Anti-hepatic fibrosis compound, preparation method and application

Abstract

The invention discloses an anti-hepatic fibrosis compound, a preparation method and application, the molecular formula of the compound is C30H26ClF3N6O3S, the compound can play a role in target enzyme ATX and play a role in preventing or treating hepatic fibrosis by inhibiting ATX, and compared with the prior art, the IC50 value of the compound is 12.86 mu M and is obviously superior to that of the compound in the prior art; in the aspect of the alkaline phosphatase index, the index values of the compound in high, medium and low doses are all lower than those in the prior art, and the compound disclosed by the invention is not dose-dependent in the aspect of the liver function index, and can generate a better drug effect in a low dose, so that the compound disclosed by the invention further improves the liver function index.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to an anti-hepatic fibrosis compound, a preparation method and application. BACKGROUND

[0002] Hepatic fibrosis is a process of diffuse excessive deposition of extracellular matrix (ECM) in the liver caused by various pathogenic factors, and is a common pathological pathway of most chronic liver diseases. Hepatic fibrosis can further develop into cirrhosis, and even further into hepatocellular carcinoma. Research has shown that hepatic fibrosis is reversible, and no clear anti-hepatic fibrosis treatment drug has been found in clinical practice. Therefore, it is of great clinical significance to find a treatment drug that can effectively reverse hepatic fibrosis. Autotaxin (ATX) is a secreted glycoprotein with phosphodiesterase and lysophospholipase D activity. Clinical case reports have found that serum ATX is positively correlated with the degree of hepatic fibrosis. Clinical studies have also found that patients with viral infection (HBV and HCV) have elevated serum ATX levels, and the incidence of cirrhosis and liver cancer is also elevated. Preliminary mechanism studies have found that the ATX activity product LPA can activate hepatic stellate cells and promote collagen secretion. Therefore, ATX is considered to be an effective serum marker for hepatic fibrosis, and also a target for the development of anti-hepatic fibrosis drugs.

[0003] In the applicant's previous research, a Chinese invention patent with publication number CN111777597A discloses an anti-hepatic fibrosis compound. Although this compound can improve liver function indicators, the improvement effect is limited, and the IC 50 value is relatively high. Therefore, how to provide another anti-hepatic fibrosis compound and further improve liver function indicators and reduce IC 50 value is a technical problem that needs to be solved by those skilled in the art. SUMMARY

[0004] In view of this, the present application provides an anti-hepatic fibrosis compound, a preparation method and application, to provide another anti-hepatic fibrosis compound and further improve liver function indicators and reduce IC 50 value.

[0005] An anti-hepatic fibrosis compound, the molecular formula of which is: 30 H 26 ClF3N6O3S, and the structural formula is as follows: .

[0006] A preparation method of an anti-hepatic fibrosis compound, comprising the following steps: Step 1: Place 2-(4-chlorophenoxy)propionylhydrazine and 1-isothiocyano-3-(trifluoromethyl)benzene in anhydrous ethanol, reflux for the first time, cool the mixture to room temperature, then filter, concentrate the filtrate to obtain methyl 4-[2-[2-(4-chlorophenoxy)propionyl]hydrazine-thioformamido]benzoate; Step 2: Methyl 4-[2-[2-(4-chlorophenoxy)propionyl]hydrazidothiocarbamate]benzoate was dissolved in sodium hydroxide solution, heated under reflux for a second time, cooled, and then acidified with HCl to pH 2-3. The white solid was collected by filtration and dried to obtain 5-[1-(4-chlorophenoxy)ethyl]-4-[3-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-3-thiol; Step 3: A mixture of 5-[1-(4-chlorophenoxy)ethyl]-4-[3-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-3-thiol and 2-chloro-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide was added to acetone along with anhydrous potassium carbonate. The mixture was refluxed for a third time, filtered, and the filtrate was evaporated to dryness. The residue was recrystallized from the filtrate using MeOH / MTBE to obtain the above-mentioned anti-liver fibrosis compound.

[0007] This invention also provides the use of the above-mentioned compounds in the preparation of drugs for treating liver fibrosis.

