A compound 5n and its application in the preparation of drugs for treating acute kidney injury

Abstract

The present invention belongs to the technical field of pharmaceutical therapeutics, and particularly relates to a compound 5n and its application in the preparation of a drug for treating acute kidney injury. The structural formula of the compound 5n is shown as general formula A: Experiments show that the compound 5n can effectively alleviate acute kidney injury in mice, protect renal function, and has the potential to be developed into a drug for treating acute kidney injury, with broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutics, and particularly relates to the use of a compound 5n in the preparation of a medicament for treating acute kidney injury. Background Art

[0002] Acute Kidney Injury (AKI) is a common critical disease in clinical practice, which can lead to incomplete repair of the kidney, persistent chronic inflammation, and progressive fibrosis. It is an important cause of secondary chronic kidney disease, renal failure, and death of inpatients.

[0003] So far, there is no effective treatment for AKI. Therefore, it is of great significance to find a renal protective drug that can reduce renal tissue injury, promote repair, and prevent the occurrence of chronic fibrosis.

[0004] Patent CN111362930A discloses a 1,2,4-oxadiazole-pyridine compound and its application, and discloses a compound 51 with an R2 group of 2-methoxycarbonyl-3-thiophene, which is an inhibitor of glycogen synthase kinase 3β (GSK3β). The patent discloses the application of this type of compound in the preparation of anti-neuroinflammatory drugs and drugs for treating Alzheimer's disease, but its pharmacological effect in preventing and treating acute kidney injury has not been reported yet. Summary of the Invention

[0005] In order to solve the above technical problems, one of the purposes of the present invention is to provide a compound 5n, and the structural formula of the compound 5n is shown in formula (A):

[0006]

[0007] The preparation route of the compound 5n in the present invention is as follows:

[0008]

[0009] The preparation route is mainly for explaining the present invention, rather than imposing any limitation on it.

[0010] a. According to the method reported in the literature, 2-chloro-4-cyanopyridine reacts with 4-trifluoromethoxyaniline through a nucleophilic substitution reaction. After adding water to precipitate, it is washed with petroleum ether to obtain compound 1;

[0011] b. Compound 1 reacts with hydroxylamine hydrochloride under the action of K2CO3 to generate compound 2;

[0012] c. Compound 2 reacts with 4-nitrobenzoyl chloride to obtain compound 3;

[0013] d. Compound 3 is subjected to a reduction reaction to obtain Compound 4;

[0014] e. Compound 4 is dissolved in a pyridine solution, and benzenesulfonyl chloride or thiophenesulfonyl chloride containing a 5-methoxycarbonyl-2-thiophene substituent is added. After the reaction is stirred at room temperature until completion, an aqueous hydrochloric acid solution is added. The reaction mixture solution is placed on ice until a solid precipitate forms, and then filtered and dried to obtain the crude product. The crude product is purified by silica gel column chromatography using a mixed system of dichloromethane and methanol or chloroform and methanol or ethyl acetate and petroleum ether as the eluent to obtain Compound 5n.

[0015] The second object of the present invention is to provide the use of Compound 5n in the preparation of a drug for treating acute kidney injury.

[0016] The present invention also provides a drug for treating acute kidney injury, which contains a pharmaceutically effective dose of Compound 5n, and the structural formula of the Compound 5n is shown as formula (A):

[0017]

[0018] The drug contains a pharmaceutically effective dose of Compound 5n and pharmaceutically acceptable excipients.

[0019] Preferably, the drug further contains a pharmaceutically acceptable carrier.

[0020] Preferably, the pharmaceutically acceptable carrier includes excipients with one or several functions such as excipients, stabilizers, antioxidants, colorants, diluents, sustained-release agents, etc.; such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.

[0021] Preferably, the drug is any one of an injection, a tablet, a pill, a capsule, a suspension or an emulsion.

