Human cathepsin S inhibitor and application

Abstract

The invention relates to a human cathepsin S inhibitor which has a structure shown as a formula (I), # imgabs0 #, wherein R1 is heterocycle substituted piperazinyl, alkane substituted piperazinyl, aromatic hydrocarbon substituted piperazinyl, aromatic substituent or benzylamine; r2 is an alkane substituent group, a benzyl group or an aromatic substituent group. The inhibitor has the application of treating and preventing liver tissue fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry and relates to an inhibitor of human cathepsin S (CTSS) and its application. Background Art

[0002] Human cysteine ​​cathepsin S (Cathepsin S), referred to as CTSS, is synthesized in the rough endoplasmic reticulum in the form of an inactive zymogen. The zymogen consists of 331 amino acids and includes three regions: signal peptide, propeptide, and mature peptide. The signal peptide is hydrolyzed and removed by proteases during transport through the lumen of the endoplasmic reticulum, and then the propeptide is glycosylated in the Golgi apparatus. In the trans-Golgi network, the glycosylated propeptide is labeled with mannose-6-phosphate and then sorted to the lysosome. In an acidic environment, the CTSS propeptide forms a mature and active CTSS by self-cleavage or with the help of other proteases. When the intracellular calcium ion level increases, the lysosomal membrane fuses with the cytoplasmic membrane, and CTSS is released into the extracellular space in the form of exocytosis.

[0003] Liver fibrosis is the result of continuous damage-repair when the liver is stimulated by external factors (such as viral hepatitis, drug-induced liver injury, non-alcoholic fatty liver disease, etc.), and is characterized by excessive deposition of extracellular matrix (ECM) components after activation and proliferation of hepatic stellate cells (HSC). In this process, fibronectin (FN) is an important molecular marker for the occurrence of fibrosis. If the damaging factors can be removed at an early stage, liver fibrosis can be reversed, thus avoiding further development into cirrhosis or even liver cancer.

[0004] The early stages of liver fibrosis formation go through the stages of liver damage and liver inflammation. The persistent inflammatory process accelerates the development of liver fibrosis. CTSS has been widely studied as a target for immune regulation, but CTSS as a key factor in early liver fibrosis and its molecular mechanism are still unknown to the industry. The applicant tested clinical serum samples of different stages of liver fibrosis and found that in early liver fibrosis, the CTSS protein content in the serum increased significantly. Both clinical early liver fibrosis puncture samples and animal experiments have shown that elevated CTSS is a key factor in early liver fibrosis. Therefore, targeting CTSS and developing CTSS inhibitors is expected to develop therapeutic drugs for early human liver fibrosis, alleviating the dilemma that there is still no clear drug target for the effective treatment of early liver fibrosis in the clinic.

[0005] In order to construct a screening model for targeted inhibition of CTSS collagen cleavage reaction and obtain early liver fibrosis therapeutic drugs, a yeast eukaryotic expression system was used to recombinantly express biologically active CTSS protein. The yeast strain is a histidine-deficient Pichia pastoris 1163 strain (his4 pep4 prb1), in which the mutations of the vacuolar protease A encoding gene pep4 and the protease B encoding gene prb1 inhibit the activity of all other proteases in the yeast vacuole, which is conducive to the complete and efficient expression of exogenous gene-encoded proteins in yeast cells and the development of purification processes. The pPICZα yeast vector uses the ethanol oxidase AOX1 promoter to drive the efficient expression of CTSS protein through methanol induction.

[0006] High-purity CTSS is the premise and basis for constructing targeted inhibition of CTSS collagen cleavage reaction and efficient and specific screening of therapeutic drugs for early liver fibrosis. The proteome is the sum of all proteins in cells and is the final result of gene transcription, translation and post-translational modification. Proteomics analysis can be used to study the abundance, function and interaction of proteins in cells in different environments. The important role of proteomics in CTSS protein purification is reflected in the identification of other impurities that may exist in the purified CTSS solution through omics analysis, thereby determining the protein purification method and improving the protein purity. The identification of the components of the CTSS protein complex is the core step in the separation and purification of CTSS protein.

[0007] The basis for using recombinant CTSS protein to screen for highly effective inhibitors targeting early liver fibrosis is to obtain highly active CTSS protein by expressing it in a yeast system and purifying it efficiently. To this end, it is necessary to establish a detection system for the activity of recombinant CTSS protein and a screening system for highly effective inhibitors.

[0008] CN103153989A provides a cathepsin S inhibitor, such as a compound of the following structure (Formula (VIII)),

[0009] Summary of the invention

[0010] One object of the present invention is to provide a novel inhibitor of human cathepsin S (CTSS protein).

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned CTSS protein inhibitor.

[0012] Another object of the present invention is to provide the use of the above CTSS protein inhibitor.

[0013] According to one aspect of the present invention, a CTSS protein inhibitor, whose target is cysteine ​​cathepsin S (CTSS protein) in the early stage of human liver fibrosis, has a structure of formula (I):

[0014]

[0015] Wherein: R1 is a piperazinyl substituted by a heterocycle, a piperazinyl substituted by an alkane, a piperazinyl substituted by an aromatic hydrocarbon, an aromatic substituent or a benzylamine; R2 is an alkane substituent, a benzyl or an aromatic substituent.

[0016] The CTSS protein inhibitor of the present invention is preferably selected from the following formulas: Compound B (Formula II), Compound C (Formula (III)), Compound D (Formula IV), Compound E (Formula V), Compound F (Formula VI) and Compound G (Formula VII):

[0017]

[0018] According to another aspect of the present invention, a pharmaceutical composition is provided, which contains the CTSS protein inhibitor of the present invention.

[0019] According to another aspect of the present invention, there is provided use of the CTSS protein inhibitor and a pharmaceutical composition containing the same in the preparation of a drug for treating and / or preventing early liver fibrosis.

[0020] The present invention also provides a method for screening highly effective CTSS protein inhibitors, which is based on a method for preparing accurately and efficiently expressed human CTSS protein.

[0021] The method for preparing human CTSS of the present invention comprises electrotransferring pPICZα-CTSS-eGFP and pPICZα-CTSS-His vectors into recipient Pichia pastoris cells to obtain recombinant yeast cells. The recombinant yeast cells are induced with 0.7% methanol to start the expression of CTSS-eGFP or CTSS-His. The present invention uses three indicators to characterize the accurate and efficient expression of recombinant CTSS protein. First, the positioning of C-terminal eGFP shows that CTSS protein is evenly distributed in the cytoplasm rather than aggregated to vacuolar hydrolysis, indicating that CTSS protein is not only accurately expressed in yeast cells, but also should be active; second, after the recombinant CTSS-His yeast whole cell lysate protein is separated by SDS-PAGE gel, methanol-induced high-abundance protein appears at the theoretical molecular weight, indicating that CTSS is efficiently expressed after induction; third, CTSS antibody hybridization confirms that the methanol-induced high-abundance protein is recombinant CTSS-His. In the present invention, different methanol concentrations are used for induction, the methanol concentration is determined by comparing the protein yield, and a concentration of 0.7% is selected for subsequent purification of CTSS-His protein.

[0022] The method for preparing high-purity CTSS-His comprises collecting the methanol-induced recombinant yeast cells, breaking the cell walls and ultrasonicating the recombinant yeast cells to obtain a whole cell lysate. The whole cell lysate is centrifuged to obtain a whole cell lysate protein. The whole cell lysate protein is subjected to nickel column affinity chromatography and eluted with imidazole of a concentration gradient, and the elution peak of the recombinant CTSS protein is collected. The sample of the elution peak is separated and purified. First, gel filtration chromatography is selected for separation, and the protein peak fraction is collected. The fraction is separated and identified for purity by SDS-PAGE gel separation. Since a high abundance of impurity proteins is detected, other methods are required to purify CTSS. An innovative point of the present invention is to use proteomics to guide the separation and purification of CTSS protein. The innovation is that the impurity protein band is digested by enzyme digestion and then subjected to mass spectrometry analysis and identification, and the characteristics of the impurity protein components are used to guide the protein purification method. The protein is subjected to isoelectric point analysis, and anion exchange chromatography is selected to separate CTSS. The separated CTSS is eluted with sodium chloride of a concentration gradient, and the elution peak of the recombinant CTSS protein is collected. The sample of the elution peak was subjected to SDS-PAGE gel separation, and only one CTSS protein band was detected, indicating that the anion exchange can efficiently separate and purify the CTSS protein. The high-purity fraction obtained by the sodium chloride elution was ultrafiltered and concentrated through a 10kD ultrafiltration tube to obtain concentrated recombinant human CTSS protein.

[0023] In the method, the nickel affinity chromatography column is GE HisTrap FF, and the column volume is 5 mL. The pH of the equilibration buffer used for enriching CTSS protein by the nickel column is 7.4, and its components include 40mM Tris-HCl and 150mM NaCl. The equilibration buffer is loaded after equilibration of the nickel column, and the flow rate is 5 mL / min. The pH of the elution buffer after the loading is 7.4, and its components include 40mM Tris-HCl, 150mM NaCl, and 0.4M Imidazole. The elution procedure is Imidazole linear gradient elution. The Imidazole elution concentration is uniformly increased from 40mM to 140mM, and fractions with elution volumes of 20-27mL and 28-43mL are collected respectively.

[0024] The gel filtration chromatography column is Superdex 200increase 10 / 300GL, and the ratio of column length to column inner diameter used in the chromatography is 300:10. The separation range of the chromatographic medium used for proteins is 10-600kDa molecular weight. The equilibrium buffer of the gel filtration chromatography column has a pH of 7.4, and its components include 40mM Tris-HCL and 150mM NaCl. The sample used in the gel filtration chromatography comes from the P2 component eluted by Imidazole. The fractions obtained by the chromatographic separation are F1 and F2.