[0008] According to the present invention, an anti-liver fibrosis compound, its preparation method, and its application are provided, and another anti-liver fibrosis compound is provided. This compound can act on the target enzyme ATX, and by inhibiting ATX, it can prevent or treat liver fibrosis. Compared with the prior art, the IC50 of this compound is higher. 50 The value was 12.86 μM, which is significantly better than the compound in CN111777597A. Regarding alkaline phosphatase, the compound showed lower values ​​at all three dosage levels (high, medium, and low) than the compound in CN111777597A. Furthermore, the compound of this invention does not exhibit dose-dependent effects on liver function indicators, producing good efficacy even at low doses. In contrast, the efficacy of the compound in CN111777597A increases with increasing drug concentration. Therefore, the compound of this invention further improves liver function indicators. Attached Figure Description

[0009] Figure 1 A process flow diagram for preparing the anti-liver fibrosis compound provided in Example 1; Figure 2 The mass spectrum of the compound obtained in Example 1; Figure 3 The compound obtained in Example 1 1 H-NMR spectrum; Figure 4 Sirius red staining of liver tissue sections from mice in the normal control group; Figure 5 Sirius red staining of liver tissue sections from mice in the model group; Figure 6 Sirius red staining of liver tissue sections from mice in the low-dose administration group; Figure 7 Sirius red staining of liver tissue sections from mice in the medium-dose administration group; Figure 8 This is a Sirius red staining image of liver tissue sections from mice in the high-dose administration group. Detailed Implementation

[0010] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain embodiments of the present invention, and should not be construed as limiting the present invention.

[0011] Example 1 Please see Figure 1 Example 1 provides a method for preparing an anti-liver fibrosis compound, comprising the following steps: Step 1: 214 mg, 1 mmol of 2-(4-chlorophenoxy)propionylhydrazine and 200 mg, 1 mmol of 1-isothiocyano-3-(trifluoromethyl)benzene were placed in 10 mL of anhydrous ethanol and refluxed for 3 hours. The mixture was then cooled to room temperature, filtered, and the filtrate was concentrated to obtain a light brown oily substance, namely methyl 4-[2-[2-(4-chlorophenoxy)propionyl]hydrazine-thioformamido]benzoate. Step 2: Dissolve 300 mg of methyl 4-[2-[2-(4-chlorophenoxy)propionyl]hydrazidothiocarbamate]benzoate in 10 mL of 2N sodium hydroxide solution, heat under reflux for 2 hours, cool, then acidify with HCl to pH 2-3, filter to collect the white solid, and dry to obtain a white solid, namely 5-[1-(4-chlorophenoxy)ethyl]-4-[3-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-3-thiol; Step 3: A mixture of 140 mg and 0.5 mmol of 5-[1-(4-chlorophenoxy)ethyl]-4-[3-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-3-thiol and 200 mg and 0.5 mmol of 2-chloro-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide was added to acetone along with 138 mg and 1 mmol of anhydrous potassium carbonate. The mixture was refluxed for 4 hours, filtered, and the filtrate was evaporated to dryness. The residue was recrystallized from MeOH / MTBE to give 60 mg of a white solid, which is a novel anti-liver fibrosis compound with the molecular formula: C 30 H 26 The structural formula of the compound ClF3N6O3S is as follows: .

[0012] like Figures 2-3 As shown, Figure 2 This is the mass spectrum of the compound. Figure 3 For this compound 1 H-NMR spectrum.

[0013] NMR data are 1 H-NMR (400MHz, DMSO-d6): δ1.60-1.61(3H,d),2.07(3H,s),3.04(3H,s),4.11(2H,s),5.61-5.63(1H,m),7.22-7.24(2H , d), 7.25-7.27(3H, t), 7.48-7.50(2H, t), 7.72-7.74(2H, d), 7.76-7.78(2H, d), 9.45(1H, s).

[0014] Example 2: Human ATX enzyme inhibition assay 1. ATX inhibition was measured using the specially labeled substrate Amplex Red reagent by ultraviolet spectrophotometry. This reagent is available by purchasing a kit.

[0015] 2. The working solution for determination shall be prepared as follows: 2.1 Buffer solution: 50mM Tris-HCl, 140mM NaCl, 5mM KCl, 1mM CaCl2, 1mM MgCl2, 0.01% Triton-X-100, pH 8.0.

[0016] 2.2 ATX solution: ATX (mouse ATX-β from Sino Biological) stock solution (128 nM, diluted with assay buffer), diluted to a final concentration of 32 nM with assay buffer before assay.