[0022] The beneficial effects of the present invention are as follows:

[0023] In the present invention, Compound 5n is an inhibitor of glycogen synthase kinase 3β (GSK3β). The results of cell and animal experiments prove that Compound 5n can effectively reduce acute kidney injury in mice and protect renal function. Its mechanism of action is related to the ability of Compound 5n to reduce the levels of inflammatory factors in cells and renal tissues. The present invention provides the use of Compound 5n in reducing acute kidney injury. Compound 5n has the potential to be developed into a drug for treating acute kidney injury, and has a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of Compound 5n of the present invention;

[0025] Figure 2 It is the test result graph of the MTT method in Example 2 of the present invention;

[0026] Figure 3 It is the test result graph of Western Blot in Example 2 of the present invention;

[0027] Figure 4 It is the semi - quantitative analysis result graph in Example 2 of the present invention;

[0028] Figure 5 It is the result of the effect of compound 5n on the levels of inflammatory factors in cisplatin - induced HK2 cells in Example 3 of the present invention. In the figure, graph a shows the mRNA level graph of TNF - α, graph b shows the mRNA level graph of IL - 1β, graph c shows the mRNA level graph of IL - 6, and graph d shows the mRNA level graph of MCP - 1;

[0029] Figure 6 It is the graph of the relationship between the contents of serum creatinine and blood urea nitrogen in a mouse acute kidney injury model with different concentrations of compound 5n in Example 4 of the present invention, where Figure 6 A is serum creatinine, Figure 6 B is blood urea nitrogen;

[0030] Figure 7 It is the test result graph of Western Blot of renal tissue in a mouse acute kidney injury model with different concentrations of compound 5n in Example 4 of the present invention;

[0031] Figure 8 It is Figure 7 the semi - quantitative analysis result of the test result of Western Blot in

[0032] Figure 9 It is the result of the levels of inflammatory factors in renal tissue in a mouse acute kidney injury model with compound 5n in Example 5 of the present invention. In the figure, graph a shows the mRNA level graph of TNF - α, graph b shows the mRNA level graph of IL - 1β, graph c shows the mRNA level graph of IL - 6, and graph d shows the mRNA level graph of MCP - 1. Detailed implementation mode

[0033] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0034] The technical solutions of the present invention will be described in more detail below in conjunction with the embodiments:

[0035] Example 1

[0036] Synthesis of compound 5n:

[0037]

[0038] 2-Chloro-4-cyanopyridine (4.14 g, 30 mmol), palladium acetate (135 mg, 0.6 mmol), BINAP (562.8 mg, 0.85 mmol), and cesium carbonate (13.68 g, 60 mmol) were placed in a three-necked flask. Under a nitrogen atmosphere, 100 mL of 1,4-dioxane was added using a syringe, and finally 4-trifluoromethoxyaniline (8.1 mL, 60 mmol) was injected using a syringe. The mixture was heated to reflux at 80 °C in an oil bath for 4 hours to undergo a nucleophilic substitution reaction. After the reaction, it was cooled to room temperature, water was added to precipitate a solid, and the solid was washed with petroleum ether to obtain Intermediate 1 (white solid, yield 78%);

[0039] Intermediate 1 (5.6 g, 20 mmol), ethanol (50 mL), hydroxylamine hydrochloride (4.2 g, 60 mmol), potassium carbonate (4.4 g, 32 mmol), and H2O (10 mL) were added to a round-bottom flask. The mixture was stirred at room temperature on a magnetic stirrer for 1 h, then refluxed in an oil bath for 3 h, cooled to room temperature, three times the amount of water was added to precipitate a solid, and the solid was filtered and dried to obtain Intermediate 2 (white solid, yield 82%);

[0040] Intermediate compound 2 (4.7 g, 15 mmol) and 4-nitrobenzoyl chloride (2.8 g, 15 mmol) were added to a round-bottom flask, and 80 mL of DMF was added. The mixture was stirred at room temperature on a magnetic stirrer for 1 h, then transferred to an oil bath and refluxed at 110 °C for 5 h. After cooling to room temperature, three times the amount of ice water was added to precipitate a solid, and the solid was filtered by suction to obtain Intermediate 3 (yellow solid, yield 65%);

[0041] Intermediate 3 (4.0 g, 9.3 mmol) was dissolved in methanol and heated to 40 °C. 80% hydrazine hydrate (14 mmol) was added, and an appropriate amount of 10% Pd / C was added. The mixture was refluxed for 40 min. After the reaction, it was filtered while hot, and purified by silica gel column chromatography to obtain Intermediate 4 (yellow solid, yield 45%);