[0025] The anion exchange chromatography column is GE Q Sepharose high performance 16 / 20, with a column volume of 5 mL. The equilibrium buffer of the ion exchange column has a pH of 7.4, and its components include a 40mM Tris-HCl buffer. The elution buffer after the anion exchange chromatography includes 1M NaCl and 40mM Tris-HCl buffer. The elution procedure is NaCl linear gradient elution. The NaCl elution concentration is uniformly increased from 120mM to 200mM, and the fractions with an elution volume of 228-240mL are collected to obtain a high-purity recombinant human CTSS protein.

[0026] The recombinant human CTSS protein is used to screen for highly effective inhibitors targeting early liver fibrosis. The activity of the recombinant CTSS is evaluated based on the enzymatic reaction of the substrate, and the affinity and catalytic efficiency of the recombinant CTSS enzyme for the substrate are characterized by comparison with the commercial CTSS Michaelis constant (Km). The substrate of the enzymatic reaction is Benzyloxycarbonyl-L-leucyl-L-arginine4-methylcoumaryl-7-amide (Z-Leu-Arg-MCA). The substrate is cut by CTSS to produce free MCA, and the amount of substrate generated can be detected by fluorescence. The calculation of the Km constant requires determining the maximum speed (Vmax) reached by the enzymatic reaction. To determine Vmax, the CTSS dosage is set to 250nM, and the concentration of the substrate (S) is 0, 1, 2, 5, 10, 20, 150, 250, 500μM. The pH of the enzymatic reaction buffer is 5.5, and the components include 50mM NaAc, 5mM EDTA, and 2mM DTT at a concentration of 50mM. The reaction condition of the enzyme digestion is to stand in an incubator at 37°C for 1 hour. The study found that the activity of yeast recombinant CTSS is basically consistent with that of commercial CTSS.

[0027] The CTSS enzyme cleavage reaction system for screening highly effective inhibitors includes substrate COL18A1 (purchased from Shanghai Institude of Biotechnology, item number 5390), recombinant CTSS protein prepared according to the above method, inhibitor LY-3000328 (purchased from MedChem Express, item number HY-15533, CAS: 1373215-15-6) and the CTSS protein inhibitor (Compound BG) described in the present invention. The enzyme cleavage reaction conditions are 37°C and the reaction is allowed to stand for 1 hour. The product Endostatin obtained by the enzyme cleavage reaction is detected by silver staining. In the enzyme cleavage system for screening inhibitors, it was found that the concentration of the LY-3000328 inhibitor had a dose-dependent correlation with the content of Endostatin generated by the enzyme cleavage reaction. In subsequent experiments, 800nM LY-3000328 or compound BG was selected for screening highly effective inhibitors.

[0028] The models used for the anti-liver fibrosis activity of the compound are the TGF-β1-induced Lx-2 cell model and the CCl4-induced liver fibrosis mouse model.

[0029] The present invention constructs a series of compounds having the function of CTSS protein inhibitors. The synthesized CTSS inhibitors are verified by in vivo and in vitro models to have the ability to inhibit CTSS protein expression and resist liver fibrosis, and can be used for the prevention and treatment of liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a DNA sequence of 6 histidine tags fused to the 3' end of the human CTSS gene; the underlined sequence is the coding sequence of the 6 histidine tags;

[0031] Figure 2 . is the DNA sequence of the eGFP (green fluorescent protein) tag fused to the 3' end of the human CTSS gene; the gray part is the DNA sequence of the green fluorescent protein tag;

[0032] Figure 3 The amino acid sequence of the human CTSS protein C-terminus fused with a 6-histidine tag; the 3' end of CTSS is fused with a 6-histidine tag. In the amino acid sequence of the encoded protein, amino acid residues 1-16 are the signal peptide sequence, amino acid residues 17-114 are the propeptide sequence (italic part), and amino acid residues 115-331 are the mature CTSS protein sequence (underlined part), and the last bold part is the 6-histidine sequence;

[0033] Figure 4The amino acid sequence of the human CTSS protein C-terminus fused with an eGFP tag, wherein amino acid residues 1-16 are the signal peptide sequence, amino acid residues 17-114 (italicized part) are the propeptide sequence, amino acid residues 115-331 are the mature CTSS protein sequence, and the last (bold part) is the eGFP protein sequence;

[0034] Figure 5 Efficient and accurate expression of human CTSS protein in yeast system. A: Construction of yeast expression vector pPICZα of CTSS-eGFP and CTSS-6×His fusion genes. In the amino acid sequence of CTSS expression, 1-16 is a signal peptide, 17-114 is a propeptide, and 115-331 is an active peptide. The functional active peptide sequence includes 5 disulfide bonds formed by cysteine. eGFP or 6×His was connected to the C-terminus of CTSS to construct a fusion gene, and methanol was used to induce the expression of CTSS fusion protein. Zeocin is an antibiotic for screening yeast transformants. AOX1 is a methanol-induced promoter. B: Expression process of CTSS fusion gene in yeast. The plasmid was linearized by restriction enzyme digestion and electroporated into yeast competent cells. After screening with the antibiotic zeocin, 3 yeast transformant clones were picked, and then CTSS expression was induced with methanol. The connected eGFP tag protein was used to characterize the localization and expression of CTSS. C: Observation of the localization of CTSS-eGFP in yeast. The localization of GFP indicates that CTSS is evenly distributed in yeast cells and does not aggregate to the vacuole for hydrolysis. The localization information of CTSS indicates that it can not only be accurately expressed in yeast cells, but also that the expressed protein may be active. D: Efficient expression of CTSS-His in yeast. 12% SDS-PAGE gel was used to separate 2μg and 0.6μg of whole-cell lysate proteins before and after methanol induction, and the band of CTSS after methanol induction was detected by silver staining. The arrow points to the protein band that meets the theoretical molecular weight of CTSS produced after methanol induction. E: The CTSS antibody was used to confirm that the band induced in D is CTSS protein. The whole-cell lysate proteins extracted from three transformants before and after methanol induction were separated by 12% SDS-PAGE gel, and the protein expression after methanol induction was detected by CTSS antibody. The arrow points to the CTSS protein band. Yeast transformant No. 2 was named pPICZα-CTSS-His-2# for subsequent CTSS-His protein expression and purification;

[0035] Figure 6 Figure 1 shows nickel column enrichment and molecular sieve purification of CTSS recombinant protein. A: Liquid chromatogram of nickel affinity chromatography and imidazole elution. The smooth curve is the protein fraction separated after elution with different concentrations of imidazole, and the two peaks are P1 and P2. The dashed line is the imidazole concentration during the elution process.

[0036] The left ordinate is the UV absorbance at 280nm, the right ordinate is the linear gradient of imidazole in the buffer solution (%), and the abscissa is the volume (mL). B: Detection of protein in liquid chromatography fractions. The P1 and P2 fractions separated in A were treated with 10mM DTT at 80℃ and then separated by SDS-PAGE gel and stained with Coomassie Brilliant Blue. In the P2 fraction, the arrow points to the band with a theoretical molecular weight of 37kD for CTSS. This fraction was used for separation and purification of molecular sieves. C: Liquid chromatography of molecular sieve purification. The protein after concentration of the P2 fraction in B was separated by Superdex200increase column, and the two peaks were F1 and F2. The coordinate is the UV absorbance at 280nm, and the abscissa is the volume (mL). D: Detection of protein in the F2 component separated by molecular sieve liquid chromatography. The F2 component of the molecular sieve in D was concentrated and separated by 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue. In addition to the CTSS theoretical molecular weight band of 37 kD, a high molecular weight (HMW) band was detected at 80 kD;

[0037] Figure 7 Proteomics guides efficient separation and purification of CTSS. A: Proteomics identification of impurities and abundance analysis. The HMW protein band in Figure 8D was cut off and digested with trypsin. Proteomics identified 108 yeast endogenous proteins. The abundance of the top 10 proteins in terms of signal intensity reached 80%, and these 10 proteins were used for subsequent isoelectric point analysis. B: Isoelectric point analysis of 10 highly abundant yeast endogenous proteins. Among these 10 proteins, 9 proteins were negatively charged in pH 7.4 solution and 1 protein was positively charged. C: Liquid chromatography of CTSS separation and purification by anion exchange column. The anion exchange column separated the proteins concentrated in the P2 fraction in Figure 5A, and the two peaks were F1 and F2. The smooth curve represents the proteins eluted based on different NaCl concentrations. The dashed line represents the conductivity based on the NaCl concentration gradient. The left ordinate is the UV absorbance at 280nm, the right ordinate is the NaCl conductivity, and the abscissa is the volume (mL). D: Purity test of F1 and F2 fractions separated by anion exchange column. The F1 and F2 fractions separated in C were treated with 10mM DTT at 80℃ and then separated by SDS-PAGE gel and stained with Coomassie Brilliant Blue. Only one protein band of CTSS was detected in the F1 fraction, indicating that anion exchange can efficiently separate and purify CTSS protein;

[0038] Figure 8To establish a recombinant CTSS protease cleavage system and screen for efficient targeted early liver fibrosis inhibitors; A: The principle of FRET reaction between CTSS and luminescent substrates. MCA released after CTSS cleaves the FRET substrate produces fluorescence under the conditions of 370nm excitation light and 460nm emission light; B: Km value to evaluate the activity of recombinant CTSS protein. 250nM CTSS enzyme and different concentrations (0, 1, 2, 5, 10, 20, 150, 250, 500μM) of FRET substrate were added to the reaction system and reacted at 37℃ for 1h. Recombinant CTS The Km value of S protein is basically consistent with that of commercial CTSS; C: The enzyme cleavage system for efficient inhibitor screening was established using recombinant CTSS; in the reaction system, COL18A1 is the reaction substrate, and Endostatin is the product of CTSS cleaving COL18A1, which is detected by silver staining results; LY3000328 is a commercial inhibitor of CTSS, and the enzyme cleavage reaction without inhibitor is the control group; the amount of Endostatin generated in the enzyme cleavage reaction with the addition of inhibitor is significantly lower than the amount of product generated without inhibitor, indicating that the system can be used for inhibitor screening;

