Use of Compound 1 in the treatment or prevention of diseases such as renal fibrosis

Abstract

The present invention relates to the use of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide (also referred to as Compound 1 in the present invention) or a pharmaceutically acceptable salt thereof in the treatment or prevention of diseases such as renal fibrosis.

Description

[Technical Field]

[0001] The present invention relates to the use of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide (also referred to as Compound 1 in the present invention) or a pharmaceutically acceptable salt thereof in the treatment or prevention of diseases such as renal fibrosis. [Background technology]

[0002] Renal fibrosis is a common pathway and the primary pathological basis for the progression of various chronic kidney diseases (CKD) to end-stage renal disease (ESRD). The main phenotypes include glomerular sclerosis and renal interstitial fibrosis, which include renal tubular cell injury, inflammatory cell infiltration, myofibroblast activation, tubular atrophy, and microvascular rarefaction. First, fibroblasts and perivascular cells acquire a myofibroblastic phenotype by expressing smooth muscle actin (α-SMA) and become the primary matrix-producing cells. Fibroblast activation is accompanied by the infiltration of inflammatory cells, such as neutrophils, lymphocytes, and macrophages. Subsequently, changes occur in renal tubular epithelial cells, leading to the progression of tubular atrophy. Meanwhile, capillary endothelial cells undergo senescence and apoptosis, resulting in microvascular rarefaction. Renal tubulointerstitial fibrosis is the primary pathological basis of all renal diseases and one of the important markers of chronic kidney disease. The degree of fibrosis is closely related to the decline in renal function, and progression leads to complete renal dysfunction. Patients are often asymptomatic in the early stages, but renal fibrosis is usually diagnosed at the end-stage. At this stage, dialysis or kidney transplantation are the only options. Slowing or preventing the progression of renal fibrosis is key to preventing and treating chronic kidney disease. Currently, there is no substantial clinical application for effectively treating chronic kidney disease or other pathological changes. To date, there are no approved commercially available drugs for renal fibrosis, and there is a significant unmet clinical need.

[0003] The two commercially available TRK inhibitors (larotrectinib and entrectinib) are both intended for patients with tumors harboring NTRK gene mutations. There are no commercially available drugs for the treatment of renal fibrosis, and no clinical or preclinical trials have been conducted for this indication. There are no commercially available EPHA6 inhibitors, and no clinical or preclinical trials have been reported for this indication. While there are preclinical trials for DDR1 in atopic pulmonary fibrosis, no drugs have yet progressed to the clinical trial stage for pulmonary fibrosis. There are also no CSF1R inhibitors that have reached the clinical trial stage for fibrotic diseases.

[0004] The murine renal fibrosis model induced by unilateral ureteral obstruction (UUO) is currently the most widely used renal fibrosis model. Unilateral ureteral obstruction (UUO) induces renal tubulointerstitial fibrosis in C57BL / 6J mice. This model causes obstruction of the mouse renal drainage system, resulting in acute changes in renal function and chronic renal structural damage, mimicking the renal interstitial damage commonly seen in clinical settings due to ureteral obstruction. Persistently elevated urinary tract pressure, reduced renal blood flow, obstruction of venous drainage, macrophage infiltration, fibrocytic proliferation, and scar formation ultimately lead to renal tubulointerstitial fibrosis, renal tubular atrophy, and renal dysfunction. This model was used to test the therapeutic effects of compounds on renal fibrosis.

[0005] CN113831344B discloses N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide. Summary of the Invention

[0006] The present invention relates to the use of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide (also referred to herein as Compound 1) or a pharmaceutically acceptable salt thereof in the treatment of renal fibrosis. In the present invention, renal fibrosis includes tubular and tubulointerstitial fibrosis, focal segmental and global glomerular sclerosis. [ka] compound 1 In a first aspect, the present invention provides the use of Compound 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment or prevention of renal fibrosis. In a second aspect, the present invention provides a method for use in treating or preventing renal fibrosis, said method comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt thereof. In a third aspect, the present invention provides a pharmaceutical composition for use in treating or preventing renal fibrosis, the pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. In a fourth aspect, the present invention provides Compound 1, or a pharmaceutically acceptable salt thereof, for use in treating or preventing renal fibrosis.

