Use of QX77, AR7 and CA77.1 in activating Nur77 activity

Abstract

The application discloses a use of a compound shown in formula (I), formula (II) or formula (III) or a tautomer, a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof in preparation of a drug for preventing and / or treating a disease related to a nuclear receptor Nur77 (nerve growth factor-induced gene B, NGFI-B). The compound shown in formula (I), formula (II) or formula (III) can be combined with Nur77, effectively activate Nur77 activity, and can effectively treat and / or prevent a disease related to Nur77.

Description

Technical Field

[0001] This invention relates to the fields of medicine and biology. Specifically, this application relates to the use of QX77, AR7, and CA77.1 in activating Nur77 activity, and more specifically, to the use of QX77, AR7, and CA77.1 in the prevention and / or treatment of Nur77-related diseases, the use of QX77, AR7, and CA77.1 in activating Nur77 activity, methods for activating Nur77 activity, combination drugs or kits and their uses, and methods for the prevention and / or treatment of Nur77-related diseases. Background Technology

[0002] Under stimuli such as stress, cytokines, infectious agents, and growth factors, Nur77 regulates the transcription and expression of downstream genes by binding to DNA response elements via its DNA barrier function (DBD). Nur77 can bind to the response element NBRE not only as a monomer but also as a homodimer or as a heterodimer with two other members of the Nur77 family, Nurr1 and Nor1. Simultaneously, under 9-cis-retinoic acid mediation, Nur77 can form a heterodimer with the retinoid X receptor (RXR) and bind to the RXR response element DR-5 to exert transcriptional activation. Furthermore, ChIP experiments have confirmed that Nur77 can synergistically interact with specificity protein (Sp) family transcription factors, such as Sp1 or Sp4, binding to GC-enriched sequences in the promoter regions of genes such as survivin, integrins (β1 / β3 / β4 isoforms), and PAX-FOX01.

[0003] Nur77 not only exerts its genotypic function at the transcription factor level by regulating the transcription and expression of downstream target genes, but also possesses non-genotypic activities. For example, Nur77 regulates biological functions through protein-protein interactions. By binding to β-Catenin, Nur77 blocks the promoters of Wnt downstream genes, and simultaneously binds to the transcription factor TCF4, enhancing TCF4's ability to recruit transcriptional co-repressors, thereby inhibiting the expression of Wnt downstream genes c-myc and cyclin D1, negatively regulating Wnt signaling pathway activity and suppressing colon cancer. Furthermore, Nur77 can interact with proteins to alter protein conformation, thereby changing the function of some proteins. Bcl-2, a protein with anti-apoptotic activity in most cases, acts as a receptor on the outer mitochondrial membrane and becomes a downstream effector of Nur77 through its interaction with its ligand-binding domain. When Nur77 binds to Bcl-2, it exposes Bcl-2's hidden pro-apoptotic BH3 domain, causing a conformational change in Bcl-2 that prevents it from inhibiting the functions of pro-apoptotic proteins Bax and Bak. Instead, it inhibits the function of the anti-apoptotic protein Bcl-xL, causing Bcl-2 to transform from an anti-apoptotic protein into a pro-apoptotic protein.

[0004] The LBD domain of Nur77 differs from the typical LBD regions of other nuclear receptors. Crystal structure analysis reveals that the ligand binding pocket (LBP) in the Nur77-LBD contains several tightly packed hydrophobic amino acids, making it difficult for the LBP to accommodate small molecule ligands. Although the endogenous ligands of Nur77 remain undetermined, several studies have reported small molecule compounds that can bind to Nur77 and activate or inhibit its transcriptional activity. These compounds have shown pharmacological activity in studies of cancer, endometriosis, metabolic and inflammatory diseases, and possess clinical application potential. In summary, binding to Nur77 can affect both genotypic and non-genotypic activities of Nur77.

[0005] Therefore, Nur77 can serve as a drug target, and it is very important to develop new small molecule ligands that target Nur77. Summary of the Invention

[0006] This application aims to at least partially address at least one of the technical problems existing in the prior art. The present invention aims to partially address at least one of the technical problems existing in the prior art. To this end, the present invention provides the use of QX77, AR7, and CA77.1 in activating Nur77 activity. QX77, AR7, and CA77.1 can target Nur77, activate Nur77 activity, and can be used for the prevention and / or treatment of Nur77-related diseases.

[0007] This invention is based on the following discoveries of the inventors:

[0008] While researching novel ligands for the nuclear receptor Nur77, the inventors of this application unexpectedly screened out QX77, which can specifically bind to Nur77 and significantly enhance the transcriptional activity of activated Nur77. Through functional screening, they discovered that QX77 can Nur77-dependently treat pulmonary fibrosis, liver fibrosis, and tumor invasion, and subsequently tested structural analogues of QX77, AR7 and CA77.1. In some embodiments of this application, QX77, AR7, and CA77.1 regulate the physiological function of the corresponding nuclear receptor by modulating Nur77, thereby enabling their use in the treatment of related diseases.

[0009] In a first aspect, the present invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of Nur77-related diseases;

[0010]

[0011] According to embodiments of the present invention, the compounds represented by formula (I), (II), or (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and modulate its activity. Therefore, drugs prepared using the compounds represented by formula (I), (II), or (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can effectively prevent and / or treat Nur77-related diseases.

[0012] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor-1-related diseases.

[0013] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0014] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0015] In a second aspect, the present invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in the prevention and / or treatment of Nur77-related diseases;

[0016]

[0017] According to embodiments of the present invention, the compounds represented by formula (I), formula (II) or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity, thereby effectively preventing and / or treating Nur77-related diseases.

[0018] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor-1-related diseases.

[0019] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0020] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0021] In a third aspect of the invention, the invention provides a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof for the prevention and / or treatment of Nur77-related diseases;

[0022]

[0023] According to embodiments of the present invention, the compounds represented by formula (I), formula (II) or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity, thereby effectively preventing and / or treating Nur77-related diseases.

[0024] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor-1-related diseases.

[0025] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0026] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0027] In a fourth aspect of the invention, the invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in the preparation of a reagent for modulating the activity of Nur77 or modulating the activity of a dimer containing Nur77.

[0028]

[0029] According to embodiments of the present invention, the compounds represented by formula (I), formula (II), or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity. Therefore, reagents prepared using the compounds represented by formula (I), formula (II), or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can effectively modulate Nur77 activity or the activity of Nur77-containing dimers, and are particularly suitable for in vitro modulation of the activity of Nur77 or Nur77-containing dimers.

[0030] According to an embodiment of the present invention, the dimer containing Nur77 includes a dimer containing Nur1 and a dimer containing Nor1.

[0031] According to an embodiment of the present invention, the dimer containing Nur77 includes Nur77 homodimer and heterodimer containing Nur77.

[0032] According to an embodiment of the present invention, the heterodimer containing Nur77 is selected from RXR / Nur77, RXR / Nurr1, and RXR / Nor1.

[0033] In a fifth aspect of the invention, the invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in modulating the activity of Nur77 or modulating the activity of a dimer containing Nur77.

[0034]

[0035] According to embodiments of the present invention, the compounds represented by formula (I), formula (II) or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77, activate Nur77 activity, and modulate the activity of dimers containing Nur77.

[0036] According to an embodiment of the present invention, the dimer containing Nur77 includes a dimer containing Nur1 and a dimer containing Nor1.

[0037] According to an embodiment of the present invention, the dimer containing Nur77 includes Nur77 homodimer and heterodimer containing Nur77.

[0038] According to an embodiment of the present invention, the heterodimer containing Nur77 is selected from RXR / Nur77, RXR / Nurr1, and RXR / Nor1.

[0039] In a sixth aspect, the present invention provides a method for regulating Nur77 activity or regulating the activity of a Nur77-containing dimer. According to an embodiment of the present invention, the method comprises: contacting a compound of formula (I), formula (II), or formula (III), or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, with cells expressing Nur77 or a Nur77-containing dimer;

[0040]

[0041]

[0042] According to embodiments of the present invention, the compound represented by formula (I), formula (II) or formula (III) above, or its tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt, can target Nur77, activate Nur77 activity, and also activate the activity of the dimer containing Nur77.

[0043] According to an embodiment of the present invention, the dimer containing Nur77 includes a dimer containing Nur1 and a dimer containing Nor1.

[0044] According to an embodiment of the present invention, the dimer containing Nur77 includes Nur77 homodimer and heterodimer containing Nur77.

[0045] According to an embodiment of the present invention, the heterodimer containing Nur77 is selected from RXR / Nur77, RXR / Nurr1, and RXR / Nor1.