[0017] 2.3 LPC substrate solution: Weigh out LPC (16:0) substrate solid and dilute it in the determination buffer to a final concentration of 250 nM.

[0018] 2.4 Detection Method: ATX was detected using the Amplex Red kit. Each reaction contained 50 μM Amplex Red reagent, 1 U / mL HRP, 0.1 U / mL choline oxidase, 250 nM LPC, and ATX diluted with 1X reaction buffer. The reaction was incubated at 37°C for 2 hours. The absorbance was measured at 562 nm using a microplate reader.

[0019] 2.5 The compound of Example 1 was diluted with DMSO to prepare a series of solutions of 5 mM, 2.5 mM, 1.25 mM, 0.5 mM, 0.25 mM, and 0.125 mM. 5 μL of each solution was added to the kit solution in 2.4, resulting in a series of solutions of the compound of Example 1 with final concentrations of 125 μM, 62.5 μM, 31.25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. The change in absorbance after adding the compound was measured according to method 2.4, and the IC50 of the compound was calculated from these data. 50 The value is 12.86 μM.

[0020] Example 3: Pharmacological Experiment Study 1. Experimental materials and animals 1.1 Drugs and reagents: Alanine aminotransferase (ALT) assay kit (R1, R2) (MIVDRAY kit), aspartate aminotransferase (AST) assay kit (R1, R2) (MIVDRAY kit), carbon tetrachloride (analytical grade), and a 20% corn oil solution prepared with corn oil before use.

[0021] 1.2 Experimental animals: Clean-grade Kunming mice, weighing 18-22g, from Henan Skbes Biotechnology Co., Ltd.

[0022] 1.3 Experimental instruments: MIVDRAY BS-380 fully automated biochemical analyzer, EX 125DZH electronic analytical balance (Ohaus Instruments (Changzhou) Co., Ltd.), HC30L8R high-speed refrigerated centrifuge (Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd.).

[0023] 1.4 Test Drugs and Processing Methods: The compound obtained from the strip preparation in Example 1 was used. For the high-dose group, it was first dissolved in DMSO and then diluted with corn oil to form a 10.0 mg / ml emulsion. For the medium-dose group, it was first dissolved in DMSO and then diluted with corn oil to form a 5.0 mg / ml emulsion. For the low-dose group, it was first dissolved in DMSO and then diluted with corn oil to form a 2.5 mg / ml emulsion. Separately, the compound from CN111777597A was used. For the high-dose control group, it was first dissolved in DMSO and then diluted with corn oil to form a 10.0 mg / ml emulsion. For the medium-dose control group, it was first dissolved in DMSO and then diluted with corn oil to form a 5.0 mg / ml emulsion. For the low-dose control group, it was first dissolved in DMSO and then diluted with corn oil to form a 2.5 mg / ml emulsion.

[0024] 2. Experimental Methods 2.1 Effects on CC14-induced liver fibrosis in mice 2.1.1 Establishment of a mouse model of CCl4-induced liver fibrosis: Except for the normal group, all other groups were injected intraperitoneally with 20% CC14 corn oil solution (5 μL / g body weight) twice a week.

[0025] 2.2.2 Seventy-five male ICR mice were randomly divided into eight groups: normal group, model group, and high-dose administration group (30 mg / kg). -1 ·d -1 ), medium-dose group (15 mg·kg) -1 ·d -1 Low-dose group (7.5 mg·kg) -1 ·d -1 ), high-dose control group (30 mg·kg) -1 ·d -1 ), medium-dose control group (15 mg·kg) -1 ·d -1 ), low-dose control group (7.5 mg·kg) -1 ·d -1 ).

[0026] 2.2.3 Normal group: No medication or modeling, normal diet and water intake. Model group: Intraperitoneal injection of 20% CC14 corn oil solution alone. (High-dose, medium-dose, low-dose) medication groups and (high-dose, medium-dose, low-dose) control groups: In addition to modeling, the corresponding test solutions prepared under section 1.4 should be injected intraperitoneally. In this experiment, both the medication group and the control group were administered medication starting from day 14 of modeling, once a day, 6 times a week. On day 42 of modeling, blood was collected from the orbital sinus of the normal group, model group, control group, and medication group, and the mice were sacrificed. The total modeling time was 42 days, and the total medication time was 28 days. After blood collection, the mice were allowed to stand for 2 hours, centrifuged at 3000 r / min at 4℃ for 15 min, and the supernatant was collected. The serum ALT, AST, albumin, and ALP levels of CC14-induced liver injury mice were determined according to the kit instructions. After blood collection, the mice were euthanized, and the livers were quickly removed. The surrounding connective tissue was removed, and a portion of the liver was stored at -80°C for later use. Another portion was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4μm thick serial sections. The liver tissue was stained with Sirius red, and the collagen fibers were observed under an optical microscope.