[0007] The present invention further relates to the use of compound 1 or a pharmaceutically acceptable salt thereof in the treatment or prevention of diseases mediated by DDR1, DDR2, CSF1R and / or EPHA6 or diseases that can be controlled by inhibition of DDR1, DDR2, CSF1R and / or EPHA6.

[0008] In one embodiment, the present invention provides the use of Compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by DDR1 or a disease that can be controlled by inhibiting DDR1.

[0009] In another aspect, the present invention provides a method for treating or preventing a disease mediated by DDR1 or a disease that can be controlled by inhibiting DDR1, the method comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof.

[0010] In another aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease mediated by DDR1 or a disease controllable by inhibition of DDR1, the pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0011] In another aspect, the present invention provides Compound 1 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of a disease mediated by DDR1 or a disease that can be controlled by inhibition of DDR1.

[0012] In each of the above-mentioned aspects, the diseases mediated by DDR1 or that can be controlled by inhibition of DDR1 are fibrosis, tumors, immune disorders, skin and connective tissue diseases, musculoskeletal diseases, genitourinary diseases, pregnancy complications, cardiovascular diseases, and neurodegenerative diseases.

[0013] In one aspect, the present invention provides the use of Compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by DDR2 or a disease that can be controlled by inhibition of DDR2.

[0014] In another aspect, the present invention provides a method for treating or preventing a disease mediated by DDR2 or a disease controllable by inhibition of DDR2, the method comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof.

[0015] In another aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease mediated by DDR2 or a disease controllable by inhibition of DDR2, the pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0016] In another aspect, the present invention provides Compound 1, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease mediated by DDR2 or a disease controllable by inhibition of DDR2.

[0017] In each of the above-described aspects, the disease mediated by DDR2 or that can be controlled by inhibition of DDR2 is fibrosis, tumors, immune system disorders, inflammatory diseases, musculoskeletal diseases, skin and connective tissue diseases, gastrointestinal diseases, cardiovascular diseases, genitourinary diseases, pregnancy complications, and neurodegenerative diseases.

[0018] In one aspect, the present invention provides the use of Compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by CSF1R or a disease that can be controlled by inhibition of CSF1R.

[0019] In another aspect, the present invention provides a method for treating or preventing a disease mediated by CSF1R or a disease controllable by inhibition of CSF1R, the method comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof.

[0020] In another aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease mediated by CSF1R or a disease controllable by inhibition of CSF1R, the pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0021] In another aspect, the present invention provides Compound 1 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of a disease mediated by CSF1R or a disease that can be controlled by inhibition of CSF1R.

[0022] In each of the above-mentioned aspects, the disease mediated by CSF1R or that can be controlled by inhibition of CSF1R is fibrosis, tumors, immune system disorders, inflammatory diseases, and neurodegenerative diseases.

[0023] In one aspect, the present invention provides the use of Compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by EPHA6 or a disease that can be controlled by inhibition of EPHA6.

[0024] In another aspect, the present invention provides a method for treating or preventing a disease mediated by EPHA6 or a disease controllable by inhibition of EPHA6, the method comprising administering to a subject in need thereof a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof.

[0025] In another aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease mediated by EPHA6 or a disease controllable by inhibition of EPHA6, the pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0026] In another aspect, the present invention provides Compound 1 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of a disease mediated by EPHA6 or a disease controllable by inhibition of EPHA6.

[0027] In each of the above-mentioned aspects, the diseases mediated by EPHA6 or that can be controlled by inhibiting EPHA6 are kidney diseases, tumors, digestive system diseases, cardiovascular diseases, and psychiatric diseases. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows changes in mouse body weight. [Figure 2] FIG. 2 shows the results of H&E staining of kidney tissue. [Figure 3A] Figure 3A shows Masson staining of collagen fibers in kidney tissue. [Figure 3B] FIG. 3B shows the results of quantitative statistical analysis of Masson staining. [Figure 4A] FIG. 4A shows the results of immunohistochemical staining of kidney tissue with α-SMA. [Figure 4B] FIG. 4B shows the results of quantitative statistical analysis of α-SMA immunohistochemical staining. [Figure 5A] FIG. 5A shows the results of F4 / 80 immunohistochemical staining of kidney tissue. [Figure 5B] FIG. 5B shows the results of quantitative statistical analysis of F4 / 80 immunohistochemical staining. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is further illustrated, but not limited, by the examples of the invention described below. Example 1: Inhibitory effect of Compound 1 on the enzyme activities of TRK, DDR, EPHA6, and CSF1R HTRF measurement principle: The kinase phosphorylates the substrate, and an antibody labeled with Eu-CryPtate binds to the phosphorylated site in the substrate. Streptavidin-XL665 binds to the biotin on the substrate. When Eu and XL665 come into close proximity, the donor Eu emits light (620 nm) after excitation with a light source (320 nm). This energy resonance transfers to the nearby XL665 receptor, which then emits light (665 nm) after excitation. The specific signal is proportional to the amount of phosphorylated substrate. Addition of an inhibitor suppresses the phosphorylation level, resulting in no detection of 665 nm emission and only detection of 620 nm emission, allowing the level of kinase activity inhibition by the compound to be assessed.