[0046] In a seventh aspect, the present invention provides a combination drug or cassette. According to an embodiment of the present invention, the combination drug or cassette comprises: a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof as an active ingredient;

[0047]

[0048]

[0049] According to embodiments of the present invention, the compounds represented by formula (I), (II), or (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity. Therefore, the combination of a reagent prepared using the compounds represented by formula (I), (II), or (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, and the ligand L of RXR can effectively modulate Nur77 activity or the activity of Nur77-containing dimers, and can effectively prevent and / or treat Nur77-related diseases.

[0050] According to an embodiment of the present invention, Nur77 includes Nurr1 and Nor1.

[0051] In an eighth aspect of the invention, the invention provides the use of the combined drug or cassette described in the seventh aspect in the preparation of a medicament for the prevention and / or treatment of Nur77-related diseases. As is known from the foregoing, the use of the aforementioned combined drug or cassette can effectively modulate Nur77 activity or the activity of Nur77-containing dimers, thereby effectively preventing and / or treating Nur77-related diseases.

[0052] According to embodiments of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor1-related diseases.

[0053] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0054] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0055] In a ninth aspect of the invention, a method for preventing and / or treating Nur77-related diseases is provided. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable dose of a compound of formula (I), formula (II), or formula (III) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, or the combination drug or cassette described in the seventh aspect;

[0056]

[0057] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor1-related diseases.

[0058] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0059] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0060] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0061] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0062] Figure 1 A represents the activation of Gal4 / DBD-Nur77 / LBD transcriptional activity by the QX77 compound and EC50, as determined by yeast one-hybrid assay in Example 1 of this invention.50 Measurement results diagram. ** p<0.01, **** p < 0.0001, n ≥ 3.

[0063] Figure 1 B represents the activation of Gal4 / DBD-Nur77 transcriptional activity by the QX77 compound and the EC50 activity, as determined by yeast one-hybrid assay in Example 1 of this invention. 50 The measurement results are shown in the figure. The ns value is not statistically significant. *** p<0.001, **** p < 0.0001, n ≥ 3.

[0064] Figure 1 C is a graph showing the activation results of the transcriptional activity of the QX77 compound on the Nur77 monomer, as measured by reporter gene assay in Example 1 of this invention. ns is not statistically significant. * p<0.05, ** p<0.01, *** p<0.001 **** p < 0.0001, n ≥ 3.

[0065] Figure 1 D is a graph showing the activation results of the transcriptional activity of the QX77 compound on the Nur77 dimer, as measured by reporter gene assay in Example 1 of this invention. ns is not statistically significant. *** p<0.001, **** p < 0.0001, n ≥ 3.

[0066] Figure 2 This invention relates to the activation of Gal4 / DBD-Nur77 / LBD transcriptional activity and EC50 activity of the QX77 analogues AR7 and CA77.1, as measured by yeast one-hybrid assay in Example 1 of this invention. 50 Measurement results diagram. ** p<0.01, **** p < 0.0001, n ≥ 3.

[0067] Figure 3 This is a graph showing the activation results of the QX77 compound on the transcriptional activities of Gal4 / DBD-RXRα / LBD, Gal4 / DBD-PPARα / LBD, Gal4 / DBD-GRα / LBD, Gal4 / DBD-ERα / LBD, and Gal4 / DBD-LXRα / LBD, as measured by yeast one-hybrid assay in Example 1 of this invention. ns is not statistically significant. **** p < 0.0001, n ≥ 3.

[0068] Figure 4Figure A shows the results of surface plasmon resonance (SPR) measurements of the binding of QX77 compound and Nur77-LBD protein in Example 2 of this invention. Different concentrations of QX77 compound were sequentially passed through a CM5 chip coupled with Nur77-LBD protein to obtain the response values ​​of binding and dissociation rates, and the KD value of QX77 and Nur77 / LBD binding was calculated.

[0069] Figure 4 Figure B shows the results of fluorescence titration of QX77 and Nur77 protein in Example 2 of this invention. The QX77 compound was gradually added to the purified Nur77-LBD protein, and the binding was detected after excitation by 280nm light.

[0070] Emission spectrum of Nur77 / LBD protein at 300-450 nm.

[0071] Figure 4 Figure C shows the results of the cell thermal displacement experiment in Example 2 of this invention, which measured the binding of QX77 and Nur77 proteins. HeLa cells were treated with QX77 at a concentration of 20 μM for 3 hours, followed by incubation of cell lysates at different temperatures. After centrifugation, the supernatant was collected and the Nur77 protein content was detected by Western blotting. The obtained bands were quantitatively analyzed using ImageJ, and the relative values ​​of Nur77 protein content at each temperature (the percentage of Nur77 protein at the lowest temperature) were calculated. A protein content change curve was plotted using GraphPad software.

[0072] Figure 5 This image shows the results of the cell scratch assay in Example 3 of this invention, which determined the inhibitory effect of QX77 on tumor cell invasion. Human lung tumor cells A549 were co-treated with TGFβ (5 ng / mL) and QX77 (10 μM) for 24 hours. Images were taken under a microscope at 0 h and 24 h, and the invasion ratio was statistically analyzed using Imagej software.

[0073] Figure 6 This image shows the results of the cell scratch assay in Example 3 of this invention, which determined the inhibitory effects of QX77 analogs AR7 and CA77.1 on tumor cell invasion. Human lung tumor cells A549 were treated with TGFβ (5 ng / mL) and either AR7 (10 μM) or CA77.1 (10 μM) for 24 hours. Microscopic images were taken at 0 h and 24 h, and the invasion rate was statistically analyzed using Imagej software.

[0074] Figure 7This is a graph showing the results of the cell scratch assay in Example 3 of this invention, which determined the inhibitory effect of QX77 on tumor cell invasion. Human breast cancer cells DA-MB-231 and mouse colon cancer cells MC38 were co-treated with TGFβ (5 ng / mL) and QX77 (10 μM) for 12 hours. The cells were photographed under a microscope at 0 h and 12 h, and the invasion ratio was calculated using Imagej software.

[0075] Figure 8 A is a schematic diagram of the MC38 mouse xenograft model in Example 4 of this invention. C57BL / 6 mice were subcutaneously injected with MC38 mouse colon cancer cells, and on the second day, QX77 (10 mg / kg) and the corresponding solvent were administered by gavage.

[0076] Figure 8 B is a schematic diagram of tumor sampling in the MC38 xenograft model in Embodiment 4 of the present invention.

[0077] Figure 8 C is the tumor volume growth curve in Example 4 of the present invention.

[0078] Figure 8 D is a bar chart of tumor mass in Example 4 of the present invention.

[0079] Figure 9 This image shows the morphological changes of lung epithelial cells under a conventional microscope as observed in Example 5 of this invention using QX77. Human lung cancer alveolar basal epithelial cells A549 and normal human lung epithelial cells Beas-2B were treated with TGFβ (5 ng / mL) or co-treated with QX77 (10 μM) for 48 hours before being photographed under a microscope.

[0080] Figure 10 Figure A shows the results of the immunoblotting experiment in Example 5 of this invention, which determined the effects of QX77 on COL1A1 and α-SMA proteins in a fibrotic cell model. Human embryonic lung fibroblasts WI-38 were co-treated with TGFβ (10 ng / mL) and QX77 (2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours.

[0081] Figure 10 Figure B shows the effect of QX77 on COL1A1 in the lung epithelial cell transdifferentiation into fibrotic cell model, as determined by the Western blot experiment in Example 5 of this invention. Human lung cancer alveolar basal epithelial cells A549 were co-treated with TGFβ (5 ng / mL) and QX77 (2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours.

[0082] Figure 10Figure C shows the effect of QX77 on COL1A1 in the lung epithelial cell transdifferentiation into fibrotic cell model, as determined by the Western blot experiment in Example 5 of this invention. Mouse alveolar epithelial cells MLE-12 were co-treated with TGFβ (10 ng / mL) and QX77 (2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours.

[0083] Figure 10 Figure D shows the effect of QX77 on COL1A1 in the lung epithelial cell transdifferentiation into fibrotic cell model, as determined by the Western blot experiment in Example 5 of this invention. Human alveolar epithelial cells Beas-2B were co-treated with TGFβ (10 ng / mL) and QX77 (2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours.

[0084] Figure 11 This is a graph showing the effect of QX77 analogues AR7 and CA77.1 on COL1A1 in a lung epithelial cell transdifferentiation into fibrotic cell model, as determined by Western blotting in Example 5 of this invention. A549 cells were co-treated with TGFβ (10 ng / mL) and AR7 (5 μM, 10 μM) or CA77.1 (5 μM, 10 μM) for 36 hours.

[0085] Figure 12 To measure the transwell migration of lung epithelial cells by QX77 as determined in Example 5 of this invention, Beas-2B and MLE-12 cells were co-treated with TGFβ (10 ng / mL) and QX77 (10 μM) for 24 hours. Hematoxylin staining was performed, and the number of migrating cells was photographed under a microscope and quantitatively counted.