[0027] 3. Experimental Results 3.1 Effects of the compound on liver function in mice The results of the serum markers in the experiment on the effect of the compound on the liver function of mice induced by CC14 are shown in Table 1 below.

[0028] Table 1. Serum data of the effects of compounds on liver function in mice.

[0029] As shown in Table 1, compared with the normal group, the liver function of mice in the carbon tetrachloride model group was significantly reduced. The detection results of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were significantly increased (p<0.01), and the detection results of serum albumin were decreased. Compared with the model group, the compounds of the present invention and the compounds of the control group could significantly reduce the levels of serum alanine aminotransferase and aspartate aminotransferase (p<0.01), and alkaline phosphatase (P<0.01 in low-dose control group, low-dose administration group and high-dose administration group; p<0.05 in medium-dose control group, high-dose control group and medium-dose administration group), while significantly increasing the level of serum albumin (p<0.01), suggesting that the compounds of the present invention and the compounds of the control group have a protective effect on liver function.

[0030] Compared with the control group, the compounds of the present invention, except for the high-dose group, showed better values ​​of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at both medium and low doses than the corresponding dose groups in the control group. Regarding alkaline phosphatase, the high, medium, and low dose groups of the present invention all showed lower values ​​than the control group, indicating superiority. Regarding albumin, the high, medium, and low dose groups of the present invention all showed higher values ​​than the control group, with the low-dose group showing significantly higher values ​​than the low-dose control group (p<0.05), indicating superiority. Table 1 also shows that the compounds of the present invention do not exhibit dose-dependent effects on liver function indicators, producing good efficacy even at low doses, while the efficacy of the control group compounds increased with increasing drug concentration. From the above results, it can be concluded that, overall, the compounds of the present invention are superior to the control group compounds in improving liver function indicators.

[0031] 3.2 Effects of the compounds on Collagen I and α-SMA in the liver tissue of mice in each group.

[0032] 3.2.1 RNA Extraction: Liver tissue was minced and lysed using 1 mL of TRIzol. 200 µL of chloroform was added, and the mixture was vigorously vortexed and incubated for 3 min. The mixture was then centrifuged at 12000 g for 15 min. An equal volume of isopropanol was added to the supernatant, and the mixture was incubated at room temperature for 10 min. The mixture was then centrifuged at 12000 g for 10 min. The supernatant was discarded, and 1 mL of 75% ethanol was added. The mixture was centrifuged at 7500 g for 5 min, and the supernatant was discarded again. The precipitate was dried and dissolved in DEPC water.

[0033] 3.2.2 RT-PCR: The extracted RNA was reverse transcribed into cRNA using the Takara Reverse Transcription Kit (RR047A). The reverse transcription system was prepared according to the instructions of the reverse transcription kit. All operations for preparing the reaction system were performed on ice. 3.2.3 Reagents were prepared according to the Takara TB Green Premix Ex TaqII (RR820A) manual for qPCR reactions. GAPDH was used as an internal control to assess the relative expression levels of Collagen-I and α-SMA.

[0034] The effects of the compounds on Collagen I and α-SMA in CC14-induced mouse liver tissue are shown in Table 2.

[0035] Table 2. Effects of the compounds on Collagen I and α-SMA in mouse liver tissue.

