation of 3-(4-aminophenyl)isoxazole[4,5-g]quinoline-4,7,9[8H]-trione (compound 52)

[0219]

[0220] 50 mg (0.15 mmol) of compound 51 was dissolved in 3 mL of ethanol, and 169 mg (0.75 mmol) of stannous chloride dihydrate was added. Under nitrogen protection, the mixture was refluxed at 80 °C for 3 h. TLC monitoring showed the reaction was complete. The solvent was evaporated to dryness, and 10 mL of 1 M hydrochloric acid was added. The mixture was extracted three times (15 mL × 5) with EA. The organic phase was dried over anhydrous sodium sulfate, and evaporated to dryness to give a crude solid. The crude solid was rinsed with a small amount of methanol to give 25 mg of a purplish-black solid, 52, in 54.3% yield. Compound 52: 1 H NMR (500MHz, DMSO-d6) δ12.60(s,1H),8.10(s,1H),7.86(s,2H),6.72(s,1H),6.68(d,J=8.4Hz,2H),5.83(s,2H). 13C NMR(126MHz,DMSO-d6)δ169.48(s),161.32(s),155.90(s),152.32(s),146.95(s),140.30(s),135. 14(s),127.92(s),124.21(s),120.24(s),117.78(s),113.61(s),112.60(s),111.06(s).ESI-HR-MS m / z[M+H] + calcd for C 16 H9N3O4,307.0593; found,307.0599.

[0221] Example 25

[0222] Synthesis and preparation of 3-(4-aminophenyl)-5-(2-morpholinylethyl)isothiazol[4,5g]quinoline-4,7,9[8H]-trione (compound 53)

[0223]

[0224] 31 mg (0.1 mmol) of compound 52 was dissolved in 3 mL of isopropanol, and 15 mg (0.1 mmol) of 4-(2-chloroethyl)morpholine and 56 mg (0.4 mmol) of potassium carbonate were added. The reaction was carried out at 80 °C for 4 h. TLC monitoring showed the reaction was complete. 20 mL of water was added, and the mixture was extracted three times (10 mL × 3) with EA. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by 200-300 mesh silica gel column chromatography. The eluent was a DCM / EA mixed solution [V(DCM):V(EA) = 5:1]. The solution was evaporated to dryness to give 10 mg of black solid 53, with a yield of 23.8%. Compound 53: 1 H NMR (500MHz, DMSO) δ8.38(d,J=8.6Hz,1H),7.87(d,J=8.4Hz,2H),7.31(d,J=8.6Hz,1H),6.69(d,J=8.3Hz ,2H),5.84(d,J=15.9Hz,2H),4.61(t,J=5.1Hz,2H),3.57(s,4H),3.49(s,2H),2.78(s,2H),2.45(s,2H). 13C NMR (126MHz, DMSO) δ177.99(s),171.83(s),167.20(s),166.30(s),160.67(s),152.31(s),147.36(s),138.49(s),130. 75(s),126.30(s),118.19(s),116.85(s),113.62(s),112.70(s),66.56(s),64.38(s),57.14(s),53.92(s).ESI-HR-MS m / z[M+H] + calcd for C 22 H 20 N4O5,420.1434; found,420.1440.

[0225] Example 26

[0226] The proliferation inhibitory activity of the compound was determined using human keratinocytes (HaCaT cells), human non-small cell lung cancer cells A549, human breast cancer cells MDA-MB-231, human liver cancer cells HepG2, and human colorectal cancer cells HT-29.

[0227] (1) Experimental methods: a. Plating: Select HaCaT cells in good growth condition, carefully aspirate the old culture medium, add 2 mL of PBS buffer to wash the culture dish, and discard it. Add 1 mL of EDTA-containing trypsin for digestion. When the cells no longer adhere to the wall, add 2 mL of fresh culture medium to stop the digestion, transfer to a 15 mL centrifuge tube, and centrifuge at 800 rpm for 5 min. Discard the supernatant, add 3 mL of culture medium, mix well, and take 50 μL of the cell suspension to dilute it in 450 μL of PBS buffer. Use a hemocytometer to count the cells and calculate the cell density in the cell suspension. Assuming a cell density of 3 × 10⁻⁶ cells / mL... 4a) Dilute the drug at a concentration of 1 mg / mL, mix thoroughly by pipetting, and add the cell-containing culture medium to each well of a 96-well plate (100 μL per well). Incubate in a cell culture incubator (37°C, 5% CO2). b) After 12 h of incubation, dilute the drug with culture medium to six concentration gradients as needed. Then, in a biosafety cabinet, aspirate the old culture medium from the 96-well plate and add the drug's culture medium dilution (100 μL per well). Incubate in an incubator (37°C, 5% CO2). c) After 48 h of incubation, add 20 μL of 5 mg / mL MTT solution to each well under light-protected conditions, and continue incubation in a cell culture incubator. d) After 4 h of incubation, carefully aspirate the liquid from the 96-well plate and add 100 μL of the solution to each well. DMSO was shaken at low speed on a shaker for 30 seconds, and the OD value of each well at 570 nm was measured using an ELISA reader; e. In the experiment, blank group (culture medium, MTT), control group (cells, culture medium, DMSO, MTT), and drug group (cells, culture medium, drug, MTT) were set up. The drug had 3 replicates at each concentration, and the experiment was repeated 3 times.