[0039] Fig. 9 Provide a synthetic route for potential CTSS inhibitors;

[0040] Fig.10 To screen for highly effective inhibitors using recombinant CTSS; A: Figure 8 The system established in C detects the inhibitory effects of 7 compounds on CTSS; B: Comparison of the inhibitory efficiency of LY3000328 and its derivatives through quantitative analysis; Based on the silver staining results in A, the grayscale quantitative analysis of the product Endostatin protein band was performed using ImageJ software, and the enzyme cleavage reaction without inhibitors was used as a control. The grayscale value of Endostatin protein without inhibitors was set to 1 to compare the inhibitory efficiency after adding inhibitors. The results showed that compound F had the highest inhibitory efficiency;

[0041] Fig.11 The effect of compound BG on the expression of FN protein in Lx-2 cells induced by TGF-β1;

[0042] Fig.12 Effects of compound F on liver function and liver fibrosis induced by CCl4 in mice. A: serum alanine aminotransferase (ALT) levels of mice in corn oil control group, CCl4 model group, LY-3000328 treatment group, and compound F treatment group; B: serum aspartate aminotransferase activity (AST) levels of mice in corn oil control group, CCl4 model group, LY3000328 treatment group, and compound F treatment group; C: WB detection of FN expression in liver tissue of mice in corn oil control group, CCl4 model group, LY3000328 treatment group, and compound F treatment group. DETAILED DESCRIPTION

[0043] The present invention is described in detail but not limited by the description of preferred embodiments below.

[0044] All materials can be purchased commercially unless otherwise specified.

[0045] The compound LY-3000328 is a commercial inhibitor of CTSS, and its structural formula is as follows. It was purchased from MedChemExpress, item number HY-15533, CAS: 1373215-15-6.

[0046]

[0047] Example 1 Synthesis of CTSS protein inhibitors

[0048] Based on the target enzyme topology and reported inhibitor structural features, compound BG( Fig. 9 ), the target compound was synthesized by organic synthesis method, and the synthetic route of compound (BG) is as follows:

[0049]

[0050] (1) Preparation of compound S2:

[0051] Measure methanol (500mL) and pour it into a 1L round-bottom flask, weigh S1 (S1 purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., 100g, 0.22mol) and dissolve it in methanol, and add NaBH4 (17g, 0.44mol) to the round-bottom flask in batches. Stir at room temperature for 0.5 hours, the reaction is over, concentrate the methanol, add 500mL of water and 300mL of ethyl acetate to the flask, pour it into a separatory funnel for separation, extract the aqueous phase twice with 600mL of ethyl acetate (300mL each time), combine the organic phases, wash once with saturated sodium chloride (500mL), separate the organic phase, dry it with anhydrous sodium sulfate, and concentrate to obtain a white solid compound S2 (94g, yield 93.2%). 1 H NMR (400MHz, CDCl3) δ7.44(d,J=2.5Hz,1H),7.28(dd,J=8.7,2.5Hz,1H),6.73(d ,J=8.7Hz,1H),4.75(q,J=5.1Hz,1H),4.31-4.16(m,2H),2.16-2.06(m,1H),2.05 -1.98(m,1H),1.89(d,J=5.1Hz,1H); 13C NMR (100MHz, CDCl3) δ153.86,132.67,132.22,126.42,119.13,112.57,63.18,62.33,30.78; ESI-MS 251.04[M+Na] + ,253.06[M+Na] +

[0052] (2) Preparation of compound S3:

[0053] Measure toluene (500 mL) and pour it into a 1L round-bottom flask. Weigh S2 (50 g, 0.22 mol) and p-toluenesulfonic acid (3.75 g, 0.022 mol) and add them to toluene. Add a water separator and a reflux condenser to the top of the round-bottom flask, stir under reflux for 7 hours, and the reaction is completed and cooled to room temperature. Add saturated sodium bicarbonate aqueous solution (300 mL) to the flask, continue stirring for 10 minutes, pour into a separatory funnel for separation, wash the organic phase twice with water (600 mL) (300 mL each time), wash once with saturated sodium chloride (500 mL), separate the organic phase, dry it with anhydrous sodium sulfate, and concentrate to obtain a colorless oil compound S3 (45 g, yield 97.8%). 1 H NMR(400MHz,DMSO)δ7.25(brs,1H),7.26-7.22(m,1H),6.70(brd,J=8.0Hz,1H ),6.53-6.44(m,1H),5.94(dt,J=9.9,3.5Hz,1H),4.81(dd,J=3.5,2.0Hz,2H); 13 C NMR (100MHz, DMSO) δ152.76,131.30,128.73,124.35,124.32,122.65,117.52,112.39,65.27.ESI-MS 233.02[M+Na] + ,235.01[M+Na] +

[0054] (3) Preparation of compound S4:

[0055] Dimethyl sulfoxide (450 mL) and water (100 mL) were measured and poured into a 1L round-bottom flask, and S3 (45 g, 0.21 mol) was weighed and added to the round-bottom flask. NBS (44 g, 0.25 mol) was added in batches in small amounts and repeatedly, and stirred at room temperature for 2 hours, and the reaction was completed. In a 5L measuring cup, saturated sodium bicarbonate aqueous solution (1 L) and dichloromethane (1 L) were added respectively, and the reaction solution was poured into the measuring cup, and the organic phase was separated. The organic phase was washed four times with water (4 L) (1 L each time), and washed once with saturated sodium chloride (500 mL), and the organic phase was separated and dried over anhydrous sodium sulfate, and concentrated to obtain a white solid compound S4 (60.6 g, yield 92.3%). 1 H NMR (400MHz, DMSO) δ7.48(d,J=2.5Hz,1H),7.38(dd,J=8.8,2.5Hz,1H),6.83(d,J=8.8Hz,1H),6 .30(d,J=5.6Hz,1H),4.66(dd,J=5.6,4.0Hz,1H),4.48-4.45(m,1H),4.44(m,1H),4.31(m,1H); 13 C NMR (100MHz, DMSO) δ152.10,132.94,131.95,124.88,118.56,111.83,67.03,65.31,48.41; ESI-MS 331.89[M+Na] + ,333.89[M+Na] +

[0056] (4) Preparation of compound S5:

[0057] Pour isopropanol (500 mL) into a 2L round-bottom flask, weigh S4 (60 g, 0.19 mol) and add it to the round-bottom flask. Add ammonia water (300 mL) dropwise at room temperature, stir at room temperature for 20 hours, and the reaction is complete. Add water (500 mL) to the reaction solution, cool the system to 0°C, and continue stirring for 0.5 hours. A large amount of white solid is generated, filtered, and the filter cake is washed twice with petroleum ether (100 mL each time) to obtain a white solid compound S5 (45 g, yield 94.6%). 1H NMR (400MHz, DMSO) δ7.52(d,J=2.6Hz,1H),7.24(dd,J=8.7,2.6Hz,1H),6.70(d,J=8.7Hz,1H),5.18(d,J=3.5Hz,1 H,-OH),4.15(dd,J=11.0,2.4Hz,1H),3.90(ddd,J=11.0,5.6,1.3Hz,1H),3.65-3.52(m,2H),2.08(brs,2H,-NH2); 13 C NMR (100MHz, DMSO) δ152.82,132.26,130.30,128.41,117.98,111.27,67.81,66.55,51.73; ESI-MS 266.15[M+Na] + ,268.26[M+Na] +

[0058] (5) Preparation of compound S6:

[0059] Take tetrahydrofuran (60mL) and water (60mL) respectively and pour them into a 250mL round-bottom flask, weigh S5 (10g, 0.04mol) and sodium bicarbonate (6.88g, 0.08mol) respectively and add them into the round-bottom flask. Cool the reaction system to 0-5°C, control the temperature below 15°C, add p-fluorobenzoyl chloride (7.14g, 0.045mol) dropwise, continue stirring for 1 hour after the addition is complete, and the reaction is completed. Take another 500mL round-bottom flask, add methyl tert-butyl ether (120mL) and water (120mL) thereto, pour the reaction solution into the above-mentioned mixed solvent, separate the organic phase, and wash the organic phase once with water (500mL) and saturated sodium chloride (500mL) respectively. The organic phase was separated and dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was slurried with DCM / PE (5 mL / 120 mL) for one hour and filtered to obtain a light yellow solid compound S6 (13.6 g, yield 90.7%). 1 H NMR (400MHz, DMSO) δ8.81(d,J=8.0Hz,1H),7.99(dd,J=8.8,5.5Hz,1H),7.36-7.25(m,4H),6.80(d,J=8.8Hz,1H),5.50(d,J =5.3Hz,1H),4.99(dd,J=8.0,5.3Hz,1H),4.21(dd,J=11.2,2.5Hz,1H),4.03(ddd,J=11.2,5.9,1.2,1H),3.96-3.91(m,1H); 13C NMR(100MHz,DMSO)δ165.13,162.72,153.60,132.09,131.13,130.51,130.24, 130.15,124.42,118.41,115.22,115.01,111.47,67.23,64.74,49.86; ESI-MS 388.15[M+Na] + ,340.29[M+Na] +

[0060] (6) Preparation of compound S7a:

[0061] Toluene (20 mL) and water (2 mL) were measured and poured into a 100 mL round-bottom flask, and S6 (400 mg, 1.09 mmol), Pd (dppf) Cl2 (60 mg, 0.11 mmol), phenylboric acid (200 mg, 1.63 mmol) and potassium carbonate (300 mg, 2.18 mol) were weighed and added to the round-bottom flask. Under nitrogen protection, the reaction was carried out at 100 ° C for 20 hours, and the reaction was completed. The reaction solution was concentrated, and the residue was eluted by silica gel column chromatography with petroleum ether / ethyl acetate (volume ratio 1:1) and concentrated to obtain a white solid compound S7a (110 mg, yield 27.8%). 1 H NMR (400MHz, DMSO) δ8.85(d,J=8.0Hz,1H),8.00(ddd,J=8.8,5.5,2.4Hz,2H),7.55-7.51(m,2H),7.48(dd,J=8.5,2.4Hz,1H),7.44-7.37(m,3H),7.28 (td,J=8.1,7.4,4.4Hz,3H),5.49(d,J=4.2Hz,1H),5.09(dd,J=8.0,5.1Hz ,1H),4.26(dd,J=11.2,2.3Hz,1H),4.11-4.03(m,1H),3.97(m,1H).ESI-MS 364.25[M+H] +

[0062] (7) Preparation of compound S7b:

[0063] Dimethyl sulfoxide (10 mL) was measured and poured into a 100 mL round-bottom flask, and S6 (1 g, 2.73 mmol), Allylpalladium (II) Chloride Dimer (100 mg, 0.273 mmol), Tri-tert-butylphosphinetetrafluoroborate (159 mg, 0.55 mmol), benzylamine (878 mg, 8.19 mmol) and sodium tert-butoxide (918 mg, 9.56 mmol) were weighed and added into the round-bottom flask. The mixture was stirred at 100 °C for 45 minutes under nitrogen protection, and the reaction was completed. The reaction solution was cooled to room temperature, and ethyl acetate (30 mL) and water (50 mL) were added for extraction. The organic phase was washed 5 times with water (250 mL) (50 mL each time), and once with saturated brine (50 mL). The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated. The residue was chromatographed on a silica gel column and eluted with petroleum ether / ethyl acetate (volume ratio of 4:1). The product was concentrated to give compound S7b (500 mg, yield 51.1%) as a white solid. 1 HNMR (400MHz, DMSO) δ8.67(d,J=8.5Hz,1H),8.00-7.81(m,3H),7.57-7.40(m,1H),7.34-7.22(m,4H),7.16(dt,J=14.7,7.1Hz,3H),6.53(d, J=8.4Hz,1H),6.45-6.35(m,2H),5.85(t,J=6.3Hz,1H),5.29(d,J=4.4Hz,1H),4.95(t,J=7.2Hz,1H),4.21-3.99(m,3H),3.93-3.72(m,2H); 13 CNMR(101MHz,DMSO)δ165.18,162.60,145.40,130.92,130.18,130.08,128.06(2×C),127.12(2× C),126.39,122.50,116.13,115.09,114.87,113.16,112.39,67.48,66.00,50.96,47.15; ESI-MS 393.54[M+H] +

[0064] (8) Preparation of compound S7c:

[0065] Dimethyl sulfoxide (1500 mL) was measured and poured into a 250 mL round-bottom flask, and S6 (8.5 g, 23.2 mmol), Allylpalladium (II) Chloride Dimer (850 mg, 2.32 mmol), Tri-tert-butylphosphine tetrafluoroborate (1.34 g, 4.64 mmol), N-Boc-piperazine (13 g, 69 mmol) and sodium tert-butoxide (7.8 g, 81 mmol) were weighed and added into the round-bottom flask. The mixture was stirred at 100 ° C for 45 minutes under nitrogen protection, and the reaction was completed. The reaction solution was cooled to room temperature, and ethyl acetate (400 mL) and water (400 mL) were added for extraction. The organic phase was washed 5 times with water (2 L) (400 mL each time), and once with saturated brine (400 mL). The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated. The residue was chromatographed on a silica gel column and eluted with petroleum ether / ethyl acetate (volume ratio of 2:1). The product was concentrated to give a light yellow solid compound S7c (4.2 g, yield 38.9%). 1 H NMR (400MHz, DMSO) δ8.76 (d, J=8.1Hz, 1H), 7.99 (dd, J=8.8, 5.6Hz, 2H), 7.29 (t, J=8.9Hz,1H),6.86(dd,J=8.9,2.9Hz,1H),6.78-6.67(m,2H),5.37(d,J=4.2Hz, 1H),4.96(dd,J=8.1,5.1Hz,1H),4.15(dd,J=11.0,2.3Hz,1H),3.98-3.91(m,1H ),3.90-3.86(m,1H),3.40(t,J=5.2Hz,4H),2.87(t,J=5.2Hz,4H),1.39(s,9H); 13 C NMR (101MHz, DMSO) δ165.10,162.64,153.77,148.43,145.34,130.80,130.22,130.13,121.79,118.36,1 17.93,116.38,115.16,114.94,78.87(2×C),66.93,65.71,50.37(2×C),50.04(2×C),28.02(3×C); ESI-MS 472.22[M+H] +

[0066] (9) Preparation of compound S7d:

[0067] DCM (100 mL) was measured and poured into a 250 mL round-bottom flask, S7c (4.2 g, 8.1 mmol) was weighed and added to the round-bottom flask, the reaction system was cooled to 0°C, TFA (27.6 g, 240 mmol) was added dropwise, and the reaction was stirred at room temperature for 1.5 hours to terminate the reaction. The reaction solution was concentrated, water (100 mL) was added, pH>14 was adjusted with 20% aqueous sodium hydroxide solution, and extracted five times with ethyl acetate (500 mL) (100 mL each time), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a light yellow solid compound S7d (3.2 g, yield 97.0%). 1 H NMR (400MHz, DMSO) δ8.76(d,J=8.2Hz,1H),7.99(dd,J=8.8,5.6Hz,2H),7.29(t,J=8.9Hz,2H),6.81(dd,J=8.9,2.9Hz,1H),6.70(d,J=8.9Hz,1H), 6.65(d,J=2.9Hz,1H),5.40(brs,1H),5.09-4.87(m,1H),4.27-4.11(m,1 H),3.91(m,2H),2.82(dd,J=6.4,3.5Hz,4H),2.76(dd,J=6.4,3.5Hz,4H); 13 C NMR (100MHz, DMSO) δ165.21,162.64,147.83,146.24,130.83,130.19,130.10,121.84,117. 66,116.83,116.28,115.18,114.97,67.11,65.75,51.04(2×C),50.62,45.69(2×C); ESI-MS 372.17[M+H] +

[0068] (10) Preparation of compound S7e:

[0069] Methanol (20 mL) was measured and poured into a 100 mL round-bottom flask, S7d (1 g, 2.68 mmol) and p-tolualdehyde (486 mg, 4.02 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 30 minutes, sodium cyanoborohydride (253 mg, 4.02 mmol) was added, stirred for 3 hours, sodium cyanoborohydride (253 mg, 4.02 mmol) was added, and stirring was continued for 3 hours, and the reaction was completed. The reaction solution was extracted with ethyl acetate (50 mL) and water (50 mL), and the aqueous phase was extracted again with ethyl acetate (50 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated, and the residue was chromatographed on a silica gel column, eluted with dichloromethane / methanol (volume ratio 20:1), and concentrated to obtain a light yellow solid compound S7e (830 mg, yield 64.8%). 1 H NMR (400MHz, DMSO) δ8.75(d,J=8.2Hz,1H),8.03-7.94(m,2H),7.28(t,J=8.9Hz,1H),7.17(d ,J=7.8Hz,2H),7.11(d,J=7.8Hz,2H),6.82(dd,J=8.9,2.9Hz,1H),6.69(d,J=8.9Hz,1H),6.6 6(d,J=2.9Hz,1H),5.36(d,J=4.1Hz,1H),4.96(dd,J=8.2,5.2Hz,1H),4.14(dd,J=10.7,2.1 Hz,1H),3.90(m,2H),3.43(s,2H),2.92(t,J=4.9Hz,4H),2.44(t,J=4.9Hz,4H),2.27(s,3H); 13 C NMR (100MHz, DMSO) δ165.15,165.09,162.62,147.92,145.46,135.91,134.93,130.82,130.80 130.19,130.10,128.82,128.70,121.75,117.62,116.98,116.29,115.16,1 14.94,66.98,65.73,61.74,52.54(2×C),50.49,49.75(2×C),20.66; ESI-MS 476.27[M+H] +

[0070] (11) Preparation of compound S7f:

[0071] Methanol (20 mL) was measured and poured into a 100 mL round-bottom flask, S7d (1 g, 2.68 mmol) and 3-oxetanone (195 mg, 2.69 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 30 minutes, sodium cyanoborohydride (170 mg, 2.69 mmol) was added, stirred for 3 hours, 3-oxetanone (195 mg, 2.69 mmol) and sodium cyanoborohydride (170 mg, 2.69 mmol) were added, and stirring was continued for 3 hours, and the reaction was completed. The reaction solution was extracted with ethyl acetate (50 mL) and water (50 mL), and the aqueous phase was extracted again with ethyl acetate (50 mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the residue was eluted with dichloromethane / methanol (volume ratio 40:1) by silica gel column chromatography, and concentrated to obtain a light yellow solid compound S7f (700 mg, yield 60.9%). 1 H NMR(400MHz,DMSO)δ8.76(d,J=8.2Hz,1H),8.04-7.94(m,2H),7.29(t,J=8.9Hz,1H),6.84(dd ,J=8.9,2.9Hz,1H),6.71(d,J=8.9Hz,1H),6.68(d,J=2.9Hz,1H),5.36(d,J=4.1Hz,1H),4.97 (dd,J=8.2,5.2Hz,1H),4.53(t,J=6.5Hz,2H),4.42(t,J=6.0Hz,2H),4.14(dd,J=10.6,2.1Hz ,1H),3.98-3.84(m,2H),3.40(p,J=6.2Hz,1H),2.95(t,J=5.0Hz,4H),2.35(t,J=5.0Hz,4H); 13 C NMR (100MHz, DMSO) δ165.17,162.64,147.98,145.40,130.83,130.20,130.11,121.81,117.64,116.9 1,116.33,115.18,114.96,74.31(2×C),67.02,65.73,58.44,50.52,49.42(2×C),49.06(2×C); ESI-MS 428.16[M+H] +