[0086] Figure 13 Figure A shows the Nur77-dependent effect of QX77 on COL1A1 in the lung epithelial cell transdifferentiation into fibrotic cell model, as determined by Western blotting in Example 5 of this invention. In Beas-2B cells, Nur77 expression was interfered with by co-treatment with TGFβ (10 ng / mL) and QX77 (5 μM) for 36 hours, and the collagen content of stellate cells was detected by Western blotting.

[0087] Figure 13 Figure B shows the Nur77-dependent effect of QX77 on COL1A1 in the lung epithelial cell transdifferentiation into fibrotic cells model, as determined by Western blotting in Example 5 of this invention. In A549 cells, Nur77 expression was interfered with by co-treatment with TGFβ (10 ng / mL) and QX77 (5 μM) for 36 hours, and the collagen content of stellate cells was detected by Western blotting.

[0088] Figure 14This figure shows the results of QX77's inhibitory effect on pulmonary fibrosis in the mouse bleomycin pulmonary fibrosis model in Example 6 of this invention. Male C57BL / 6 mice were intrabronchial infusion of bleomycin (2 mg / kg), and after 7 days, they were randomly divided into two groups: a solvent group and a drug group. The drug group received QX77 (20 mg / kg dissolved in corn oil) daily by gavage, while the solvent group received corn oil daily by gavage. Samples were collected after 14 days of treatment. Mouse body weight was recorded daily (A), lung tissue was weighed upon collection, and the lung-to-body ratio was statistically analyzed (B). The collagen content of the lung tissue was detected using a hydroxyproline assay kit (C).

[0089] Figure 15 This is a graph showing the results of HE and Sirius red staining of a mouse bleomycin-induced pulmonary fibrosis model in Example 6 of this invention, indicating the effect of QX77 on improving lung tissue and reducing collagen.

[0090] Figure 16 This figure shows the results of qPCR assays in Example 6 of the present invention, which determined the inhibitory effect of QX77 on fibrosis in a mouse bleomycin-induced pulmonary fibrosis model. Liver tissue was lysed and mRNA was extracted for RT-PCR detection of the mRNA levels of liver fibrosis-related markers COL1A1, COL1A31, SPP1, CTGF, and Timp1. β-actin was used as an internal reference gene and normalized with the control. * p<0.05, ** p<0.01, **** p<0.01, n=6.

[0091] Figure 17 This image shows the results of qPCR assays in Example 6 of this invention, which determined the regenerative effect of QX77 on alveolar epithelial cell damage in a mouse bleomycin-induced pulmonary fibrosis model. Liver tissue was lysed and mRNA was extracted for RT-PCR detection of AT1 markers (Ager, Hpox, caveolin1) and the important differentiation-regulating transcription factor Cebpa. β-actin was used as an internal reference gene and normalized with controls. ns showed no statistical significance. * p<0.05, ** p<0.01, n=6.

[0092] Figure 18This image shows the results of microscopic observation of the effects of QX77 on cell morphology, lipid droplets, and the fibrosis marker α-SMA in rat fibroblasts (CFSCs) in Example 7 of this invention. CFSC cells were co-treated with TGFβ (5 ng / mL) and QX77 (10 μM) for 24 hours. Images were taken under a regular microscope or after Oil Red O staining and microscopic imaging. The intensity of Oil Red O staining was statistically analyzed using ImageJ software, and the fluorescence intensity of α-SMA was detected using immunofluorescence staining. *p<0.05, **p<0.01 *** p < 0.001, n = 3.

[0093] Figure 19 Figure A shows the degradation effects of QX77 on α-SMA and COL1A1 proteins in a rat hepatic stellate cell (CFSC) fibrosis model, as determined by Western blotting, in Example 7 of this invention. Rat hepatic stellate cells (CFSCs) were co-treated with TGFβ (5 ng / mL) and different concentrations of QX77 for 24 hours.

[0094] Figure 19 Figure B shows the results of the immunoblotting experiment in Example 7 of this invention, which determined the degradation effect of QX77 on COL1A1 protein in a human hepatic stellate cell (LX2) fibrosis model. Human hepatic stellate cells (LX2) were co-treated with TGFβ (5 ng / mL) and different concentrations of QX77 for 24 hours.

[0095] Figure 20 This figure shows the results of qRT-PCR assays in Example 7 of this invention, which showed the downregulation of COL1A1 and α-SMAm RNA levels in a fibrotic cell model by QX77. CFSC cells were co-treated with TGFβ (5 ng / mL) and QX77 (1 μM, 5 μM, 10 μM, 20 μM) for 24 hours. qRT-PCR was used to detect the mRNA expression levels of stellate cell activation markers. β-actin was used as an internal reference gene and normalized with controls. ** p<0.01, *** p<0.001, **** p < 0.0001, n = 3.

[0096] Figure 21 Figure A shows the effect of QX77 on liver morphology and color as measured in the CCl4 liver fibrosis model of Example 8 of this invention. C57BL / 6 mice were intraperitoneally injected with CCl4 (2 mL / kg, dissolved in corn oil) and the corresponding solvent three times a week for five weeks. In the third week after modeling, the mice were injected intraperitoneally and simultaneously administered the solvent or QX77 (10 mg / kg) by gavage for two weeks. In the fifth week, the mice were collected, and the appearance of the isolated liver was photographed at the time of collection.

[0097] Figure 21Figure B shows the effect of QX77 on liver function as measured by the CCl4 liver fibrosis biochemical detection kit in Example 8 of this invention. After enucleation of the above mice, blood was collected by centrifugation, and serum levels of ALT and AST in the serum were detected. * P<0.05, ** P<0.01, *** P<0.001.

[0098] Figure 22 Figure A shows the results of QX77 inhibiting liver fibrosis in the mouse CCl4 liver fibrosis model of Example 8 of this invention. For the above mice, liver tissue was collected, paraffin sections were stained with Sirius red, and positive areas were quantitatively analyzed using ImageJ and plotted using Graphad. *** P<0.001, **** P<0.0001.

[0099] Figure 22 Figure B shows the results of immunoblotting analysis of QX77, a liver fibrosis marker used in the mouse CCl4 liver fibrosis model of Example 8 of this invention, to determine its effect on inhibiting fibrosis. The expression levels of COL1A1 and α-SMA were detected by immunoblotting after liver tissue lysis and protein extraction.

[0100] Figure 23 This figure shows the results of qPCR assays in Example 8 of this invention, which determined the effect of QX77 on liver fibrosis markers that inhibit fibrosis in a mouse CCl4 liver fibrosis model. Liver tissue was lysed and mRNA was extracted for RT-PCR to detect the mRNA levels of liver fibrosis-related markers TGFβ, α-SMA, COL1A1, and TIMP1. β-actin was used as an internal reference gene and normalized with controls. **p<0.01, ***p<0.001, n=6. Detailed Implementation

[0101] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0102] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0103] Detailed description of the invention

[0104] Definitions and General Terms

[0105] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0106] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0107] In this document, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutically acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0108] In this document, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that retains the bioavailability of the free acid without other side effects. In addition to pharmaceutically acceptable salts, other salts are also contemplated in this invention. They may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts, or may be used for the identification, characterization, or purification of the compounds of this invention.

[0109] In this document, the term "amine salt" refers to the product obtained by neutralizing an alkyl primary amine, secondary amine, or tertiary amine with an acid. The acid includes the inorganic or organic acids described in this application.

[0110] In this paper, the term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, non-corresponding isomers, and conformational isomers.

[0111] Depending on the choice of raw materials and methods, the compounds of the present invention can exist as one or a mixture of possible isomers, for example as purely optical isomers, or as mixtures of isomers, such as racemic and diastereomeric mixtures, depending on the number of asymmetric carbon atoms. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to the chiral centers (or multiple chiral centers) in the molecule. The prefixes D and L or (+) and (-) are symbols used to specify the plane-polarized rotation induced by the compound, where (t) or L indicates that the compound is levorotatory. Compounds with the prefix (+) or D are dextrorotatory.

[0112] When the bonds of the chiral carbon in the formulas of this invention are depicted as straight lines, it should be understood that both the (R) and (S) configurations of the chiral carbon and the resulting enantiomerically pure compounds and mixtures thereof are included within the scope of the general formula. The illustration of racemic or enantiomerically pure compounds in this document is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. The absolute configuration of a stereocenter is represented by wedge-shaped and dashed bonds.

[0113] In this document, the term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom between two positions within a molecule. The compounds of this invention can exhibit tautomerism. Tautomers can exist in two or more interconvertible forms. Proton-transfer tautomers arise from the migration of covalently bonded hydrogen atoms between two atoms. Tautomers generally exist in equilibrium form, and attempts to isolate a single tautomer typically yield a mixture whose physicochemical properties are consistent with those of the mixture of compounds. The equilibrium position depends on the intramolecular chemical characteristics. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form is dominant; while in phenols, the enol form is dominant. This invention encompasses all tautomeric forms of the compounds.