[0228] Relative cell viability = (OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0229] The cell inhibition rate of the compound = 100% - survival rate

[0230] Using software to process data and calculate the IC of compounds 50 value.

[0231] (2) Experimental results: The inhibitory activity of the target compounds on the proliferation of human keratinocytes (HaCaT) is shown in Table 5. The results show that naphthoquinone derivatives have strong inhibitory activity on the in vitro proliferation of HaCaT cells, and the activity of some compounds reaches the nanomolar level, which is significantly better than that of the control.

[0232] Table 5. Inhibitory activity (ICP-C) of the target compounds against the proliferation of A549, MDA-MB-231, HepG2, HT-29, and HaCaT cells. 50 (μM) and LDH release activity

[0233]

[0234]

[0235]

[0236]

[0237]

[0238] Example 27

[0239] The compounds were selected for Western blotting experiments to determine their effect on p-STAT3 levels.

[0240] (1) Experimental principle: Polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate sample proteins and transfer them to a solid support in which proteins are adsorbed in a non-covalent form. The proteins or peptides on the solid support are used as antigens and antibodies are used as "probes" for specific binding. Then, the primary antibody is bound with enzyme- or isotope-labeled secondary antibody. The expression level of the specific target protein separated by electrophoresis is detected by substrate color development or autoradiography.

[0241] (2) Experimental methods: a. Plating: Select HaCaT cells in good growth condition, wash with PBS, digest with trypsin, centrifuge, discard the supernatant, add 3 mL of culture medium, mix well, take 50 μL of the liquid and add it to 450 μL of PBS buffer, mix well, count the cells using a hemocytometer, and calculate the cell density of the liquid. Then, with a cell density of 2.5 × 10⁻⁶ cells / mL... 5 a) Dilute to 1 mL / well, mix thoroughly by pipetting, add the cell-containing culture medium to a 6-well plate (2 mL per well), and incubate in a cell culture incubator (37℃, 5% CO2); b) Drug administration: After 12 h of incubation, dilute the target compound with good cell activity to the required concentration gradient with culture medium. Then, in a biosafety cabinet, aspirate the culture medium from the 6-well plate, add the drug's culture medium dilution (2 mL per well), and incubate in an incubator (37℃, 5% CO2). c) Sample protein preparation: After 24 h of incubation, transfer the 6-well plate to ice, aspirate the cell culture medium, wash twice with pre-chilled PBS, add cell lysis buffer RIPA, phosphatase inhibitor, and protease inhibitor, lyse for 30 min, scrape off cells with pre-chilled cell scraping, centrifuge at 13800 rpm for 20 min at 4℃, transfer the supernatant to a 1.5 mL EP tube, and detect protein concentration using a BCA kit. Adjust all protein samples to equal concentration, mix thoroughly, add 4× loading buffer, boil in a metal bath at 100℃ for 10 min, then load the samples. d. Prepare SDS-PAGE gels, load 20 μg of sample protein into each well, and electrophoresis at 100V for 90 min. Then transfer to an NC membrane at 100V for 90 min, cut out the target band, block p-STAT3 with 5% BSA at room temperature for 1 h, block STAT3 and GAPDH with 5% skim milk powder at room temperature for 1 h, incubate with primary antibody at 4℃ overnight, wash three times with PBST for 10 min each time, incubate with secondary antibody at room temperature for 1 h, wash three more times with PBST for 10 min each time, and then develop and detect.

[0242] Image Lab 4.0.1 was used to detect the bound immune complexes.

[0243] (3) Experimental results: such as Figure 1 As shown, Figure 1 The representative compounds shown all significantly downregulated p-STAT3 expression levels in HaCaT cells, while having no significant effect on total STAT3 protein levels. Only the data for compound 18 is shown in the figure. Therefore, the compounds of this invention are effective inhibitors of STAT3 phosphorylation.

[0244] Example 28

[0245] Compounds with good cell viability were selected for LDH release activity assay to determine the effect of the target compounds on the cell membrane.

[0246] (1) Experimental Principle: The lactate dehydrogenase cytotoxicity assay kit was used for detection. Cell membrane structure disruption caused by apoptosis or necrosis leads to the release of enzymes from the cytoplasm into the culture medium, including lactate dehydrogenase (LDH), which has relatively stable activity. LDH release is considered an important indicator of cell membrane integrity. Under the action of LDH, NAD+ is reduced to NADH. NADH and INT are then catalyzed by lipoamide dehydrogenase to generate NAD+ and formazan, a Johnson & Johnson chromogen, producing an absorption peak at a wavelength of 490 nm. The absorbance is linearly positively correlated with LDH activity, and can be used to quantify LDH activity.