[0036] The effect of this compound on liver fibrosis in mice was detected by RT-qPCR. Table 2 shows that, compared with the normal group, the gene transcription levels of Collagen I and α-SMA in the model group were significantly increased (p<0.01), indicating that the gene transcription levels of Collagen I and α-SMA increase during the progression of CCl4-induced liver fibrosis in mice. Regarding Collagen I gene transcription, after treatment with the compound of this invention, except for a slight increase at low doses, medium doses (p<0.05) and high doses (p<0.01) both downregulated Collagen I gene transcription in a dose-dependent manner. In contrast, only low doses (p<0.01) downregulated Collagen I gene transcription in the control group, while the transcription levels at medium and high doses were higher than those in the model group, with the high dose reaching three times that of the model group. Regarding α-SMA gene expression, both the compounds of this invention and the control group downregulated α-SMA gene expression (low-dose control group p<0.05, other groups p<0.01), but the three dosage groups of the compounds of this invention were superior to the corresponding dosage groups of the control compounds. These results indicate that the compounds of this invention can improve CCl4-induced liver fibrosis in mice by downregulating the gene transcription levels of Collagen I and α-SMA, and are superior to the control group compounds.

[0037] 3.3 Effects of the compound on CCL4-induced morphology of mouse liver tissue like Figures 4-8 The images show staining patterns of mouse liver tissue in the normal group, model group, and different doses of the compound of this invention, respectively. It can be seen that the model group exhibits obvious reticular fibrosis. The high-dose and low-dose treatment groups showed significantly improved liver pathology compared to the model group, with a significant reduction in hepatic fibrosis and connective tissue proliferation. The gold standard for tissue sections demonstrates that this compound has a definite preventive effect against the formation of liver fibrosis in mice.

[0038] In summary, based on the anti-liver fibrosis compound, preparation method, and application described in the above embodiments, another anti-liver fibrosis compound is provided. This compound can act on the target enzyme ATX, and by inhibiting ATX, it can prevent or treat liver fibrosis. Compared with the prior art, the IC50 of this compound is significantly higher. 50 The value was 12.86 μM, which is significantly better than the compound in CN111777597A. Regarding alkaline phosphatase, the compound showed lower values ​​at all three dosage levels (high, medium, and low) than the compound in CN111777597A. Furthermore, the compound of this invention does not exhibit dose-dependent effects on liver function indicators, producing good efficacy even at low doses. In contrast, the efficacy of the compound in CN111777597A increases with increasing drug concentration. Therefore, the compound of this invention further improves liver function indicators.

[0039] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A compound that combats liver fibrosis, characterized in that, The molecular formula is: C 30 H 26 ClF3N6O3S, with the following structural formula: 。 2. The method for preparing the anti-liver fibrosis compound according to claim 1, characterized in that, Includes the following steps: Step 1: Place 2-(4-chlorophenoxy)propionylhydrazine and 1-isothiocyano-3-(trifluoromethyl)benzene in anhydrous ethanol, reflux for the first time, cool the mixture to room temperature, then filter, concentrate the filtrate to obtain methyl 4-[2-[2-(4-chlorophenoxy)propionyl]hydrazine-thioformamido]benzoate; Step 2: Methyl 4-[2-[2-(4-chlorophenoxy)propionyl]hydrazidothiocarbamate]benzoate was dissolved in sodium hydroxide solution, heated under reflux for a second time, cooled, and then acidified with HCl to pH 2-3. The white solid was collected by filtration and dried to obtain 5-[1-(4-chlorophenoxy)ethyl]-4-[3-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-3-thiol; Step 3: A mixture of 5-[1-(4-chlorophenoxy)ethyl]-4-[3-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-3-thiol and 2-chloro-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide was added to acetone along with anhydrous potassium carbonate. The mixture was refluxed for a third time, filtered, and the filtrate was evaporated to dryness. The residue was recrystallized from the filtrate using MeOH / MTBE to obtain the anti-liver fibrosis compound of claim 1.

3. The preparation method according to claim 2, characterized in that, In step 1, the molar ratio of 2-(4-chlorophenoxy)propionylhydrazine and 1-isothiocyano-3-(trifluoromethyl)benzene is 1:

1.

4. The preparation method according to claim 2, characterized in that, In step 1, the first timeframe is 3 hours.

5. The preparation method according to claim 2, characterized in that, In step 2, the second time is 2 hours.

6. The preparation method according to claim 2, characterized in that, In step 3, the molar ratio of 5-[1-(4-chlorophenoxy)ethyl]-4-[3-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-3-thiol and 2-chloro-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide is 1:

1.

7. The preparation method according to claim 2, characterized in that, In step 3, the third time is 4 hours.

8. The use of the compound of claim 1 in the preparation of a drug for treating liver fibrosis.

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