[0114] In this document, the term "solvent" refers to a compound of the present invention or a salt thereof comprising a stoichiometric or nonstoichiometric solvent bound by intermolecular noncovalent forces, and a hydrate when the solvent is water.

[0115] In this document, the term "treatment" refers to the use of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with it; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any use of a drug to treat, cure, alleviate, improve, reduce, or suppress a disease in an individual, including but not limited to the administration of the drugs described herein to individuals in need.

[0116] In this article, the terms "cancer" or "tumor" can refer to any unregulated cell growth. Examples include colon cancer, rectal cancer, kidney cancer, skin cancer (especially cutaneous T-cell lymphoma), and so on.

[0117] In this paper, the term "regulation" includes positive regulation (e.g., maintenance, upregulation, or activation) and negative regulation (e.g., downregulation or inhibition).

[0118] Detailed description of the use of QX77, AR7, and CA77.1 in activating Nur77 activity according to the present invention

[0119] This invention proposes the use of QX77, AR7, and CA77.1 in the prevention and / or treatment of Nur77-related diseases, the use of QX77, AR7, and CA77.1 in activating Nur77 activity, a method for activating Nur77 activity, a combination drug or kit and its use, and a method for the prevention and / or treatment of Nur77-related diseases, which will be described in detail below.

[0120] use

[0121] In a first aspect, the present invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of Nur77-related diseases;

[0122]

[0123] In this document, the term "Nur77-related disease" should be interpreted broadly, including Nur77-mediated related diseases as well as non-Nur77-mediated related diseases that are affected by Nur77 activity (e.g., diseases that can be treated by targeting Nur77 and activating Nur77 activity). The specific type of disease is not limited, and any disease related to Nur77 is within the scope of protection of this application.

[0124] In some embodiments of this application, QX77, AR7, and CA77.1 regulate the physiological functions of the corresponding nuclear receptors by modulating Nur77 (which has activities such as dimer transcription), thereby enabling their use in the treatment of related diseases.

[0125] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor-1-related diseases.

[0126] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0127] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0128] According to embodiments of the present invention, the dosage of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts is about 1.0 mg to 100.0 mg, for example 1.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, 25.0 mg, 30.0 mg, 35.0 mg, 40.0 mg, 45.0 mg, 50.0 mg, 55.0 mg, 60.0 mg, 65.0 mg, 70.0 mg, 75.0 mg, 80.0 mg, 85.0 mg, 90.0 mg, 95.0 mg, 100.0 mg, or any two values ​​therebetween as endpoints, exemplarily 20.0 mg to 100.0 mg.

[0129] In this document, the term "about" or "approximately" refers to an acceptable error for a particular value, as determined by those skilled in the art, which depends in part on how the value is measured or determined. In some embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In some embodiments, the term "about" or "approximately" refers to within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

[0130] In this document, the dosage of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates or pharmaceutically acceptable salts is approximately 20.0 mg to 100.0 mg. The dosing frequency can be adjusted according to actual needs, including but not limited to once a week, once every two days, once a day, twice a day (i.e., BID), three times a day, etc. The dosage is set based on the dosage of a person of normal weight and can be adjusted according to the weight and condition of different people. All of these are within the protection scope of this invention.

[0131] In a second aspect, the present invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in the prevention and / or treatment of Nur77-related diseases;

[0132]

[0133] According to embodiments of the present invention, the compounds represented by formula (I), formula (II) or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity, thereby effectively preventing and / or treating Nur77-related diseases.

[0134] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor-1-related diseases.

[0135] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0136] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0137] According to embodiments of the present invention, the dosage of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates or pharmaceutically acceptable salts is about 1.0 mg to 100.0 mg, exemplarily 20.0 mg to 100.0 mg.

[0138] In a third aspect of the invention, the invention provides a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof for the prevention and / or treatment of Nur77-related diseases;

[0139]

[0140] According to embodiments of the present invention, the compounds represented by formula (I), formula (II) or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity, thereby effectively preventing and / or treating Nur77-related diseases.

[0141] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor-1-related diseases.

[0142] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0143] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0144] According to embodiments of the present invention, the dosage of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates or pharmaceutically acceptable salts is about 1.0 mg to 100.0 mg, exemplarily 20.0 mg to 100.0 mg.

[0145] In a fourth aspect of the invention, the invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in the preparation of a reagent for modulating the activity of Nur77 or modulating the activity of a dimer containing Nur77.

[0146]

[0147] According to embodiments of the present invention, the compounds represented by formula (I), formula (II), or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity. Therefore, reagents prepared using the compounds represented by formula (I), formula (II), or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can effectively modulate Nur77 activity or the activity of Nur77-containing dimers, and are particularly suitable for in vitro modulation of the activity of Nur77 or Nur77-containing dimers.

[0148] In an optional embodiment of the present invention, activating Nur77 activity includes, but is not limited to, activating Nur77 transcriptional activity, activating or inhibiting Nur77 binding activity with DNA and RNA, activating or inhibiting Nur77 binding activity with proteins, activating or inhibiting Nur77 binding activity with lipids, and activating or inhibiting Nur77 localization activity in cells.

[0149] According to an embodiment of the present invention, the dimer containing Nur77 includes a dimer containing Nur1 and a dimer containing Nor1.

[0150] According to an embodiment of the present invention, the dimer containing Nur77 includes Nur77 homodimer and heterodimer containing Nur77.

[0151] According to an embodiment of the present invention, the heterodimer containing Nur77 is selected from RXR / Nur77, RXR / Nurr1, and RXR / Nor1.

[0152] According to embodiments of the present invention, the final concentration of the compound represented by formula (I), formula (II), or formula (III), or its tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts thereof, is approximately 0.1 μM to 50.0 μM, for example 0.1 μM, 0.5 μM, 1 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM, 5.5 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10 μM, etc. 0.0 μM, 11.0 μM, 12.0 μM, 13.0 μM, 14.0 μM, 15.0 μM, 16.0 μM, 17.0 μM, 18.0 μM, 19.0 μM, 20.0 μM, 21.0 μM, 22.0 μM, 23.0 μM, 24.0 μM, 25.0 μM, 30.0 μM, 35.0 μM, 40.0 μM, 45.0 μM, 50.0 μM, or a range of any two values ​​between them, for example, 2.0 μM to 50.0 μM.

[0153] In this document, the term "final concentration used" refers to the final concentration added to achieve the purpose of modulating Nur77 activity or modulating the activity of Nur77-containing dimers. For example, when culturing cells in vitro, this final concentration used is the final concentration in the cell culture medium.

[0154] According to embodiments of the present invention, the final concentration of the compound represented by formula (I), formula (II) or formula (III) or its tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt is approximately 2.5 μM to 20.0 μM.

[0155] In a fifth aspect of the invention, the invention provides the use of a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate or pharmaceutically acceptable salt thereof in modulating the activity of Nur77 or modulating the activity of a dimer containing Nur77.

[0156]

[0157] According to embodiments of the present invention, the compounds represented by formula (I), formula (II) or formula (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77, activate Nur77 activity, and modulate the activity of dimers containing Nur77.

[0158] According to an embodiment of the present invention, the dimer containing Nur77 includes a dimer containing Nur1 and a dimer containing Nor1.

[0159] According to an embodiment of the present invention, the dimer containing Nur77 includes Nur77 homodimer and heterodimer containing Nur77.

[0160] According to an embodiment of the present invention, the heterodimer containing Nur77 is selected from RXR / Nur77, RXR / Nurr1, and RXR / Nor1.

[0161] In an optional embodiment of the present invention, when used to regulate Nur77 activity in vitro or to regulate the activity of a dimer containing Nur77, the final concentration of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts thereof is approximately 0.1 μM to 50.0 μM.

[0162] In an optional embodiment of the present invention, when used to regulate Nur77 activity in vitro or to regulate the activity of Nur77-containing dimers, the final concentration of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts thereof is about 2.5 μM to 20.0 μM.

[0163] Methods for regulating Nur77 activity or regulating the activity of Nur77-containing dimers

[0164] In a sixth aspect, the present invention provides a method for regulating Nur77 activity or regulating the activity of a Nur77-containing dimer. According to an embodiment of the present invention, the method comprises: contacting a compound of formula (I), formula (II), or formula (III), or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, with cells expressing Nur77 or a Nur77-containing dimer;

[0165]

[0166] According to embodiments of the present invention, the compound represented by formula (I), formula (II) or formula (III) above, or its tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt, can target Nur77, activate Nur77 activity, and also activate the activity of the dimer containing Nur77.