[0072] (12) Preparation of compound B (S8a):

[0073] Tetrahydrofuran (10 mL) was measured and poured into a 100 mL round-bottom flask, S7a (110 mg, 0.3 mmol) and CDI (122 mg, 0.75 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 1 hour, the reaction solution was cooled to -5 ° C, methylamine aqueous solution (47 mg, 0.6 mmol) was slowly added dropwise, and stirring was continued for 15 minutes to terminate the reaction. 20% sodium hydroxide aqueous solution (10 mL) was added to the reaction system, stirred at room temperature for 10 minutes, extracted with ethyl acetate (50 mL), and the organic phase was washed once with 20% sodium hydroxide aqueous solution (10 mL), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, and the crude product was slurried with ethyl acetate (5 mL) to obtain a white solid compound B (S8a) (110 mg, yield 88.7%). 1 H NMR(400MHz, DMSO)δ9.01(d,J=7.7Hz,1H),7.98(dd,J=8.7,5.6Hz,2H),7.61-7.48(m,4H),7.40(t,J=7.7Hz,2H),7.32-7.26(m,3H), 7.23(q,J=4.6Hz,1H),6.98(d,J=8.7Hz,1H),5.17(dd,J=8.0,3.7Hz,1H),4.97(q,J=2.2Hz,1H),4.36(dd,J=11.9,1.8Hz,1H),4.28(m 1H),2.56(d,J=4.6Hz,3H); 13 CNMR(100Hz,DMSO)δ165.26,164.97,162.79,155.60,153.85,139.69,133.13,130.37,130.28,128.91(2×C) ,128.58,127.36,126.85,126.18(2×C),120.85,116.98,115.27,115.06,67.84,64.11,46.98,26.93; ESI-MS 443.10[M+Na] +

[0074] (13) Preparation of Compound C (S8b):

[0075] Tetrahydrofuran (10 mL) was measured and poured into a 100 mL round-bottom flask, S7b (250 mg, 0.63 mmol) and CDI (207 mg, 1.27 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 3 hours, the reaction solution was cooled to -5 ° C, methylamine aqueous solution (40 mg, 1.27 mmol) was slowly added dropwise, and stirring was continued overnight to terminate the reaction. 20% sodium hydroxide aqueous solution (10 mL) was added to the reaction system, stirred at room temperature for 10 minutes, extracted with ethyl acetate (50 mL), and the organic phase was washed once with 20% sodium hydroxide aqueous solution (10 mL), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, and the crude product was slurried with ethyl acetate (10 mL) to obtain a white solid compound C (S8b) (220 mg, yield 76.9%). 1 H NMR(400MHz,DMSO)δ8.86(d,J=8.2Hz,1H),7.93(dd,J=8.7,5.6Hz,2H),7.37-7.26(m,4 H),7.23-.11(m,4H),6.59(d,J=8.7Hz,1H),6.47(dd,J=8.8,2.8Hz,1H),6.41(d,J=2.8H z,1H),5.91(t,J=6.2Hz,1H),5.03(dd,J=8.2,4.7Hz,1H),4.84(td,J=5.0,2.2Hz,1H),4 .19(dd,J=11.6,2.2Hz,1H),4.16-.12(m,2H),4.07-3.98(m,1H),2.53(d,J=4.6Hz,3H); 13 C NMR(100MHz,DMSO)δ165.19,164.93,162.72,155.69,145.21,143.09,140.44,130.53,130.22,128.10(2×C),12 7.17(2×C),126.45,121.06,116.53,115.20,114.98,113.82,112.46,68.68,64.13,47.41,47.05,26.87; ESI-MS 450.17[M+H] +

[0076] (14) Preparation of compound D (S8d):

[0077] Tetrahydrofuran (10 mL) was measured and poured into a 100 mL round-bottom flask, S7c (500 mg, 1.06 mmol) and CDI (300 mg, 1.85 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 3 hours, the reaction solution was cooled to -5 ° C, methylamine aqueous solution (148 mg, 1.91 mmol) was slowly added dropwise, and stirring was continued for 15 minutes to terminate the reaction. 20% sodium hydroxide aqueous solution (20 mL) was added to the reaction system, stirred at room temperature for 10 minutes, and then extracted with ethyl acetate (100 mL), and the organic phase was washed once with 20% sodium hydroxide aqueous solution (20 mL), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, and the crude product was slurried with ethyl acetate (20 mL) to obtain a gray solid compound S8c (520 mg, yield 92.9%). 1 H NMR (400MHz, DMSO) δ8.93 (d, J=7.8Hz, 1H), 7.98 (dd, J=8.8, 5.6Hz, 2H), 7.29 (t, J=8.9Hz, 1H),7.19(q,J=4.5Hz,1H),6.90(dd,J=9.0,2.9Hz,1H),6.78(d,J=6.0Hz,1H),6.76(s,1H ),5.08–5.01(m,1H),4.87(td,J=4.0,1.8Hz,1H),4.26(dd,J=11.8,1.8Hz,1H),4.20–4.1 0(m,1H),3.41(t,J=5.0Hz,4H),2.90(t,J=5.0Hz,4H),2.54(d,J=4.5Hz,3H),1.39(s,9H); 13 C NMR (100MHz, DMSO) δ165.71,165.39,163.24,156.11,154.26,148.68,146.14,130.97,130.78,120.92,119.29,1 18.49,117.21,115.71,115.50,79.37(2×C),68.71,64.31,50.39(2×C),47.66(2×C),28.52(3×C),27.38; ESI-MS 529.26[M+H] +

[0078] Measure dichloromethane (10 mL) and pour it into a 25 mL round-bottom flask, weigh S8c (250 mg, 0.47 mmol). Cool the reaction system to 0 ° C, add TFA (1.6 g, 14.2 mmol) dropwise, stir at room temperature for 1.5 hours, and the reaction is completed. Concentrate the reaction solution, add water (100 mL), adjust the pH>14 with 20% sodium hydroxide aqueous solution, extract five times with ethyl acetate (500 mL) (100 mL each time), separate the organic phase, dry it with anhydrous sodium sulfate, and concentrate to obtain a light yellow solid compound D (S8d) (150 mg, yield 74.3%); 1 H NMR (400MHz, DMSO) δ8.92(d,J=4.5Hz,1H),8.00-7.93(m,2H),7.30(t,J=8.6Hz,2H),7.17(d,J=5.6Hz,1H),6.95-6.88(m,1H),6.79(d, J=9.6Hz,2H),5.07(d,J=4.7Hz,1H),4.90(s,1H),4.27(dd,J=11.7Hz,4.2Hz,1H),4.16(dd,J=11.7,4.2Hz,1H),2.54(d,J=4.5Hz,3H); 13 C NMR (100MHz, DMSO) δ165.72,165.41,163.25,156.11,148.77,145.70,130.92,130.76,121.12,119 .10,118.15,117.31,115.73,115.52,68.68,64.44,48.56(2×C),47.69,44.16(2×C),27.38; ESI-MS 429.19[M+H] +

[0079] (15) Preparation of compound E (S8e):

[0080] Tetrahydrofuran (30 mL) was measured and poured into a 100 mL round-bottom flask, S7e (500 mg, 1.05 mmol) and CDI (256 mg, 1.57 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 12 hours, the reaction solution was cooled to -5 ° C, methylamine aqueous solution (225 mg, 2.1 mmol) was slowly added dropwise, and stirring was continued overnight to terminate the reaction. 20% sodium hydroxide aqueous solution (20 mL) was added to the reaction system, stirred at room temperature for 1 hour, extracted with ethyl acetate (100 mL), and the organic phase was washed once with 20% sodium hydroxide aqueous solution (20 mL), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, and the crude product was slurried with ethyl acetate (20 mL) to obtain a white solid compound E (S8e) (290 mg, yield 45.3%). 1 H NMR (400MHz, DMSO) δ8.93(d,J=7.9Hz,1H),7.96(dd,J=8.7,5.7Hz,2H),7.90(t,J=6.2Hz,1H),7.35-7 .24(m,4H),7.23-7.15(m,5H),7.11(d,J=7.8Hz,2H),6.86(dd,J=9.0,2.9Hz,1H),6.76(d,J=8.9Hz,1 H),6.72(d,J=2.8Hz,1H),5.08(dd,J=8.0,4.0Hz,1H),4.92(td,J=4.3,2.0Hz,1H),4.26(dd,J=11.8, 1.9Hz,1H),4.21-4.10(m,3H),3.43(s,2H),2.95(t,J=5.0Hz,4H),2.45(t,J=4.9Hz,4H),2.27(s,3H); 13 C NMR(100MHz,DMSO)δ165.21,165.00,162.74,155.44,147.68,145.81,139.54, 135.93,134.92,130.48,130.28,128.84(2×C),128.71(2×C),128.21(2×C),126 .97(2×C),126.74,120.47,118.04,117.00,116.66,115.22,115.00,68.45,63 .91,61.76,52.53(2×C),49.63(2×C),47.38,43.75,20.67; ESI-MS609.36[M+H] +

[0081] (16) Preparation of compound F (S8f):