[0042] Intermediate 4 (205.6 mg, 0.5 mmol) was dissolved in pyridine solution, and benzenesulfonyl chloride or thiophenesulfonyl chloride containing 5-methoxycarbonyl-2-thiophene substituent was added. The mixture was stirred at room temperature for 8 h. After the reaction was monitored by TLC and completed, aqueous hydrochloric acid was added. The reaction mixture solution was placed on ice until a solid precipitate separated out, and then the crude product was filtered and dried. The crude product was purified by silica gel column chromatography using a mixed system of dichloromethane and methanol or chloroform and methanol or ethyl acetate and petroleum ether as the eluent to obtain compound 5n. The systematic name of 5n is methyl 3-(N-(4-(3-(2-((4-trichloromethoxyphenyl)amino)pyridin-4-yl)-1,2,4-oxadiazol-5-yl)phenyl)aminosulfonyl)phenothiazine-2-carboxylate.

[0043] 1 H NMR (400 MHz, DMSO-d6) δ 11.0 (s, 1H, NH), 9.6 (s, 1H, NH-SO2), 8.3 (d, J = 5.2 Hz, 1H, pyridine), 8.1 (d, J = 8.9 Hz, 2H, Ar-H), 8.0 (d, J = 5.3 Hz, 1H, thiophene), 7.8 (d, J = 9.2 Hz, 2H, Ar-H), 7.6 (d, J = 5.3 Hz, 1H, thiophene), 7.5 (s, 1H, pyridine), 7.4 (d, J = 8.8 Hz, 2H, Ar-H), 7.3 (dd, J = 5.3, 1.3 Hz, 1H, pyridine), 7.3 (d, J = 8.5 Hz, 2H, Ar-H), 3.9 (s, 3H, CH3) 13 C NMR (100 MHz, DMSO-d6) δ 175.4, 167.0, 159.6 (CO), 156.2, 148.6, 142.3, 141.8, 141.7, 140.1, 134.6, 133.0, 132.3, 130.6, 129.4 (2C), 121.5 (2C), 119.1 (2C), 118.9 (q, J = 255.5 Hz, OCF3), 118.6, 117.8, 111.3, 108.7, 53.2 (CH3).

[0044] HRMS (ESI): m / z 618.0647 [M + H] + , calcd for C 26 H 18 F3N5O6S2, 618.0651.

[0045] Example 2

[0046] Protective effect of compound 5n on cisplatin-induced injury of renal tubular epithelial cells

[0047] MTT method: Human renal tubular epithelial cells (HK2) were seeded in 96-well plates at a seeding density of approximately 4000 cells / well. After culturing for 24 h, the cells were switched to serum-free medium and starved for 12 h. Then, cisplatin and compound 5n were added to the groups, which were the normal group (NC), the model group (Cisplatin 20 μM), and the treatment groups (Cisplatin 20 μM + compound 5n). Among them, the concentrations of compound 5n were 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, and 64 μM) in sequence, and the cells were cultured for another 24 h. After the culture was completed, 20 μL of MTT solution at 5 g·L -1 was added to each well, and the cells were incubated for another 4 h. The medium was aspirated, and 150 μL of DMSO was added to each well, followed by shaking and mixing. The OD values of each well were measured at 492 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the results were recorded. The cell viability was plotted against the dose. Result calculation:

[0048] Cell viability = (OD value of cells in the experimental group - OD value of cells in the blank group) / (OD value of cells in the control group - OD value of cells in the blank group) × 100%.

[0049] Western Blot: HK2 cells in the logarithmic growth phase were seeded in 6-well plates and divided into the normal group (NC), the control group (compound 5n 32 μM), the model group (Cisplatin 20 μM), the low-dose group (Cisplatin 20 μM + compound 5n 16 μM), the medium-dose group (Cisplatin 20 μM + compound 5n 32 μM), and the high-dose group (Cisplatin 20 μM + compound 5n 64 μM). Each group was repeated 3 - 4 times. The seeding density was approximately 1.0×105 cells / ml. After incubating for 24 h and starving for 12 h with serum-free medium, the stimuli and drugs were added respectively. The cells were cultured for another 24 h. The cells were washed three times with PBS, collected, and total protein was extracted. The protein expression of kidney injury molecule 1 (KIM1) was detected by Western Blot, and semi-quantitative analysis was performed.