[0167] According to an embodiment of the present invention, the dimer containing Nur77 includes a dimer containing Nur1 and a dimer containing Nor1.

[0168] According to an embodiment of the present invention, the dimer containing Nur77 includes Nur77 homodimer and heterodimer containing Nur77.

[0169] According to an embodiment of the present invention, the heterodimer containing Nur77 is selected from RXR / Nur77, RXR / Nurr1, and RXR / Nor1.

[0170] According to embodiments of the present invention, when used to regulate the activity of Nur77 in vitro or to regulate the activity of dimers containing Nur77, the final concentration of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts is approximately 0.1 μM to 50.0 μM.

[0171] According to embodiments of the present invention, when used for in vitro regulation of Nur77 activity or regulation of Nur77-containing dimer activity, the final concentration of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts is approximately 2.5 μM to 20.0 μM.

[0172] Combined drugs or pillboxes and their uses

[0173] In a seventh aspect, the present invention provides a combination drug or cassette. According to an embodiment of the present invention, the combination drug or cassette comprises: a compound of formula (I), formula (II) or formula (III) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof as an active ingredient;

[0174]

[0175] According to an embodiment of the present invention, ligand L serves as a second active ingredient, said ligand L being used to modulate the nuclear receptor activity that forms a dimer with Nur77.

[0176] According to embodiments of the present invention, the compounds represented by formula (I), (II), or (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, can target Nur77 and activate Nur77 activity. Therefore, reagents prepared using the compounds represented by formula (I), (II), or (III) above, or their tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts, in combination with RXR ligand L can effectively modulate Nur77 activity or the activity of Nur77-containing dimers, and can effectively prevent and / or treat Nur77-related diseases.

[0177] According to an embodiment of the present invention, Nur77 includes Nurr1 and Nor1.

[0178] According to an embodiment of the present invention, the ligand L is selected from ligands of RXR.

[0179] According to an embodiment of the present invention, the ligand L is selected from at least one of bexarotine, LGD1069, ALRT1058, UAB30, NET-4IB, and LG100268.

[0180] In an eighth aspect of the invention, the invention provides the use of the combined drug or cassette described in the seventh aspect in the preparation of a medicament for the prevention and / or treatment of Nur77-related diseases. As is known from the foregoing, the use of the aforementioned combined drug or cassette can effectively modulate Nur77 activity or the activity of Nur77-containing dimers, thereby effectively preventing and / or treating Nur77-related diseases.

[0181] According to embodiments of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor1-related diseases.

[0182] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0183] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0184] Methods for preventing and / or treating Nur77-related diseases

[0185] In a ninth aspect of the invention, a method for preventing and / or treating Nur77-related diseases is provided. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable dose of a compound of formula (I), formula (II), or formula (III) or a tautomer, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, or the combination drug or cassette described in the seventh aspect;

[0186]

[0187]

[0188] According to an embodiment of the present invention, the Nur77-related diseases include Nurr1-related diseases and Nor1-related diseases.

[0189] According to embodiments of the present invention, the Nur77-related diseases include fibrotic diseases, tumors or cancers, and lung injury-related diseases.

[0190] According to embodiments of the present invention, the Nur77-related diseases include at least one of the following: liver fibrosis, cirrhosis, liver cancer, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, pulmonary embolism, emphysema, pulmonary infectious diseases, renal fibrosis, myocardial fibrosis, skin fibrosis, systemic sclerosis, kidney tumors, breast cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, gastric cancer, prostate cancer, esophageal cancer, skin cancer, leukemia, lymphoma, sarcoma, glioma, and melanoma.

[0191] According to embodiments of the present invention, the dosage of the compound represented by formula (I), formula (II) or formula (III) or its tautomers, stereoisomers, solvates or pharmaceutically acceptable salts is about 1.0 mg to 100.0 mg, exemplarily 20.0 mg to 100.0 mg.

[0192] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0193] The pBIND-Nur77 / LBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-Nur77 / LBD fusion protein.

[0194] The pBIND-Nur77 plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-Nur77 fusion protein.

[0195] The pBIND-RXRαLBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-RXRα / LBD fusion protein.

[0196] The pBIND-PPARαLBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-PPARα / LBD fusion protein.

[0197] The pBIND-GRαLBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-GRα / LBD fusion protein.

[0198] The pBIND-ERαLBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-ERα / LBD fusion protein.

[0199] The pBIND-LXRαLBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-LXRα / LBD fusion protein.

[0200] The pGL6-TA-NBRE-luciferase plasmid mentioned in this article refers to a reporter gene plasmid that responds to Nur77 or Nurr1, Nor1 monomers and is used to detect the transcriptional activity of Nur77, Nurr1 or Nor1 monomers. NBRE refers to the DNA binding element of Nur77, Nurr1 or Nor1 monomers.

[0201] The pGL6-TA-NurRE-luciferase plasmid mentioned in this article refers to a reporter gene plasmid that responds to Nur77 / Nur77 homodimers or Nur77 / Nurr1 and Nur77 / Nor1 heterodimers, and is used to detect the transcriptional activity of Nur77 / Nur77 homodimers, where NurRE refers to the DNA binding element of the Nur77 dimer.

[0202] The renilla-luciferase plasmid mentioned in this article refers to the sea cucumber luciferase plasmid, which is used as an internal control in reporter gene experiments.

[0203] The Gene IDs of the nucleotide sequences in the examples described in this article are shown in the table below:

[0204] name Gene ID name Gene ID Nur77 3164 PPARα 5465 Nurr1 4929 GRα 2908 Nor1 8013 RXRα 6256 ERα 2099 LXRα 10062

[0205] The amino acid or nucleotide sequences in the embodiments described herein are shown in the table below:

[0206]

[0207]

[0208] Note: N in the table above represents a random nucleotide, which can be A, C, G, or T.

[0209] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0210] The materials and methods used in the following embodiments are as follows:

[0211] 1. Experimental Materials

[0212] 1.1 Cell lines

[0213] The human kidney epithelial cell line HEK293T and human lung fibroblast WI-38 were obtained from the American Type Culture Collection (ATCC) in the United States. The rat hepatic stellate cells CFSC, human bronchial epithelial cells Beas-2B, and human hepatic stellate cells LX2 were obtained from the Chinese Academy of Sciences Cell Bank.

[0214] 1.2 Compounds QX77, AR7 and CA77.1 were purchased from MedChemExpress.

[0215] 1.3 Plasmids

[0216] The pCMV-myc-Nur77 plasmid, pBIND-Nur77LBD plasmid, pBIND-ERαLBD plasmid, pBIND-LXRα plasmid, pBIND-PPARαLBD plasmid, pBIND-GRαLBD plasmid, pGL6-TA-NBRE-luciferase reporter gene plasmid, and pGL6-TA-NurRE-luciferase reporter gene plasmid were all constructed and preserved in our laboratory.

[0217] 1.4 Laboratory Animals

[0218] The animals used in this study were male C57BL / 6 mice, 6-8 weeks old, SPF grade, purchased from Hangzhou Medical College and housed in the SPF environmental laboratory of the Experimental Animal Center of Xiamen University. All animal experimental procedures complied with the "National Regulations on the Management of Experimental Animals" and the "Xiamen University Regulations on the Management of Experimental Animals (Trial Implementation)" and other relevant regulations and management systems.

[0219] The 1.5qRT-PCR primer sequences are shown in the table below.

[0220]

[0221]

[0222] 2. Data Statistics and Analysis

[0223] All data in this paper are expressed as mean ± standard error (SEM). GraphPad Prism software was used for data processing, analysis, and visualization. Statistical analysis was performed using t-tests or one-way ANOVA. p < 0.05 (*) was considered statistically significant, p < 0.01 (**) was highly significant, p < 0.001 (***) and p < 0.0001 (****) were extremely significant, and ns indicated no significance.

[0224] Example 1: Effects of QX77 and its structural analogues AR7 and CA77.1 on Nur77 transcriptional activity

[0225] 1. Effects of yeast two-hybrid assay and reporter gene assay on the transcriptional activity of QX77 and its structural analogs AR7 and CA77.1 on Nur77.

[0226] (1) Effect of yeast two-hybrid assay on transcriptional activity of Nur77

[0227] HEK 293T cells were transfected with pG5-luciferase and pBIND-Nur77-LBD or pBIND-Nur77 plasmids for 24 hours, followed by treatment with DMSO (as a negative control) and QX77 (7.8125 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) for 24 hours. Reporter gene activity was measured using a firefly and Renidae dual-luciferase system, and EC2 activation of Nur77 activity was fitted using GraphPad. 50 Values, results are shown below Figure 1 A and 1B.

[0228] The results showed that QX77 significantly activated the transcriptional activity of Gal4 / DBD-Nur77 / LBD and Gal4 / DBD-Nur77.