[0082] Tetrahydrofuran (20 mL) was measured and poured into a 100 mL round-bottom flask, S7f (350 mg, 0.82 mmol) and CDI (200 mg, 1.22 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 2 hours, the reaction solution was cooled to -5 ° C, benzylamine (176 mg, 1.62 mmol) was slowly added dropwise, and stirring was continued overnight to terminate the reaction. 20% sodium hydroxide aqueous solution (10 mL) was added to the reaction system, stirred at room temperature for 1 hour, extracted with ethyl acetate (50 mL), and the organic phase was washed once with 20% sodium hydroxide aqueous solution (10 mL), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, and the crude product was slurried with hot methanol (40 mL) to obtain white solid compound F (S8f) (200 mg, yield 43.6%). 1 H NMR (400MHz, DMSO) δ8.94(d,J=7.9Hz,1H),7.97(dd,J=8.7,5.6Hz,2H),7.91(t,J=6.2Hz,1H),7.39-7.25(m,4 H),7.23-7.18(m,3H),6.89(dd,J=9.0,2.9Hz,1H),6.77(d,J=8.9Hz,1H),6.74(d,J=2.9Hz,1H),5.09(dd,J=7 .9,4.1Hz,1H),4.93(td,J=4.3,1.9Hz,1H),4.54(t,J=6.5Hz,2H),4.43(t,J=6.0Hz,2H),4.27(dd,J=11.9,2. 0Hz, 1H), 4.17 (td, J = 7.6, 6.2, 4.1Hz, 3H), 3.41 (p, J = 6.3Hz, 1H), 2.98 (t, J = 4.8Hz, 4H), 2.36 (t, J = 4.8Hz, 4H); 13 C NMR (100MHz, DMSO) δ165.22,164.98,162.75,155.43,147.72,145.73,139.54,130.48,130.27,128.21(2×C),126.96(2×C),126.74, 120.51,118.03,116.95,116.68,115.23,115.01,74.32(2×C),68.42,63.92,58.44,49.28(2×C),49.05(2×C),47.39,43.75; ESI-MS 561.24[M+H] +

[0083] (17) Preparation of compound G (S8g):

[0084] Tetrahydrofuran (20 mL) was measured and poured into a 100 mL round-bottom flask, S7f (350 mg, 0.82 mmol) and CDI (200 mg, 1.22 mmol) were weighed and added to the round-bottom flask, stirred at room temperature for 1 hour, the reaction solution was cooled to -5 ° C, isopropylamine (100 mg, 1.63 mmol) was slowly added dropwise, and stirring was continued overnight to terminate the reaction. 20% sodium hydroxide aqueous solution (10 mL) was added to the reaction system, stirred at room temperature for 1 hour, extracted with ethyl acetate (50 mL), and the organic phase was washed once with 20% sodium hydroxide aqueous solution (10 mL), the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, and the crude product was slurried with ethyl acetate (10 mL) to obtain a white solid compound G (S8g) (280 mg, yield 66.7%). 1 H NMR (400MHz, DMSO) δ8.93(d,J=7.8Hz,1H),7.97(dd,J=8.8,5.6Hz,2H),7.30(t,J=8.9Hz,2H),7.24(d,J=7. 7Hz,1H),6.89(dd,J=9.0,2.9Hz,1H),6.76(d,J=8.9Hz,1H),6.74(d,J=2.9Hz,1H),5.04(d,J=7.6Hz,1H),4 .87(s,1H),4.54(t,J=6.5Hz,2H),4.43(t,J=6.0Hz,2H),4.25(dd,J=11.9,1.8Hz,1H),4.18-4.12(m,1H),3 .63-3.53(m,1H),3.41(p,J=6.3Hz,1H),2.98(t,J=5.0Hz,4H),2.36(t,J=4.9Hz,4H),1.01(t,J=6.7Hz,6H); 13 C NMR (100MHz, DMSO) δ165.22,164.91,162.74,154.24,147.76,145.70,130.48,130.28,120.38,118.06,117.11,116. 68,115.22,115.01,74.32(2×C),67.99,63.83,58.44,49.30(2×C),49.05(2×C),47.26,42.36,22.43,22.38.ESI-MS 513.23[M+H] +

[0085] Example 2 Screening for highly effective CTSS protein inhibitors targeting early liver fibrosis

[0086] 2.1 Expression of highly active recombinant CTSS protein

[0087] 1. Construction of yeast recombinant CTSS expression vector

[0088] The recombinant human CTSS protein gene sequence of the present invention is the same as the gene bank GenBank No. NM_004079.5. A coding sequence of a 6-histidine tag (6×His) or a coding sequence of a green fluorescent protein (eGFP) tag is introduced at its 3′ end. Figure 1 and 2 The amino acid sequence of the protein encoded by CTSS3' end after fusion of 6 histidine tags is as follows Figure 3 The amino acid sequence of the protein encoded by CTSS3' end after fusion of eGFP tag is as follows Figure 4 .

[0089] After the fusion gene was inserted into the AOX1 promoter of the pPICZα vector (Thermo Fisher, Catalog No. V19020), the recombinant human CTSS yeast expression vectors were obtained and named pPICZα-CTSS-His and pPICZα-CTSS-eGFP ( Figure 5 A).

[0090] 2. Construction of yeast recombinant CTSS engineering strain

[0091] Take 30 μg of recombinant pPICZα-CTSS-His and pPICZα-CTSS-eGFP plasmids respectively, and use Pmel endonuclease (R0560S, BioLabs) to digest at 37°C for 3 h according to the company's instructions. Use DNA recovery kit (DP209-03, Tiangen) to recover the digested DNA, add 20 μL of water to elute, and divide the DNA recovery solution into two parts and store at 4°C.

[0092] The yeast strain 1163 (Invitrogen, Genbank accession no. XM_002489831) preserved in the laboratory was inoculated into YPD (Yeast extract Peptone Dextrose: 1% yeast extract, 2% peptone, 2% glucose) medium and cultured at 30°C, 200 rpm shaking on a shaker for 20-24 h. On the day of transformation, 100 OD 600Yeast liquid, centrifuge at 2500rpm for 5min. Add 30mL YPD medium to resuspend the yeast precipitate, add 6mL pH 8.0, 1M HEPES buffer, and then add 1mL 1M DTT, mix well, and let stand in a 30℃ incubator for 15min. Centrifuge at 2500rpm for 5min, add 25mL 1M precooled sorbitol solution to the precipitated yeast cells to wash, repeat once. Finally, add 500μL 1M Sorbitol to resuspend the cell precipitate for transformation.

[0093] Take 10 μL of linearized DNA plasmid and add it to 50 μL of competent cells of Pichia pastoris 1163 strain, flick to mix, and let stand on ice for 5 minutes; transfer the yeast competent cells containing plasmid to a pre-cooled electroporation cup; set the BTX electroporation instrument conditions (2kV, 5ms), and electroporate once; after electroporation, immediately add 600 μL of pre-cooled Sorbitol to the electroporation cup, transfer all the liquid to a 1.5mL EP (Eppendorf) tube, and let it stand and culture in a 30℃ incubator for 1 hour; then add 500 μL YPD medium to the same tube and culture it in a 30℃ shaking incubator for 1 hour; take 20 μL and spread it on a YPD plate containing 500 μg / mL Zeocin, and culture it in a 30℃ incubator for 3 to 4 days until the clone morphology is good. Perform clonal PCR verification on the single clone to obtain a yeast positive clone strain containing the recombinant human CTSS gene.

[0094] 3. Research on biologically active CTSS yeast recombinant expression engineering bacteria

[0095] The DNA sequence encoding the complete reading frame of CTSS was used to construct a recombinant yeast expression plasmid. The translation products include the signal peptide, propeptide and mature peptide of human cysteine ​​cathepsin S. The signal peptide may be hydrolyzed and removed by proteases during transport through the lumen of the endoplasmic reticulum, and the remaining protein fragments are then glycosylated by the propeptide in the Golgi apparatus. The glycosylated propeptide is labeled with mannose-6-phosphate and can be sorted into the lysosomal vacuole. Once CTSS reaches the lysosomal vacuole, it can also be degraded by endogenous proteases in the cell, resulting in the failure of recombinant expression. Therefore, the distribution of recombinantly expressed CTSS in the cell determines the success or failure of CTSS expression. In order to investigate the distribution of recombinantly expressed CTSS in the cell, we constructed pPICZα-CTSS-eGFP.

[0096] Three yeast transformants transformed with pPICZα-CTSS-eGFP were selected and cloned into 3 mL of BMG medium (0.1 M phosphate buffer (pH 6.0), 1.2% glycerol, 1.34% YNB (Yeast Nitrogen Base), 0.04% Biotin) containing 200 μg / mL Zeocin, and cultured at 30°C, 200 rpm shaking on a shaker for 24 h ( Figure 5 B). The cells were obtained by centrifugation, and 2 mL of BMM medium (0.1 M phosphate buffer (pH 6.0), 1.2% glycerol, 1.34% YNB, 0.04% Biotin) containing a final concentration of 1% methanol was added to resuspend the cells, and the cells were induced in a shaking incubator at 25°C for 24 hours. The yeast cells before and after methanol induction were collected by centrifugation. The C-terminus of CTSS was fused with eGFP to locate the location of the CTSS protein in the cell. The yeast strain expressing CTSS in pPICZα-CTSS-eGFP induced by methanol was observed under a fluorescence microscope, and it was found that the recombinant protein was evenly distributed in the cytoplasm instead of being aggregated in the vacuole that is prone to specific protein degradation ( Figure 5 C), indicating that the CTSS-eGFP fusion protein is not only accurately expressed in yeast cells, but also active. This lays the technical foundation for the development of biologically active CTSS recombinant proteins.