[0050] The MTT results are as Figure 2 shown. It can be seen from Figure 2 that after the HK2 cells stimulated by cisplatin were treated with compound 5n at low, medium, and high concentrations, the relative cell viability values increased from 56.67% in the cisplatin-stimulated group to 70.38%, 86.23%, and 76.01% respectively, and the viability increased by about 1.52 times. This indicates that compound 5n has a good protective effect on renal tubular epithelial cells stimulated by cisplatin.

[0051] The Western Blot results are asFigure 3 As shown, the semi - quantitative analysis results are as Figure 4 shown. From Figure 3 , Figure 4 it can be seen that after the HK2 cells stimulated by cisplatin were treated with compound 5n, the protein expression level of kidney injury molecule KIM1 was significantly inhibited, proving that compound 5n can play a protective role and avoid the injury of renal tubular epithelial cells caused by cisplatin.

[0052] Example 3

[0053] Inhibitory effect of compound 5n on cisplatin - induced inflammatory factors

[0054] HK2 cells were seeded in 12 - well plates and divided into normal group (NC), control group (compound 5n 32 μM), model group (Cisplatin 20 μM), low - dose group (Cisplatin 20 μM + compound 5n 16 μM), medium - dose group (Cisplatin 20 μM + compound 5n 32 μM), and high - dose group (Cisplatin 20 μM + compound 5n 64 μM). Each group was repeated 3 - 4 times. The seeding density was about 0.5×105 cells / well. After incubation for 24 hours and starvation with serum - free medium for 12 hours, stimuli and drugs were added respectively. Then continue to culture for 24 hours. Wash three times with PBS, collect cells, extract RNA, reverse - transcribe, and amplify.

[0055] The results of Real - time PCR are as Figure 5 shown. Figures a, b, c, and d in the figure respectively show the levels of inflammatory factors TNF - α mRNA, IL - 1β mRNA, IL - 6 mRNA, and MCP - 1 mRNA in renal tubular epithelial cells. From Figure 5 it can be seen that cisplatin significantly induced the increase in the levels of TNF - α mRNA, IL - 1β mRNA, IL - 6 mRNA, and MCP - 1 mRNA in renal tubular epithelial cells, while the expression of TNF - α mRNA, IL - 1β mRNA, IL - 6 mRNA, and MCP - 1 mRNA in the compound 5n treatment group was significantly inhibited, indicating that compound 5n can significantly inhibit the inflammatory response induced by cisplatin.

[0056] Example 4

[0057] Protective effect of compound 5n on renal function in acute kidney injury model

[0058] 6-8-week-old C57BL / 6 mice were adaptively cultured for 1-2 days and divided into a normal control group (NC), a model group (Cisplatin 20 mg / kg), a low-dose group (Cisplatin 20 mg / kg + Compound 5n 12.5 mg / kg), a medium-dose group (Cisplatin 20 mg / kg + Compound 5n 25 mg / kg), and a high-dose group (Cisplatin 20 mg / kg + Compound 5n 50 mg / kg), with 6-10 mice in each group. The mice were intraperitoneally injected with 20 mg / kg cisplatin to establish an acute kidney injury model and were injected with low, medium, and high doses of Compound 5n for drug intervention. After 3 days, serum samples and kidney tissues were collected under anesthesia.

[0059] According to the instructions of the creatinine and urea nitrogen kits, the contents of creatinine and urea nitrogen in the serum of the animal models were detected (purchased from Nanjing Jiancheng Bioengineering Institute). The creatinine detection method is shown in Table 1, and the urea nitrogen detection method is shown in Table 2. Additionally, a homogenizer was used to grind the kidney tissues on ice to extract the total proteins in the kidney tissues. The protein expression of kidney injury molecule 1 (KIM1) in the kidney tissues was detected by Western Blot and semi-quantitative analysis was performed.