[0229] (2) Effects of AR7 and CA77.1 on Nur77 transcriptional activity determined by yeast two-hybrid assay

[0230] HEK293T cells were co-transfected with pG5-luciferase and pBIND-Nur77-LBD or pBIND-Nur77 plasmid for 24 hours, followed by treatment with DMSO (as a negative control), AR7, or CA77.1 (7.8125 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) for 24 hours. Reporter gene activity was measured using a firefly and kidney dual-luciferase system, and EC2 activation of Nur77 activity was fitted using GraphPad. 50 Values, results are shown below Figure 2 A and 2B.

[0231] The results showed that AR7 and CA77.1 significantly activated the transcriptional activity of Gal4 / DBD-Nur77 / LBD.

[0232] (2) Reporter gene assay to determine the effect of QX77 and its analogues AR7 and CA77.1 on the transcriptional activity of Nur77 monomers.

[0233] HEK293T cells were co-transfected with pGL6-TA-NBRE-luciferase, renilla-luciferase, and pCMV-myc-Nur77 plasmids for 24 hours, followed by treatment with DMSO (as a negative control), 1.25 μM, 2.5 μM, 5 μM, and 10 μM QX77 for 24 hours. Reporter gene activity was measured using a firefly and Renalis dual-luciferase system. This example exemplifies the results of QX77 treatment; the results are shown in [link to results]. Figure 3 .

[0234] The results showed that the QX77 compound could significantly activate the transcriptional activity of Nur77 monomers.

[0235] (3) Reporter gene assay to determine the effect of QX77 and its analogues AR7 and CA77.1 on the transcriptional activity of Nur77 dimer.

[0236] HEK293T cells were co-transfected with pGL6-TA-NurRE-luciferase, renilla-luciferase, and pCMV-myc-Nur77 plasmids for 24 hours, followed by treatment with DMSO (as a negative control), 1.25 μM, 2.5 μM, 5 μM, and 10 μM QX77 for 24 hours. Reporter gene activity was measured using a firefly and Renalis dual-luciferase system. This example exemplifies the results of QX77 treatment; the results are shown in [link to results]. Figure 3 .

[0237] The results showed that all QX77 compounds significantly activated the transcriptional activity of Nur77 / Nur77 homodimers.

[0238] (4) Yeast two-hybrid assay to determine the effects of QX77 and its analogues AR7 and CA77.1 on the transcriptional activity of other nuclear receptors.

[0239] HEK 293T cells were transfected with pG5-luciferase and pBIND-Nur77LBD plasmid, or pBIND-PPARαLBD plasmid, or pBIND-GRαLBD plasmid, or pBIND-ERαLBD plasmid, or pBIND-LXRαLBD plasmid for 24 hours. Subsequently, the cells were treated for 24 hours with DMSO (as a negative control), QX77, AR7, and CA77.1 compounds (0.1 μM, 1 μM, 10 μM), or 0.1 μM CD3254, 10 μM MHY14643, 10 μM DXSN, 0.01 μM E2, and 2 μM T0901317 (as a positive control). Reporter gene activity was measured using a firefly and Renalis dual-luciferase system. This example exemplifies the results of QX77; the results are shown in [link to results]. Figure 3 .

[0240] The results showed that the QX77 compound could not activate Gal4 / DBD-Nur77 / LBD, Gal4 / DBD-PPARα / LBD, Gal4 / DBD-ERα / LBD, Gal4 / DBD-GRα / LBD, or Gal4 / DBD-LXRα / LBD.

[0241] Example 2: Experimental determination of the direct binding of QX77 compound and its analogues AR7 and CA77.1 to Nur77 protein.

[0242] (1) Surface plasmon resonance (SPR) was used to determine the binding of QX77 and its analogues AR7 and CA77.1 to Nur77-LBD protein.

[0243] The extracted and purified Nur77 / LBD protein was coupled to a CM5 chip compatible with Biacore 8K, with a response of over 10,000 protein units. Concentration gradients were prepared (see details). Figure 4 The QX77 solution, AR7 solution, and CA77.1 solution (see the annotation in the upper right corner) are passed sequentially through the chip, and the response values ​​are recorded. The data is analyzed using Biacore 8K analysis software, and graphs are generated using Graphpad software. This embodiment exemplarily demonstrates the results of QX77, as shown below. Figure 4 A.

[0244] The results showed that QX77 can bind to Nur77, and the Kd value of QX77 and Nur77 binding was 2.56 μM.

[0245] (2) Fluorescent titration was used to determine the binding of QX77 compound and its analogues AR7 and CA77.1 to Nur77 protein.

[0246] Using different concentrations (see details) Figure 4 Compounds QX77, AR7, CA77.1 (see note B to the right) and purified 2 μM Nur77 / LBD protein were incubated. The emission spectra of Nur77 / LBD protein excited by 280 nm excitation light in the 290-450 nm range were detected and plotted using Origin software. This example exemplifies the results for QX77. The results are as follows: Figure 4 B.

[0247] The results showed that the concentration gradient of the QX77 compound reduced the fluorescence intensity of the Nur77 / LBD protein, indicating that the QX77 compound can bind to Nur77.

[0248] (3) Cell thermal displacement assay to determine the binding of QX77 and its analogues AR7 and CA77.1 to Nur77 protein.

[0249] HeLa cells were treated with DMSO and QX77, AR7, and CA77.1, respectively. Lysates were collected and subjected to gradually increasing temperatures (see details). Figure 4 Cell lysis buffer was incubated at the temperature indicated in the first row (C). The supernatant from the lysate was then used for Western blotting to detect undenatured Nur77 protein. The obtained bands were quantitatively analyzed using ImageJ software, and the percentage of Nur77 protein at each temperature was calculated. A protein content change curve was plotted using Graphpad software. This example demonstrates the results for QX77. Figure 4 C.

[0250] The results showed that QX77 treatment significantly enhanced the thermal denaturation of Nur77 protein, indicating that QX77 may increase its thermal stability by binding to Nur77 protein.

[0251] Example 3: Effects of QX77 and its analogues AR7 and CA77.1 on a tumor cell invasion model

[0252] (1) QX77 and its analogues AR7 and CA77.1 inhibit the invasion of lung tumor cells A549.

[0253] A549 cells were seeded in 12-well plates. When the cell density reached approximately 90% confluence, a cross-shaped scratch was made in each well using a 10 μL pipette tip, applying even pressure to avoid uneven scratch width. The original culture medium was aspirated, and floating cells were washed with PBS. Immediately after replacing the culture medium, images were acquired under a microscope as the start of the experiment (T0), and the cells were marked. After treating the cells with TGFβ, QX77 (10 μM), AR7 (10 μM), or CA77.1 (10 μM) for 24 hours, images were acquired again (T24). The scratch area was calculated using ImageJ software. Cell migration rate = (T0 scratch area - T24 scratch area) / T0 scratch area * 100. Results are as follows: Figure 5 and 6 As shown, compared with the control, TGFβ significantly promoted the migration of lung cancer cells, while QX77 and its analogues AR7 and CA77.1 significantly inhibited TGFβ-induced lung cancer cell migration.

[0254] (2) QX77 and its analogues AR7 and CA77.1 compounds inhibit the invasion of MDA-MB-231 breast tumor cells.

[0255] MDA-MB-231 cells were seeded in 12-well plates. When the cell density reached 90% confluence, a cross-shaped scratch was made in each well using a 10 μL pipette tip. The original culture medium was aspirated, and floating cells were washed with PBS. Immediately after replacing the culture medium, images were acquired under a microscope as the start of the experiment (T0). Cells were then treated with TGFβ, QX77 (10 μM), AR7 (10 μM), or CA77.1 (10 μM) for 12 hours, and images were acquired again (T24). The scratch area was calculated using ImageJ software. Cell migration rate = (T0 scratch area - T24 scratch area) / T0 scratch area * 100. This example demonstrates the results for QX77; the results are shown in [link to image]. Figure 7 The results showed that, compared with the control, TGFβ significantly promoted the migration of breast cancer cells, while QX77 inhibited the migration of breast cancer cells induced by TGFβ.

[0256] (3) QX77 and its analogues AR7 and CA77.1 inhibited the invasion of mouse colon cancer cells MC-38.

[0257] MC38 cells were seeded in 12-well plates. Once the cell density reached 90% confluence, a cross-shaped scratch was made in each well using a 10 μL pipette tip. The existing culture medium was aspirated, and floating cells were washed with PBS. Immediately after replacing the culture medium, images were acquired under a microscope as the start of the experiment (T0). Cells were then treated with TGFβ, QX77 (10 μM), AR7 (10 μM), or CA77.1 (10 μM) for 12 hours, and images were acquired again (T24). The scratch area was calculated using ImageJ software. Cell migration rate = (T0 scratch area - T24 scratch area) / T0 scratch area * 100. This example demonstrates the results for QX77; the results are shown in [link to image]. Figure 7 The results showed that, compared with the control, TGFβ significantly promoted the migration of colon cancer cells, while QX77 inhibited TGFβ-induced colon cancer cell migration.