[0097] 4. Screening of engineered strains that efficiently express CTSS

[0098] Three yeast transformants were selected and cloned into 3 mL of BMG medium (0.1 M phosphate buffer (pH 6.0), 1.2% glycerol, 1.34% YNB (Yeast Nitrogen Base), 0.04% Biotin) containing 200 μg / mL Zeocin, and cultured at 30°C, 200 rpm shaking for 24 h ( Figure 5 B). The cells were obtained by centrifugation, and 2 mL of BMM medium (0.1 M phosphate buffer (pH 6.0), 1.2% glycerol, 1.34% YNB, 0.04% Biotin) containing a final concentration of 1% methanol was added to resuspend the cells, and the cells were induced in a shaking incubator at 25°C for 24 h. Yeast cells before and after methanol induction were collected by centrifugation.

[0099] RIPA lysis buffer (50 mM Tris-HCl (PH7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail) containing glass beads was added to the collected cells, and the yeast cells were lysed by shaking for 2 minutes, and the total cell protein was extracted.

[0100] 2 μg and 0.6 μg of whole cell protein before and after induction were taken, respectively, separated by 10% SDS-PAGE gel, and detected by silver staining. An additional high-abundance protein band produced after methanol induction appeared at about 40 kD ( Figure 5 D), which is consistent with the theoretical molecular weight of complete CTSS-His, indicating that we have successfully expressed CTSS-His in genetically recombinant yeast and achieved high-efficiency expression.

[0101] Furthermore, we used anti-CTSS antibody (ab134157, Abcam) for protein hybridization and confirmed that the protein band that appeared after induction was CTSS ( Figure 5 E).

[0102] The study found that among the three clones selected, only two # , 3 # The clones were successfully expressed human CTSS protein after methanol induction. # The strain with the highest expression level of CTSS was named CTSS-His-2 # strain ( Figure 5 E) for subsequent research.

[0103] 5. Study on conditions for efficient expression of CTSS

[0104] In order to determine the methanol induction concentration with the highest protein expression, we used four methanol concentrations, namely 0.1%, 0.3%, 0.7%, and 1%, to induce CTSS-His-2 # CTSS protein expression in strains. CTSS antibody was used to detect protein expression induced by different methanol concentrations, and the grayscale quantitative analysis of protein bands was performed using ImageJ software. Table 1 shows the yield of CTSS-His protein induced by different methanol concentrations.

[0105] Table 1. CTSS expression induced by different methanol concentrations*

[0106]

[0107] *Table 1. Yield of CTSS-His protein induced by different methanol concentrations. Figure 5 After the pPICZα-CTSS-His-2# yeast transformant cells screened in E were induced with different concentrations of methanol, the protein expression was detected by CTSS antibody, and the grayscale quantitative analysis of the protein bands was performed using ImageJ software.

[0108] The ratio of the protein gray value of all methanol concentrations to the value corresponding to 0.1% was used to characterize the protein yield under methanol concentration induction. The results showed that 0.7% methanol induced the highest CTSS expression. Therefore, 0.7% methanol concentration was selected for subsequent CTSS-His protein purification.

[0109] 2.2. Efficient purification of recombinant CTSS protease

[0110] 1. Nickel column affinity chromatography of CTSS Take 20g 0.7% methanol to induce recombinant expression of CTSS-His-2 # The bacteria were resuspended in PBS buffer (0.008M Na2HPO4, 0.002M KH2PO4, 0.136M NaCl, 0.026M KCl) at a ratio of 1g:10mL (w / v). The yeast cells were homogenized and disrupted by high pressure using an APV-1000 homogenizer (SPX FLOW, Inc.). The homogenization pressure was set to 1000 bar and the cells were disrupted twice in succession. The disrupted bacterial solution was ultrasonicated (30% power, working for 1s, pausing for 10s, and the total time was set to 20min). The disrupted bacterial solution was centrifuged at 4°C and 12000g for 30min. The supernatant was collected, the pH was adjusted to 7.3-7.4, and DTT was added to a final concentration of 2mM.

[0111] The supernatant was injected into the SCG purification system (HisTrapFF 5mL pre-packed column, GE healthcare, catalog number 17-5255-01) for nickel affinity chromatography. The column was pre-equilibrated with equilibrium buffer A (40mM Tris-HCl, 0.15M NaCl, pH7.4) before sample loading at a flow rate of 5mL / min. After loading, gradient elution was performed with elution buffer B (40mM Tris-HCl, 0.15M NaCl, 0.4M imidazole, pH 7.4) containing 0.4M imidazole. The elution gradient was set to a 40mM-200mM imidazole linear gradient, a flow rate of 5mL / min, and an elution time of 4min ( Figure 6 A), collect the elution peak of recombinant human CTSS protein (elution volume 20-27mL is P1 component, 28-43mL is P2 component), collect 7mL and 15mL. Chromatography and electrophoresis detection results are shown in Figure 6 B, CTSS is mainly in the P2 component.

[0112] 2. Recombinant CTSS protease purified by nickel column cannot be purified by traditional gel filtration

[0113] The P2 fraction enriched by the nickel column was first subjected to gel filtration chromatography (Superdex 200increase 10 / 300GL, Sigma-Aldrich, catalog number GE28-9909-44). The separation range of the chromatographic medium used in the gel filtration chromatography covers 10-600 kD. The column was pre-equilibrated with equilibrium buffer A (40 mM Tris-HCl, 0.15 M NaCl, pH 7.4) and then the sample was loaded. After elution with buffer A, two protein peaks F1 and F2 were separated ( Figure 6 C), where F2 is the major chromatographic peak.

[0114] The F2 fraction was separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue. We found that the target protein band could be detected at the theoretical molecular weight position of CTSS. However, we also detected two bands at the high molecular weight gel electrophoresis position, with molecular weights of 80kD and 220kD ( Figure 6 D) These protein bands are much higher than CTSS and are impurities. Their abundance is comparable to that of the target protein band of CTSS, indicating that the Superdex 200increase 10 / 300GL gel filtration chromatography we selected could not remove the impurities and CTSS could not be effectively purified.

[0115] 3. Proteomic identification of impurities in recombinant CTSS purified by molecular sieve

[0116] If the impurity protein can be identified, a specific purification technology can be selected according to the physicochemical properties of the impurity protein to achieve rapid and efficient purification of the target protein. To this end, we cut the high molecular weight protein band in Example 2.2 from the gel, digested it with trypsin, and performed LC-MS analysis.

[0117] The peptides digested by trypsin were analyzed by LC-MS, and the identification conditions were as follows: mass spectrometer: LTQ Orbitrap Velos; liquid phase: Easy-nLC 1000; mobile phase: acetonitrile solution containing 0.1% formic acid; flow rate: 400nL / min; resolution: 30000. A total of 108 proteins were identified by mass spectrometry, of which the top ten proteins in abundance accounted for more than 80% of the abundance of all identified proteins ( Figure 7 A), is the main protein. The top ten proteins in terms of abundance are listed in Figure 7 B. Analysis revealed that except for a few individual proteins, these impurities all had low isoelectric points and were suitable for purification by anion exchange chromatography.

[0118] 4. Proteome-driven purification of recombinant CTSS target protein by anion exchange chromatography

[0119] The P2 component enriched by the nickel column in Example 2.2 was purified by anion exchange chromatography (HiTrapQ HP 5mL pre-loaded column, GE healthcare product number 17-1154-01). The column was pre-equilibrated with equilibrium buffer C (20mM Tris, pH 7.5) and then loaded with the sample. After loading, the sample was eluted in a linear elution mode with an elution buffer containing 1M sodium chloride (20mM Tris, 1M NaCl, pH 7.5). The elution gradient was set to a linear gradient of 120mM-200mM NaCl, a flow rate of 5mL / min, and an elution time of 3min, and two protein elution peaks of F1 and F2 were separated ( Figure 7 C).

[0120] The two protein elution peaks obtained by separation were subjected to SDS-PAGE electrophoresis analysis to detect the purity of the recombinant CTSS protein. It was found that the F1 obtained by anion exchange chromatography had only one protein band with a molecular weight of 37kD, which was consistent with the target molecular weight of CTSS; the band was deeply stained and large in amount, and there were no other visible protein bands, indicating that we had purified high-purity CTSS. The F2 component lane showed a 70kD main band, which was consistent with the target molecular weight of CTSS. Figure 6 In addition, several faintly visible protein bands at 50 kD and so on were also observed. These results fully demonstrate that the purification target of CTSS under the guidance of proteomics is clear and the purification is efficient.

[0121] The F1 fraction was concentrated by ultrafiltration using a 10 kD ultrafiltration tube (Amicon Ultra-15 mL, 10 kD Centrifugal Filter Unit) to obtain a recombinant human CTSS protein solution with a concentration of 5 mg / mL.

[0122] 2.3. Biological activity of highly purified recombinant CTSS protease

[0123] Z-Leu-Arg-MCA (N-Methoxysuccinyl-L-Alanyl-L Alanyl-L-Prolyl-L-Valine4-Methyl-Coumaryl-7-Amide, n-methoxysuccinyl-l-propionyl-l-propionyl-l-valine 4-methyl-coumarin-7-amide, purchased from Peptide Institute Inc, catalog number 3210-v) is a luminescent substrate. CTSS can cleave the arginine residue of Z-Leu-Arg-MCA to release MCA (4-methyl-coumarin-7-amide) ( Figure 8 A). Free MCA emits fluorescence under excitation light at 370nm and emission light at 460nm, which can be detected by a microplate reader.