[0060] Table 1 Creatinine Detection

[0061]

[0062]

[0063] Note: Dilution factor K = (sample volume + volume of enzyme solution A) / (sample volume + volume of enzyme solution A + volume of enzyme solution B) = 186 / 246;

[0064] Creatinine content (μmol / L) = [(measured A2 - K * measured A1) - (blank A2 - K * blank A1)] /

[0065] [(standard A2 - K * standard A1) - (blank A2 - K * blank)] * standard concentration (442 μmol / L).

[0066] Table 2 Urea Nitrogen Detection

[0067]

[0068] Urea nitrogen content (mmol / L) = (measured OD value - blank measured value) / (standard OD value - blank OD value) * standard concentration (10 mmol / L) * dilution factor before sample testing.

[0069] The results are as Figure 6 shown, from Figure 6It can be seen that the serum creatinine level in the cisplatin-induced model group increased significantly, indicating renal function deterioration, while compound 5n at different concentrations effectively reduced the serum creatinine level in the model group; from Figure 6 Similarly, it can be seen that compound 5n at different concentrations effectively reduced the urea level in the model group, indicating that compound 5n has a protective effect on renal function during acute kidney injury.

[0070] The results of Western Blot are as Figure 7 shown, and the results of semi-quantitative analysis are as Figure 8 shown. It can be seen that the expression level of kidney injury molecule 1 (KIM1) protein in renal tissues of the cisplatin-induced acute kidney injury mouse model increased significantly, indicating kidney injury in the model group; after injection of compound 5n, the expression level of KIM1 protein in renal tissues of the cisplatin-induced acute kidney injury mouse model decreased in a dose-dependent manner. This demonstrated that compound 5n protected against cisplatin-induced acute kidney injury in mice.

[0071] Example 5

[0072] Inhibitory effect of compound 5n on inflammation in an acute kidney injury model

[0073] 6- to 8-week-old C57BL / 6 mice were adaptively cultured for 1-2 days and divided into a control group (NC), a model group (Cisplatin 20 mg / kg), a low-dose group (Cisplatin 20 mg / kg + compound 5n 12.5 mg / kg), a medium-dose group (Cisplatin 20 mg / kg + compound 5n 25 mg / kg), and a high-dose group (Cisplatin 20 mg / kg + compound 5n 50 mg / kg), with 6-10 mice in each group. The mice were intraperitoneally injected with 20 mg / kg cisplatin to establish an acute kidney injury model and injected with low, medium, and high doses of compound 5n. After 3 days, blood samples and renal tissues were collected under anesthesia, and tissue RNA was extracted, reverse-transcribed, and amplified.

[0074] The results of Real-time PCR are as Figure 9 shown. Panels a, b, c, and d in the figure respectively show the levels of inflammatory factors TNF-α mRNA, IL-1β mRNA, IL-6 mRNA, and MCP-1 mRNA in the acute kidney injury model. Figure 9 It can be seen that the levels of inflammatory factors TNF-α mRNA, IL-1β mRNA, IL-6 mRNA, and MCP-1 mRNA in the acute kidney injury model increased significantly, while compound 5n could significantly reduce the levels of inflammatory factors and improve inflammation. This demonstrated that compound 5n could reduce the levels of inflammatory factors in acute kidney injury, revealing that the mechanism of action of compound 5n may be related to reducing the levels of inflammatory factors.

[0075] The above are only the preferred practical examples of the present invention and are not used to limit the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a compound 5n in the preparation of a medicament for treating acute kidney injury, wherein the structural formula of the compound 5n is shown as formula (A): 。 2. The application according to claim 1, characterized in that, The medicament contains a pharmaceutically effective dose of the compound 5n and also contains a pharmaceutically acceptable carrier.

3. The application according to claim 2, wherein The pharmaceutically acceptable carrier includes a carrier having one or several functions among excipients, stabilizers, antioxidants, colorants, diluents, sustained-release agents.

4. The application according to claim 2, characterized in that, The medicament is any one of an injection, a tablet, a pill, a capsule, a suspension or an emulsion.

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