[0258] Example 4: Inhibitory effect of QX77 and its analogues AR7 and CA77.1 on mouse tumor models

[0259] Mouse colon cancer cell line MC38 was digested and resuspended in PBS; 100 μL / 500,000 cells were seeded into the posterior axilla of mice. The next day, mice were randomly assigned to groups and administered the appropriate solvent and QX77 (10 mg / kg), AR7 (10 mg / kg), or CA77.1 (10 mg / kg) by gavage. The mice were observed daily to monitor tumor growth. When the tumor reached approximately the size of a small mung bean, the maximum and minimum lengths of the tumor were measured every two days using calipers. The volume was calculated using the formula: V = 1 / 2 × (length × width × width). When the tumor reached 800 mm... 3 When the sample is large, it is photographed and weighed upon collection. This embodiment exemplarily demonstrates the results of QX77, as shown in the following example. Figure 8 .

[0260] The results showed that QX77 could significantly inhibit tumor growth.

[0261] Example 5: Effects of QX77 and its analogues AR7 and CA77.1 on a pulmonary fibrosis cell model

[0262] (1) Effects of QX77 on TGFβ-induced mesenchymal transition phenotype in lung epithelial cells

[0263] Epithelial-mesenchymal transition (EMT) is considered a key characteristic of pulmonary fibrosis, with its crucial pathophysiological change being the dysregulation that occurs during the repair process following epithelial injury. Under the induction of various fibrotic factors, airway epithelial cells transdifferentiate into mesenchymal cells (fibroblasts, myofibroblasts, etc.). The connections, adhesion structures, and polarity between epithelial cells disappear, replaced by increased synthesis of type I collagen and fibronectin, accompanied by abnormal organelle distribution and cellular framework reorganization. A549 lung cancer epithelial cells and Beas-2B human bronchial epithelial cells were treated with TGFβ (5 ng / mL) alone or co-treated with QX77 (10 μM) for 48 hours. Cell morphology was observed under a microscope, and the results were as follows: Figure 9 The results showed that TGFβ induced a transformation of cells from polygonal to spindle-shaped, and QX77 inhibited the TGFβ-induced cell morphology transformation.

[0264] (2) Effects of QX77 on TGFβ-induced activation of gene expression in lung fibroblasts

[0265] Human lung fibroblasts (WI-38 cells) were co-treated with TGFβ (10 ng / mL) and different concentrations (2.5 μM, 5 μM, 10 μM, 20 μM) of QX77 for 48 hours. The expression of COL1A1 and α-SMA proteins was detected by Western blotting. Results are shown in [Figure number missing]. Figure 10 The result is as follows Figure 10 A showed that QX77 could inhibit the expression of TGFβ-induced activated COL1A1 and α-SMA proteins in a concentration-dependent manner.

[0266] (3) Effect of QX77 on TGFβ-induced collagen expression in lung epithelial cells undergoing mesenchymal transition

[0267] Lung cancer epithelial cell line A549 was treated with TGFβ (5 ng / mL) and different concentrations (2.5 μM, 5 μM, 10 μM, 20 μM) of QX77 for 48 hours. Western blotting was used to detect the expression of COL1A1 protein. Results are shown below. Figure 10 B. The results showed that QX77 could inhibit the expression of TGFβ-induced activated COL1A1 protein.

[0268] Mouse alveolar epithelial cells (MLE-12) were co-treated with TGFβ (10 ng / mL) and QX77 (1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours. Western blotting was used to detect the expression of COL1A1 protein. Results are shown below. Figure 10 C. The results showed that QX77 could inhibit the expression of TGFβ-induced activated COL1A1 protein by concentration gradient.

[0269] Human bronchial epithelial cells (Beas-2B) were treated with TGFβ (10 ng / mL) and different concentrations (1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) of QX77 for 48 hours. Western blotting was used to detect the expression of COL1A1 protein. Results are shown below. Figure 10 D. The results showed that QX77 could inhibit the expression of TGFβ-induced activated COL1A1 protein by concentration gradient.

[0270] (4) AR7 and CA77.1 inhibit collagen expression during interstitial transition in lung epithelial cells.

[0271] Lung cancer epithelial cell line A549 was treated with TGFβ (5 ng / mL) and different concentrations (5 μM, 10 μM) of AR7 and CA77.1 for 48 hours. The expression of COL1A1 and p-Smad3 proteins was detected by Western blotting. Results are shown in [Figure number missing]. Figure 11 The results showed that AR7 and CA77.1 could inhibit the expression of TGFβ-induced activated COL1A1 protein and the phosphorylation of Smad3.

[0272] (5) QX77 inhibits cell migration during the transdifferentiation of lung epithelial cells.

[0273] MLE-12 and Beas-2B cells were seeded into 24-well plate inserts and co-treated with TGFβ (10 ng / mL) and QX77 (10 μM). After 24 hours of incubation, the cells were fixed in 4% polyformaldehyde for 20 minutes and stained with 0.1% crystal violet for 30 minutes. Three regions were randomly selected, and cells were counted using Image-J software. The results are shown in [link to Image-J software]. Figure 12 The results showed that QX77 inhibited the migration of lung epithelial cells.

[0274] (6) QX77 inhibits collagen expression in lung epithelial-mesenchymal transition in a Nur77-dependent manner.

[0275] In this embodiment, Nur77 expression was inhibited in Beas-2B and A549 cells using siRNA, and TGFβ was co-treated with QX77 (5 μM) for 36 hours. Western blotting was used to detect the expression of COL1A1, a marker of hepatic stellate activation in stellate cells. Results are shown below. Figure 13 The results showed that QX77 could inhibit the expression of TGFβ-induced activated COL1A1, while this effect disappeared in Nur77-interfered cells, indicating that the inhibition of COL1A1 expression by QX77 is Nur77-dependent.

[0276] Example 6: Inhibitory effects of QX77 and its analogues AR7 and CA77.1 on fibrosis and regenerative effects of lung epithelial cell damage in a mouse bleomycin-induced pulmonary fibrosis model.

[0277] Bleomycin (BLM) is a multi-component antibiotic composed of basic glycopeptides produced by *Streptomyces verticillata*. It has antitumor activity, but one of its toxic side effects is pulmonary fibrosis. BLM can induce DNA breaks, generate free radicals, induce oxidative stress, cause apoptosis or necrosis, and induce inflammatory responses and fibrosis. Eight-week-old SPF-grade male C57BL / 6 mice were purchased and administered bleomycin (2 mg / kg) intrabronchially. After 7 days, they were randomly divided into two groups: a solvent group and a treatment group. The drug (QX77, AR7, or CA77.1) was dissolved in corn oil at 20 mg / kg and administered by gavage daily. The solvent group received corn oil by gavage daily. Samples were collected after 14 days of treatment.

[0278] (1) During this modeling period, the body weight was measured daily, and graphs were plotted using GraphPad. Figure 14 A. This embodiment exemplifies the results of QX77. The results show that, compared with the control group, the bleomycin group significantly reduced the body weight of mice, and QX77 could improve the bleomycin-induced weight loss. Lung tissue was weighed at the time of sample collection, and the results are as follows... Figure 14 B showed that bleomycin induced a significant increase in lung-to-body ratio compared to the control group, while QX77 significantly reduced the lung-to-body ratio in mice. Lung tissue was collected, and the hydroxyproline content in the lung tissue was detected, with the following results: Figure 14 C showed that QX77 significantly reduced bleomycin-induced collagen deposition.

[0279] (2) Paraffin sections of mouse lung tissue were prepared and then stained with hematoxylin and eosin (HE) and Sirius red. This example demonstrates the results of QX77 staining. The results are shown in [link to results]. Figure 15 HE results showed that in the control group, the alveoli had normal morphology and no inflammatory cell infiltration in the interstitium. In contrast, the model group showed extensive consolidation of the lung parenchyma, significant thickening of the alveolar septa, disordered lung structure, and extensive consolidation of the lung parenchyma with abundant inflammatory cell infiltration. The QX77-treated group significantly reduced these pathological changes in the lung tissue. Sirius red staining results showed that most areas of the lung tissue in the control group were pale red with light staining, indicating normal lung structure and no obvious abnormalities. In contrast, the model group showed a significant increase in red staining areas and a significantly deeper staining depth, suggesting significant collagen fiber proliferation in the lung tissue. The QX77-treated group significantly increased the red area and depth, indicating that QX77 inhibited collagen deposition in the liver of the model group mice.