[0124] The CTSS concentration was set to 250nM, and the substrate concentrations were 0, 1, 2, 5, 10, 20, 150, 250, and 500μM; a sample without CTSS was also set as a control. The reaction buffer was 50mM NaAc, 5mM EDTA, 2mM DTT, pH 5.5, the total volume was 100μL, and 3 replicates were set for each group. The reaction was allowed to stand in a 37°C incubator for 1h, and the fluorescence intensity was detected using a microplate reader (Biotek). The curve was drawn using Prism7 (GraphPad Prism version 7.0.0for Windows (GraphPad Software, San Diego, California, USA, www.graphpad.com), and the Michaelis-Menten constant (Km) value of the recombinant CTSS protein was calculated to be 29.3nM using the Michaelis-Menten equation function. This is basically consistent with the commercial CTSS Michaelis constant (Km) value (20nM) ( Figure 8 B) further proves that we have purified yeast recombinant CTSS with high purity and high biological activity.

[0125] LY-3000328 is a known CTSS inhibitor. To verify that the CTSS we prepared has the ability to cleave collagen 18A1 (COL18A1) to produce endostatin (Endostatin), we reconstructed the in vitro biochemical reaction system of CTSS cleaving collagen 18A1 with our purified CTSS, and used the specific inhibition of the CTSS inhibitor LY-3000328 to prove the specificity of CTSS activity. We set up three concentration gradients of LY-3000328 (400, 800, 1500nM), and determined the dosage of LY-3000328 by the Endostatin production. The results showed that we can indeed use CTSS to cleave collagen 18A1 to obtain Endostatin, and the production of Endostatin is dose-dependently correlated with the concentration of the inhibitor LY-3000328, indicating that the inhibitor can specifically inhibit CTSS activity ( Figure 8 C, Table 2).

[0126] We analyzed the data and found that 800 nM LY-3000328 concentration basically inhibited 50% of CTSS activity (Table 2). This dose laid the foundation for the activity evaluation of the subsequent series of CTSS synthesis inhibitors.

[0127] Table 2. Comparison of Endostatin production inhibited by different LY-3000328 concentrations**

[0128]

[0129] **Table 2. Comparison of Endostatin production inhibited by different LY-3000328 concentrations. Comparison of Endostatin production generated by CTSS cleavage of collagen COL18A1 using different concentrations (400, 800, 1500 nM) of LY-3000328 on CTSS inhibition. The results showed that Endostatin production was dose-dependent with inhibitor concentration, indicating that the inhibitor could specifically inhibit CTSS, and 800 nM of LY-3000328 or the synthesized compound (BG) was used for subsequent inhibitor screening.

[0130] Example 3. Determination of the inhibitory activity of compound BG on CTSS

[0131] The positive control LY-3000328 (Control) and the synthetic inhibitor compounds B, C, D, E, F and G of Example 1 were added to the established in vitro reaction system of CTSS-cleaved collagen 18A1, and it was found that the positive control CTSS inhibitor LY-3000328 could inhibit the production of Endostatin to a certain extent, indicating that compounds BG are inhibitors of CTSS ( Fig.10 A).

[0132] Comparing the production of Endostatin, it was further found that the activity of compound DF was significantly better than that of the positive control compound LY-3000328; when compound F was added, the amount of Endostatin produced by CTSS cleaving COL18A1 was minimal, and its inhibition efficiency on CTSS was the highest, reaching more than 80%, which was 1.3 to 6.8 times higher than the inhibitory activity of other compounds ( Fig.10 B), indicating that compound F is the strongest inhibitor of CTSS and a potential candidate drug for treating early liver fibrosis, and is named Secoendostatin F.

[0133] Example 4. Evaluation of the anti-liver fibrosis activity of CTSS inhibitors in vitro

[0134] Experimental method: Lx-2 cells (cell bank of Chinese Academy of Sciences) in good logarithmic growth phase were prepared into cell suspension and counted with a counting plate. 5Cell / well was planted in a 6-well plate and cultured at 37°C and 5% CO2 until the cells grew to 70-80%. Lx-2 cells were pretreated with human recombinant protein TGF-β1 (5μg / mL) for 2h, and then LY3000328 (10μM), compound BG (10μM) and DMSO were added to each well, cultured for 24h, and total cell protein was extracted for Western Blot analysis. Western Blot analysis: Proteins from Lx-2 cells were lysed in cold RIPA buffer containing protease inhibitors and phosphatase inhibitors. According to the instructions, the total protein concentration was determined using the BCA protein concentration assay kit and denatured by boiling at 100°C for 10min. Protein samples (30-40 μg) were separated by SDS-PAGE and transferred to PVDF membranes, blocked with milk, and incubated with primary and secondary antibodies, Fibronectin (1:2000, ab2413, Abcam), GAPDH (1:6000, #2118, CST) and Rabbit IgG HRP Linked antibody (1:10000, #7074, CST), followed by TCL luminescence and ImageJ analysis of protein expression.

[0135] Experimental results: The activation of hepatic stellate cells (HSCs) and the excessive deposition of extracellular matrix (ECM) are key steps in the process of liver fibrosis. During the activation of HSCs, hepatic stellate cells lose adhesion molecules and acquire mesenchymal cell markers. Among them, fibronectin (FN) is an important molecular marker for the occurrence of fibrosis. Fig.11 As shown, TGF-β1 induced a significant increase in the expression of FN protein in Lx-2 cells, while compounds BG reduced FN protein expression to varying degrees. In particular, compounds D, F and G had a significantly stronger inhibitory effect on FN protein expression than the inhibitor LY3000328. The results showed that the compounds had an inhibitory effect on liver fibrosis.

[0136] Example 5. Evaluation of the anti-liver fibrosis activity of CTSS inhibitors in vivo

[0137] Experimental methods: 24 8-week-old SPF male C57BL / 6 mice weighing 21-25 g (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were divided into four groups (6 mice in each group) after one week of adaptation to the laboratory as follows: (i) Control group: mice were intraperitoneally injected with 500 μl corn oil three times a week for 6 weeks, and blank solvent was injected intraperitoneally once a day in the last 2 weeks of the experiment; (ii) Model group: mice were intraperitoneally injected with 500 μl CCl4 / corn oil mixture (v / v, 1:4) three times a week for 6 weeks to induce liver fibrosis in mice, and blank solvent was injected intraperitoneally once a day in the last 2 weeks of the experiment; (iii) LY3000328 intervention group: mice were treated with CCl4 for 6 weeks according to the model group treatment method, and LY3000328 (5 mg / kg) was injected intraperitoneally once a day in the last 2 weeks of the experiment for 2 weeks; (iv) Compound F intervention group: mice were treated with CCl4 for 6 weeks according to the model group treatment method, and compound F (5 mg / kg) was injected intraperitoneally once a day in the last 2 weeks of the experiment for 2 weeks. Liver function test: ELISA method was used to detect serum alanine aminotransferase (ALT) and aspartate aminotransferase activities (C010-2-1 and C009-2-1, Nanjing Jiancheng Bioengineering Institute). The absorbance value (OD) of each blood sample was read at a wavelength of 510 nm, and the liver enzyme activity was calculated according to the instructions. Western Blot analysis: Take liver tissue blocks (100 mg) and use an ultrasonicator at 400 W for 30 seconds to fully break the liver tissue into a uniform tissue homogenate. The protein of the liver tissue was lysed in cold RIPA buffer containing protease inhibitors and phosphatase inhibitors. Western Blot analysis was performed according to the above cell experiment.

[0138] Experimental results: ALT and AST in serum are commonly used and relatively sensitive liver function indicators, which can directly reflect the degree of liver disease and liver cell damage. Fig.12 A and B show that compared with the control treatment, serum ALT and AST in mice with CCl4-induced liver fibrosis were significantly increased, but after the intervention of compound LY3000328 and compound F in mice, the levels of ALT and AST in serum were significantly reduced. The results show that the compounds have a significant alleviating and protective effect on CCl4-induced liver fibrosis.

[0139] This study further examined the effect of compound F on the expression of FN protein in CCl4-induced mouse liver. Fig.12C shows that the expression level of FN protein in the liver of mice with liver fibrosis induced by CCl4 is significantly increased, but after the intervention of compound F and compound LY3000328 in mice, the expression level of FN protein in the liver is significantly reduced; the effect of compound F is significantly better than that of compound LY3000328. The results show that the compound has the ability to inhibit CCl4-induced liver fibrosis in mice, thereby exerting a liver protective effect.

Claims

1. A human cathepsin S inhibitor having the structure of formula (I): in: R1 is a piperazinyl substituted by a heterocycle, a piperazinyl substituted by an alkane, a piperazinyl substituted by an aromatic hydrocarbon, an aromatic substituent or a benzylamino group; R2 is an alkane substituent, a benzyl group or an aromatic substituent.

2. The human cathepsin S inhibitor according to claim 1, characterized in that The inhibitor is selected from the following compounds B, C, D, E, F and G having structures of formula (II) to (VII):

3. A pharmaceutical composition comprising the human cathepsin S inhibitor according to claim 1.

4. Use of the human cathepsin S inhibitor according to claim 1 or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating and / or preventing liver fibrosis.

5. The use according to claim 4, wherein the liver fibrosis disease is early liver fibrosis.

6. A method for screening the human cathepsin S inhibitor according to claim 1, comprising using recombinant human cathepsin S prepared by a yeast expression system as a target protein to screen the inhibitor.

7. The method according to claim 6, wherein the recombinant human cathepsin S prepared by the yeast expression system comprises the following preparation steps: 1) The fusion gene expressing CTSS-eGFP is operably connected to the yeast expression vector pPICZα; and the expression vector is transferred into Pichia pastoris competent cells, and a yeast strain expressing the recombinant CTSS protein is obtained through antibiotic selection; 2) Cultivate the strain in step 1) and add 0.7% methanol to induce the expression of CTSS-eGFP. 3) Isolate and purify the expressed CTSS protein.

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