[0030] TRKA enzyme activity assay: Compound 1 was gradient diluted with DMSO. Compound 1 was diluted 4-fold from an initial concentration of 1 μM, for a total of 10 dilutions, and the porous assay was performed. Using an Echo 665, 25 nL of compound was dispensed into a 384-well reaction plate (product number 784075, Greiner). A 2x kinase solution was prepared using 1x kinase reaction buffer (5x Buffer, 5 mM MgCl2, 1 mM DTT, HO, 1 mM MnCl2). 2.5 μL of TRKA (1.5 nM, 08-186, Carna) solution was dispensed into the 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2x biotin-labeled tyrosine kinase substrate (1 μM) (product number 61TK0BLE, Cisbio-PerkinElmer) and ATP (25 μM) were mixed in the kinase reaction buffer. The reaction was initiated by adding 2.5 μL of a mixed solution of substrate and ATP to the reaction plate and centrifuging at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. Five μL of a mixed solution of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a BMG microplate reader. The ratio of the fluorescence signals in each well, i.e., (665 nm / 620 nm) × 10,000, was calculated, and this ratio represented the degree of kinase activity.

[0031] TRKB enzyme activity assay: Compound 1 was gradient diluted using DMSO. Compound 1 was diluted 4-fold from an initial concentration of 1 μM, resulting in a total of 10 dilutions for the porous assay. Using an Echo 665, 25 nL of compound was dispensed into a 384-well reaction plate. A 2x kinase solution was prepared using 1x kinase reaction buffer (5x Buffer, 5 mM MgCl2, 1 mM DTT, HO, 1 mM MnCl2). 2.5 μL of TRKB (1.4 nM, 08-187, Carna) solution was dispensed into a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2x mixture of biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. The reaction was initiated by adding 2.5 μL of a mixed solution of substrate and ATP to the reaction plate and centrifuging at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. Five μL of a mixed solution of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a BMG microplate reader. The ratio of the fluorescence signals in each well, i.e., (665 nm / 620 nm) × 10,000, was calculated, and this ratio represented the degree of kinase activity.

[0032] TRKC enzyme activity assay: Compound 1 was gradient diluted using DMSO. Compound 1 was diluted 4-fold from an initial concentration of 1 μM, resulting in a total of 10 dilutions for the porous assay. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× Buffer, 5 mM MgCl2, 1 mM DTT, HO, 1 mM MnCl2). 2.5 μL of TRKC (1.5 nM, 08-197, Carna) solution was dispensed into a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using kinase reaction buffer. 2.5 μL of the substrate and ATP mixture was added to the reaction plate to initiate the reaction, followed by centrifugation at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. Five microliters of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1,000 rpm for 1 minute and then incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a BMG microplate reader. The ratio of the fluorescence signals in each well, i.e., (665 nm / 620 nm) x 10,000, was calculated, and this ratio represented the degree of kinase activity.

[0033] DDR1 enzyme activity measurement: Compound 1 was gradient diluted with DMSO. Compound 1 was diluted 4-fold from an initial concentration of 1 μM, for a total of 10 dilutions, for the porous assay. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× Buffer, 5 mM MgCl2, 1 mM DTT, HO, 1 mM MnCl2). 2.5 μL of DDR1 (2.7 nM, 08-113, Carna) solution was dispensed into a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using kinase reaction buffer. 2.5 μL of the substrate and ATP mixture was added to the reaction plate to initiate the reaction, followed by centrifugation at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. Five microliters of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1,000 rpm for 1 minute and then incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a BMG microplate reader. The ratio of the fluorescence signals in each well, i.e., (665 nm / 620 nm) x 10,000, was calculated, and this ratio represented the degree of kinase activity.