[0280] (3) QX77 and its analogues AR7 and CA77.1 compounds can improve the expression of fibrosis-related genes in bleomycin-induced mouse lung tissue.

[0281] Lung tissue was collected from the mice described above, and the expression levels of COL1A1, COL1A31, SPP1, CTGF, and Timp1 were detected by qRT-PCR. This example demonstrates the results for QX77, and the results are shown in [link to results]. Figure 16 .

[0282] The results showed that QX77 could inhibit the expression of bleomycin-induced pulmonary fibrosis markers COL1A1, COL1A31, and profibrotic factors SPP1, CTGF, and Timp1.

[0283] (4) QX77 and its analogues AR7 and CA77.1 compounds can repair and regenerate bleomycin-induced lung epithelial cell damage.

[0284] The differentiation of AT2 to AT1 cells from lung epithelial cells is a key mechanism in lung repair and regeneration, playing a crucial role in maintaining alveolar structure and repairing damage. AT2 cells (alveolar type II epithelial cells) are the stem cell population of alveolar epithelium. During differentiation, AT2 cells gradually lose their markers (such as SFTPC, proSPC, etc.) and upregulate markers of AT1 cells (such as Ager, Hpox, caveolin1, etc.). The TRβ ligand GC-1 promotes the differentiation of AT2 to AT1 cells by upregulating Cebpa, thereby promoting lung damage repair and regeneration.

[0285] The above-mentioned mouse tissues were used for qRT-PCR detection of AT1 cell markers (Ager, Hpox, caveolin1) and the important differentiation-regulating transcription factor Cebpa. This example demonstrates the results of QX77, and the results are shown in [link to results]. Figure 17 .

[0286] The results showed that bleomycin promoted damage to AT1 cells, while compound QX77 could promote the regeneration and repair of AT1 cells.

[0287] Example 7: Effects of compound QX77 and its analogues AR7 and CA77.1 on a liver fibrosis cell model.

[0288] (1) Effects of QX77 and its analogues AR7 and CA77.1 on TGFβ-induced activation phenotype of hepatic stellate cells

[0289] Hepatic stellate cells (CFSCs) are polygonal in their resting state, with cytoplasm containing lipid droplets rich in vitamin A. Upon activation, they transform into myofibroblasts, exhibiting a narrow, spindle-shaped cell morphology and a reduction or disappearance of lipid droplets. Rat hepatic stellate cells (CFSCs) were treated with TGFβ (5 ng / mL) alone or co-treated with QX77 (10 μM), AR7 (10 μM), or CA77.1 (10 μM) for 24 hours. Cell morphology was observed under a microscope or stained with Oil Red O. This example exemplifies the results obtained with QX77. Figure 18 The results showed that TGFβ induced a transformation of cells from polygonal to spindle-shaped and reduced cellular lipid droplets, while QX77 inhibited TGFβ-induced cell morphological changes and significantly restored intracellular lipid droplet content.

[0290] CFSC cells were co-treated with TGFβ (5 ng / mL) and QX77 (10 μM), AR7 (10 μM), or CA77.1 (10 μM) for 24 hours, respectively. Immunofluorescence was used to detect the fluorescence intensity of α-SMA, an indicator of stellate cell activation. The results showed that QX77 could inhibit the expression of α-SMA induced by TGFβ.

[0291] (2) Effects of QX77 and its analogues AR7 and CA77.1 on TGFβ-induced fibrosis markers in hepatic stellate cells

[0292] Rat hepatic stellate CFSC cells were co-treated with TGFβ (5 ng / mL) and different concentrations (1 μM, 5 μM, 10 μM, 20 μM) of QX77, AR7 (1 μM, 5 μM, 10 μM, 20 μM), and CA77.1 (1 μM, 5 μM, 10 μM, 20 μM) for 24 hours. The expression of COL1A1 and α-SMA proteins was detected by Western blotting and quantitatively analyzed using Imagej. This example exemplifies the results for QX77; the results are shown in [link to results]. Figure 19 A. The results showed that QX77 could inhibit the expression of TGFβ-induced activated COL1A1 and α-SMA proteins in a concentration-dependent manner.

[0293] Human hepatic stellate LX2 cells were co-treated with TGFβ (5 ng / mL) and different concentrations (1 μM, 5 μM, 10 μM, 20 μM) of QX77, AR7 (1 μM, 5 μM, 10 μM, 20 μM), and CA77.1 (1 μM, 5 μM, 10 μM, 20 μM) for 24 hours. Western blotting was used to detect COL1A1 protein expression. This example demonstrates the results for QX77; the results are shown below. Figure 19 B. The results showed that QX77 could inhibit the expression of TGFβ-induced activated COL1A1 protein by concentration gradient.

[0294] CFSC cells were co-treated with TGFβ (5 ng / mL) and QX77 (1 μM, 5 μM, 10 μM, 20 μM), AR7 (1 μM, 5 μM, 10 μM, 20 μM), and CA77.1 (1 μM, 5 μM, 10 μM, 20 μM) for 24 hours. The mRNA expression levels of stellate cell activation markers COL1A1 and α-SMA were detected by qRT-PCR. β-actin was used as an internal reference gene and normalized with controls. This example demonstrates the results for QX77; the results are shown in [link to results]. Figure 20 The results showed that QX77 could inhibit the expression levels of TGFβ-induced activated COL1A1 and α-SMA mRNA.

[0295] Example 8: Effects of QX77 and its analogues AR7 and CA77.1 on fibrosis-related indices in a mouse CCl4 liver fibrosis model.

[0296] A CCl4-induced liver fibrosis model was established by randomly dividing approximately 8-week-old SPF-grade C57BL / 6 male mice into a control group and a model group. CCl4 and corn oil were thoroughly mixed at a 1:3 ratio. During the modeling process, mice in the model group were intraperitoneally injected with a 25% CCl4-corn oil solution three times a week, while mice in the control group were intraperitoneally injected with corn oil at a dose of 2 mL / kg. The modeling period lasted five weeks. Three weeks after modeling, mice were intraperitoneally injected with corn oil or 25% CCl4-corn oil while simultaneously being administered a solvent or QX77 (10 mg / kg), AR7 (10 mg / kg), or CA77.1 (10 mg / kg) by gavage for two weeks. Samples were then collected from the mice.

[0297] (1) In this embodiment, mouse livers were collected and their appearance was observed. This embodiment exemplarily demonstrates the results of QX77, and the results are shown in [link to documentation]. Figure 21 A. As shown in the figure, the livers of the control group were reddish, smooth, and soft in texture; compared with the control group, the livers of mice in the CCl4 model group had obvious granular texture, reduced luster, and hardened texture, exhibiting obvious liver fibrosis phenotype. QX77 at a dose of 10 mg / kg significantly improved the above-mentioned liver symptoms.

[0298] (2) After blood was collected from mice using the CCl4 liver fibrosis model, serum was collected by centrifugation, and serum liver function indicators ALT and AST were detected. This embodiment exemplarily demonstrates the results of QX77, and the results are shown in [link to results]. Figure 21 B. As shown in the figure, QX77 can downregulate the levels of ALT and AST in the serum of mice in the model group, indicating that QX77 can improve CCl4-induced liver function damage.

[0299] (3) QX77 and its analogues AR7 and CA77.1 compounds can improve CCl4-induced liver tissue morphology and collagen deposition in mice.

[0300] In this embodiment, mouse livers were sectioned in paraffin and stained with Sirius red. The results are shown in the figure. Figure 22 A, the scale bar in the figure is 100 μm; the fiber area represented by Sirius red staining was statistically analyzed using ImageJ software. The results showed that, compared with the control group, the liver of the model group mice had significant collagen deposition. This example demonstrates the results of QX77, which can inhibit collagen deposition in the liver of the model group mice.

[0301] (4) QX77 and its analogues AR7 and CA77.1 compounds can improve the expression of fibrosis-related genes in CCl4-induced mouse liver tissue.

[0302] In this embodiment, liver tissue from mice with a CCl4 liver fibrosis model was used for immunoblotting to detect the protein expression levels of COL1A and α-SMA. The results are shown in [Figure number missing]. Figure 22 B. RNA was also extracted from liver tissue, and the transcriptional levels of α-SMA, COL1A1, TGFβ, and TIMP1 were detected using qRT-PCR. β-actin was used as an internal reference gene and normalized with a control. This example demonstrates the results of QX77. The results are shown in [link to results]. Figure 23 .

[0303] The results showed that QX77 could inhibit the expression of CCl4-induced liver fibrosis markers α-SMA, COL1A1, TGFβ and TIMP1.

[0304] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0305] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. Use of a compound of formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of Nur77-related diseases; (I) (II) (III); The diseases associated with Nur77 are at least one of lung cancer, breast cancer, and colon cancer.

2. Use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of Nur77-related diseases; (I) The disease associated with Nur77 is liver fibrosis.

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