[0247] (2) Experimental methods: a. Plating: Select HaCaT cells in good growth condition, wash with PBS, digest with trypsin, centrifuge, discard the supernatant, add 3mL of culture medium, mix well, take out 50μL of liquid and add it to 450μL of PBS buffer, mix well, count the cells with a hemocytometer and calculate the cell density of the liquid. Then, dilute the drug at a cell density of 5 × 10⁴ cells / mL, mix well by pipetting, and add to 96-well plates (100 μL per well). Incubate in a cell culture incubator (37°C, 5% CO₂). b. After incubation for 12 h, dilute the drug with serum-free 1640 medium to the required concentration, carefully remove the old medium from the 96-well plate, add the drug's medium dilution (150 μL per well), and incubate in a cell culture incubator (37°C, 5% CO₂). c. After incubation for 3 h, add 15 μL of LDH release agent to the maximum LDH release well, and continue incubation in a cell culture incubator (37°C, 5% CO₂) for 1 h. At the same time, prepare the detection working solution according to the kit instructions. d. Centrifuge the 96-well plate at 800 rpm for 5 min in a multi-well centrifuge. Carefully transfer 120 μL of the supernatant from the old 96-well plate to the new 96-well plate. Add 60 μL of working solution in the dark and incubate at room temperature in the dark for 30-40 min. Measure the absorbance at 490 nm using a microplate reader. e. The experiment includes a blank group (no cells, culture medium), a control group (cells, culture medium, DMSO), a maximum release well (cells, culture medium, DMSO, LDH release agent), and a drug group (cells, culture medium, drug). Two replicates are set up for each drug concentration.

[0248] A calibration curve was plotted using LDH standards and the corresponding measured absorbance values. The LDH enzyme activity of the sample was calculated based on the standard curve formula and the actual absorbance value of the sample, and the data were expressed as mU.

[0249] (3) Experimental Results: The LDH-releasing activity of the target compounds against human keratinocytes (HaCaT) is shown in Table 5. The LDH-releasing activity of quinone compounds is basically consistent with their antiproliferative activity, that is, the higher the antiproliferative activity, the more LDH is released and the greater the cytotoxicity. Surprisingly, the antiproliferative activity of compound 18 is 26 times that of BBI608, but its LDH-releasing activity is smaller, that is, its cytotoxicity is smaller. This indicates that the compounds of the present invention can be a potential candidate drug for treating psoriasis.

Claims

1. A compound, characterized in that, Its structural formula is shown below: ； Wherein, X is selected from oxygen or sulfur atoms; R10 and R11 are independently selected from H and C1-C8 alkyl groups, respectively; R12 is selected from C1-C8 straight-chain or branched alkyl, phenyl, or substituted phenyl groups; the substituents on the substituted phenyl group are selected from halogen, amino, nitro, or hydroxyl groups; the number of substituents on the substituted phenyl group is 1, and any substitution is at position 1-5.

2. A compound, characterized in that, Its structural formula is shown below: ； Wherein, X is selected from oxygen or sulfur atoms; R10 and R11 are independently selected from H and C1-C8 alkyl groups, respectively; R12 is selected from C1-C8 straight-chain or branched alkyl, C3-C8 cycloalkyl, phenyl, substituted phenyl; the substituent on the substituted phenyl is selected from halogen or nitro; the number of substituents on the substituted phenyl is 1, and any substitution is at position 1-5; C3-C8 cycloalkyl groups are: pyrrole and piperidinyl.

3. A compound, characterized in that, It is a compound with the following structure or a pharmaceutically acceptable salt thereof. 。 4. The method for preparing the compound according to claim 1 or 2, characterized in that, When X is an oxygen atom, the following steps are included: S1. Raw material 4 undergoes a substitution reaction under the action of acetic anhydride and sodium hydroxide, and then undergoes an oxidation reaction under the action of hydrochloric acid and potassium dichromate to generate intermediate 5. S2, raw material 3 and hippuric acid react in the presence of sodium acetate with acetic anhydride as solvent to obtain intermediate 6; S3, intermediate 5 and intermediate 6 undergo a ring-closing reaction to obtain the compound; The structural formulas of the raw materials and intermediates are as follows: ； R10 and R11 are both hydrogen; R12 is selected from phenyl or substituted phenyl; the substituents on the substituted phenyl are selected from halogen or nitro; the number of substituents on the substituted phenyl is 1, and any substitution is at position 1-5.

5. The use of the compound according to any one of claims 1-3 in the preparation of an anti-psoriasis or anti-tumor medicament, characterized in that, The tumors mentioned are non-small cell lung cancer, breast cancer, liver cancer, and colorectal cancer.

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