[0034] DDR2 enzyme activity measurement: Compound 1 was gradient diluted with DMSO. Compound 1 was diluted 4-fold from an initial concentration of 1 μM, for a total of 10 dilutions, for the porous assay. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× Buffer, 5 mM MgCl2, 1 mM DTT, HO, 1 mM MnCl2). 2.5 μL of DDR2 (1.3 nM, 08-114, Carna) solution was dispensed into a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using kinase reaction buffer. 2.5 μL of the substrate and ATP mixture was added to the reaction plate to initiate the reaction, followed by centrifugation at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. Five microliters of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1,000 rpm for 1 minute and then incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a BMG microplate reader. The ratio of the fluorescence signals in each well, i.e., (665 nm / 620 nm) x 10,000, was calculated, and this ratio represented the degree of kinase activity.

[0035] EPHA6 enzyme activity measurement: Compound 1 was gradient diluted with DMSO. Compound 1 was diluted 4-fold from an initial concentration of 50 μM, resulting in a total of 10 dilutions for the porous assay. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× Buffer, 5 mM MgCl2, 1 mM DTT, HO, 1 mM MnCl2). 2.5 μL of EPHA6 (0.5 nM, 08-125, Carna) solution was dispensed into a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. 2.5 μL of the substrate and ATP mixture was added to the reaction plate to initiate the reaction, followed by centrifugation at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1,000 rpm for 1 minute and then incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a BMG microplate reader. The ratio of the fluorescence signals in each well, i.e., (665 nm / 620 nm) × 10,000, was calculated, and this ratio represented the degree of kinase activity.

[0036] CSF1R enzyme activity measurement: Compound 1 was gradient diluted with DMSO. Compound 1 was diluted 4-fold from an initial concentration of 10 μM, for a total of 10 dilutions, for the porous assay. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× Buffer, 5 mM MgCl2, 1 mM DTT, HO, 1 mM MnCl2). 2.5 μL of CSF1R (1.3 nM, 08-114, Carna) solution was dispensed into a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. 2.5 μL of the substrate and ATP mixture was added to the reaction plate to initiate the reaction, and the plate was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1,000 rpm for 1 minute and then incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a BMG microplate reader. The ratio of the fluorescence signals in each well, i.e., (665 nm / 620 nm) × 10,000, was calculated, and this ratio represented the degree of kinase activity.

[0037] Table 1 shows the IC values ​​of Compound 1 for DDR1, DDR2, TRKA, TRKB, TRKC, EPHA6, and CSF1R obtained by the above-mentioned measurements. 50 Shows. Table 1. IC of Compound 1 against DDR1, DDR2, TRKA, TRKB, TRKC, EPHA6, and CSF1R 50 [Table 1]

[0038] Example 2: Evaluation of the therapeutic effect of Compound 1 on UUO-induced renal fibrosis in mice method This efficacy study was designed with 7 groups, each of which received the following treatments: Group 1: Sham group, sham operation group; Group 2: vehicle group; Group 3: Compound 1, 3 mg / kg QD (hereafter, mg / kg is abbreviated as mpk); Group 4: Compound 1,10mg / kg QD; Group 5: Compound 1,30mg / kg QD; Group 6: Compound 1,100mg / kg QD; Group 7: positive drug pirfenidone, 250 mg / kg BID (hereinafter pirfenidone is abbreviated as PFD). UUO model mice: C57 mice (male, 8 weeks old, weighing 20-25 g) were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. After anesthesia, a 1-2 cm incision was made in the left renal region of the abdomen, and the intestine was carefully removed to expose the ureter. The ureter was ligated with a double suture and cut midway between the ligated site. The intestine was then restored, and the incision was sutured. Sham group mice: After anesthetizing C57 mice, a 1-2 cm incision was made at the kidney region on the left side of the abdomen, the intestines were carefully removed, the ureters were exposed, and the intestines were restored without ligation, and the incision was sutured. All animals received oral administration on the day of surgery. The sham and vehicle groups received the test drug solvent (5% DMSO + 5% Tween 80 + 20% PEG 400 + 70% HO). All drug solutions were prepared immediately before use. The experiment was terminated 10 days after administration. Table 2. Pharmacodynamic grouping and administration regimens [Table 2]

[0039] Preparation of test compounds: Free base Compound 1 powder was weighed and added to 5% DMSO, followed by vortex mixing until the solution became clear. Next, 5% Tween-80 was added and vortex mixed. 20% PEG400 was added and vortex mixed. 70% purified water was added and vortex mixed before storage. Preparation of positive drug: Pirfenidone was weighed in a mortar, a small amount of 0.5% CMC-Na was added, and the mixture was thoroughly and homogenously ground. The remaining 0.5% CMC-Na was added, and the mixture was mixed thoroughly before use. At the end of the experiment, the left kidney was removed and fixed in 4% PFA. Kidneys were routinely sectioned and stained 24 hours after fixation.

[0040] H&E staining: Sections were deparaffinized and hydrated according to standard methods, then stained with hematoxylin for 20 minutes, separated with 1% HCl in ethanol, and treated with running water for 20 minutes to restore the blue color. Next, sections were treated with 70% ethanol, 80% ethanol, and 90% ethanol for 3 minutes each, and stained with eosin for 10 seconds. Sections were then treated with 95% ethanol and 100% ethanol for 10 minutes each, cleared with xylene, mounted in neutral balsam, and observed under a conventional light microscope.

[0041] Masson staining: Sections were deparaffinized and hydrated according to standard procedures, then stained with a nuclear stain for 1 minute and washed with a cleaning solution for 30 seconds. Next, they were stained with a cytoplasmic stain for 10 seconds and washed with a cleaning solution for 30 seconds. They were then differentiated in differentiation solution for 8 minutes. After discarding the differentiation solution, they were directly stained with a counterstain solution for 3 minutes. To remove unfixed dye, they were washed with 100% ethanol, cleared with xylene, mounted in neutral balsam, and observed under a conventional light microscope.

[0042] α-SMA immunohistochemical staining: Paraffin sections of kidney tissue were deparaffinized and hydrated, then heated with Tris-EDTA antigen retrieval solution. After removing endogenous hydrogen peroxide, sections were blocked at room temperature for 1 hour. The primary antibody α-SMA (1:500) was incubated at room temperature for 1 hour, followed by the secondary antibody for 1 hour. After developing the stain with DAB for 3-5 minutes, sections were stained with hematoxylin for 3 minutes, differentiated with differentiation solution for 30 seconds, and then blue-reverted with blue-reverting solution. The sections were dehydrated, cleared, and mounted in neutral balsam.

[0043] F4 / 80 immunohistochemical staining: Paraffin sections of kidney tissue were deparaffinized and hydrated, then heated with Tris-EDTA antigen retrieval solution. After removing endogenous hydrogen peroxide, sections were blocked at room temperature for 1 hour. The primary antibody F4 / 80 (1:1000) was incubated at room temperature for 1 hour, followed by the secondary antibody for 1 hour. After 3-5 minutes of DAB development, sections were stained with hematoxylin for 3 minutes, differentiated in differentiation solution for 30 seconds, and then blue-reverted with blue-reverting solution. The sections were then dehydrated, cleared, and mounted in neutral balsam.

[0044] Five to nine fields were randomly selected from each sample, and the area of ​​the Masson-stained positive region was analyzed using ImageJ. The mean value of the calculated values ​​for the fields selected from each sample was used as the final statistical data for that sample. Statistical analysis of the results was performed using GraphPad_Prism 8 software, and data analysis was performed using one-way ANOVA. A significant difference was defined as p<0.05.

[0045] result As shown in Figure 1 and Table 3, the relative changes in mouse body weight in each group were within the normal range. Table 3. Relative body weight changes in mice [Table 3] Relative weight change = weight on day n / weight on day 0 * 100%

[0046] H&E staining showed that mice in the sham group had healthy kidneys with clear tubular and glomerular structures. In the vehicle group, significant tubular atrophy, tubular flattening, glomerular contraction, and thickening of the glomerular mesangium were observed in the kidneys, and free staining material appeared within the tubules. Compared with the vehicle group, the compound 1 group showed reduced tubular atrophy, a more relaxed glomerular structure, and no significant thickening of the tubular basement membrane. Compared with the vehicle group, the pirfenidone group showed a more relaxed glomerular structure and no significant deposition of material in the tubular lumen (Figure 2).

[0047] Masson collagen fiber staining revealed minimal positive staining in the sham group, primarily in the tubular lumen and brush border of the proximal tubule. In the vehicle group, extensive positive staining was observed in the intertubular space, indicating tubulointerstitial collagen deposition, a typical pathological feature of tubulointerstitial fibrosis. Compound 1 significantly attenuated intertubular collagen deposition in the 3 mpk, 10 mpk, 30 mpk, and 100 mpk groups (Figure 3A). Quantitative analysis showed that compound 1 significantly reduced the Masson staining area at the 3 mpk dose, with a dose-dependent effect observed at different doses (Figure 3B). In particular, the 30 mpk QD dose achieved comparable efficacy to the 250 mpk BID dose of pirfenidone, demonstrating significant advantages in terms of dosage and frequency of administration.

[0048] Alpha-smooth muscle actin (α-SMA) is a marker of the extracellular matrix during fibrosis and serves as a direct indicator of the level of fibrosis. The expression level of α-SMA in the kidney after intervention in each group was evaluated by immunohistochemistry (Figure 4A). The results showed a significant increase in the histochemical area of ​​α-SMA in the vehicle group compared with the sham group (P<0.01), indicating a marked increase in fibrosis. Compared with the vehicle group, the compound-treated groups showed a significant decrease in the α-SMA-positive area at 3 mpk (P<0.05), and at 10 mpk, 30 mpk, and 100 mpk, with a dose-dependent decrease (Figure 4B).

[0049] Macrophages (especially M2 macrophages) are known to play an important role in the progression of tissue fibrosis and can serve as a reference for inflammatory responses. F4 / 80 is a marker for mature macrophages. Figures 5A and 5B show the results of immunohistochemical analysis of macrophage expression in renal tissue after various interventions. In the sham group, macrophage expression was rare in the kidney, with tissue-resident macrophages primarily localized in the tubulointerstitium. In the UUO model group (vehicle group), macrophage expression levels were significantly increased and accumulated around the damaged tubules. Compared to the vehicle group, macrophage expression levels were reduced in each compound-treated group, with the reduction being particularly pronounced in the 30mpk and 100mpk-treated groups.

[0050] conclusion In this experiment, collagen, fibrotic factors, and macrophages were significantly increased in the vehicle group, indicating successful creation of a mouse renal fibrosis model. Test compound 1 significantly reduced the production of collagen (Masson staining) and fibrotic factors (α-SMA) in the kidney and significantly suppressed macrophage expression levels (F4 / 80 staining), demonstrating effective suppression of renal fibrosis and inflammatory progression, and demonstrated a reciprocal dose-dependent effect. Test compound 1 clearly alleviated renal fibrosis at a dose of 3 mg / kg, which is considered to be an effective dose for the treatment of renal fibrosis. Test compound 1 has significant advantages over the active drug pirfenidone in terms of dosage and frequency of administration. NOTE: In Figures 3B, 4B and 5B, ns indicates no obvious difference, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0051] The foregoing examples and description of several embodiments should be considered illustrative, rather than limiting, the invention as defined by the claims. It will be readily understood that many variations and combinations of the features described above can be used without departing from the invention as set forth in the claims. All such variations are intended to be within the scope of the present invention. All cited references are incorporated herein by reference in their entirety.

Claims

1. 1. Use of Compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of renal fibrosis, wherein Compound 1 has the following structure: 【Chemistry 1】

2. 1. Use of compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by DDR1 or a disease controllable by inhibiting DDR1, wherein compound 1 has the following structure: 【Chemistry 2】

3. 1. Use of compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by DDR2 or a disease controllable by inhibiting DDR2, wherein compound 1 has the structure: 【Transformation 3】

4. Use of compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by EPHA6 or a disease that can be controlled by inhibiting EPHA6, wherein compound 1 has the following structure: 【Chemistry 4】

5. 1. Use of compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of a disease mediated by CSF1R or a disease that can be controlled by inhibiting CSF1R, wherein compound 1 has the following structure: 【Transformation 5】

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