Targeting 5-HT2AR degradation agent based on natural glucosamine and preparation method and anti-hepatic fibrosis application thereof

Abstract

The invention discloses a targeted 5-HT2AR degradation agent based on natural glucosamine and a preparation method of the targeted 5-HT2AR degradation agent. Disclosed are LYTAC compounds having a structure represented by formula I or pharmaceutically acceptable salts thereof. According to the present invention, the LYTAC compound or the pharmaceutically acceptable salt thereof can significantly degrade the target protein 5-HT2AR in the targeting manner, and can degrade the 5-HT2AR through the gridding protein-lysosome pathway, such that the secretion of the hepatic stellate cell LX2 activating factor TGF-beta is reduced, and the activation of the hepatic stellate cell is prevented; and meanwhile, the compound has a remarkable cell proliferation inhibition effect on activated hepatic stellate cells, and regulates apoptosis-related proteins so as to induce apoptosis of the hepatic stellate cells in an activated state. The invention discloses an application of an LYTAC compound or a pharmaceutically acceptable salt thereof in preparation of a targeted 5-HT2AR degradation agent or a medicine for treating 5-HT2AR protein related diseases, wherein the 5-HT2AR protein related diseases are hepatic fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to a targeted 5-HT based on natural glucosamine. 2A R-degrading agents, their preparation methods, and their applications in anti-liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathophysiological process in the progression of many chronic liver diseases. Its main characteristic is the activation of hepatic stellate cells (HSCs) triggered by long-term liver injury; these HSCs are normally in a dormant state. During the progression of liver fibrosis, activated HSCs gradually transform into fibroblasts and continuously release large amounts of extracellular matrix (ECM), leading to excessive ECM deposition in liver tissue. Inhibiting HSC activation or converting them to an inactive state is considered one of the key mechanisms for reversing liver fibrosis and a potential therapeutic strategy for liver fibrosis.

[0003] Serotonin (5-HT) receptors are upregulated in activated hepatic stellate cells (HSCs) and therefore play an important role in HSC activation. HSCs can store and release 5-HT and respond to neurotransmitters in a pro-fibrotic manner. The pro-fibrotic activity of 5-HT is mediated by the activation of 5-HT receptors. 2A It occurs via the R-TGF-β-Smad pathway. 5-HT 2A R is a Gaq-coupled GPCR (G protein-coupled receptor), in which the third loop in its intramural region couples with the downstream protein Gaq, activating 5-HT. 2A R can trigger the disintegration of the downstream Gaq protein and its β-γ subunit, activating phosphokinase C (PLC), promoting the release of diglyceride (DAG) and inositol bisphosphonate (IP3), and activating protein kinase C (PKC), thereby initiating downstream reactions. 5-HT 2A R-antagonists significantly improved plasma transaminase levels, reduced hepatic 5-HT concentration, and improved liver fibrosis in a mouse model of liver fibrosis. 2A R antagonists inhibit hepatic stellate cell activation and promote apoptosis through the TGF-β and Smad 2 / 3 pathways. Antagonists that selectively target 5-HT receptors can be used as anti-fibrotic drugs.

[0004] Acetyl-D-galactosamine (GalNAc) is a natural amino sugar and a specific ligand for the desialylate glycoprotein receptor (ASGPR). As a liver-specific transmembrane glycoprotein, ASGPR recognizes glycoproteins carrying either GalNAc or galactose (Gal) ligands and internalizes them via clathrin-mediated endocytosis. After internalization, ASGPR releases GalNAc or Gal and circulates back to the plasma membrane, while the glycoprotein enters the lysosome.

[0005] Lysosome-targeting chimeras (LYTACs) promote lysosomal degradation of extracellular or cell membrane targets by hijacking lysosomal transport receptors (cation-independent mannose-6-phosphate receptors CIM6PR / ASGPR). Compared with inhibitors, LYTACs can directly degrade target proteins, substantially eliminating downstream kinase signaling, making them worthy of in-depth research and promising for clinical applications. Lysosome-targeting chimeras are bifunctional molecules with two binding domains, capable of simultaneously binding to the extracellular domains of extracellular or membrane proteins and the lysosomal targeting receptor (TLR) on the cell surface, forming a ternary complex. Subsequently, the protein is internalized and degraded through endocytosis, forming a transport vesicle. The vesicle transports the complex to the lysosome, ultimately degrading the target protein. The liver is the main site of protein catabolism, and ASGPR ligands are highly expressed in hepatocytes. By designing LYTAC molecules that target lysosomes, LYTACs can degrade 5-HT in the liver. 2A R can achieve a certain therapeutic effect.

[0006] The incidence of chronic liver disease is rising globally, and the treatment needs are far from being met: currently, the only treatment options are liver transplantation or elimination of the underlying cause. However, only a few studies have been conducted on the anti-fibrotic effects of 5-HT on HSCs. Summary of the Invention

[0007] The purpose of this invention is to achieve anti-liver fibrosis by combining the centrally acting drug risperidone with GalNaC based on lysosomal chimera (LYTAC) technology, thereby addressing the issue from two aspects: Firstly, by relying on the transporter protein ASGPR specifically expressed on the surface of hepatocytes to form LYTAC molecules, which degrade the highly expressed 5-HT. 2A R receptors reduce the level of stellate cell activating factor TGF-β; on the other hand, due to the hydrophilicity of GalNaC, the central nervous system drug risperidone exerts its effect on the highly expressed 5-HT on the surface of hepatic stellate cells. 2A The inhibitory effect of R inhibits the activation and proliferation of stellate cells.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] LYTAC compounds or their pharmaceutically acceptable salts, as shown in Formula I, are as follows:

[0010]

[0011] Another object of the present invention is to provide a method for preparing the LYTAC compound, the synthetic route of which is as follows:

[0012]

[0013]

[0014] include:

[0015] Step (1): Under a nitrogen atmosphere, using dichloromethane as the reaction solvent, 4-dimethylaminopyridine as the catalyst, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride as the condensing agent, compound H-4 undergoes an amidation reaction with carboxylated polyethylene glycol carboxyethyl to obtain compound H-5.

[0016] Step (2): Under a hydrogen atmosphere, with methanol as the reaction solvent and 10% palladium carbon as the catalyst, compound Z-12 was deprotected at room temperature by removing the Cbz protecting group (benzyloxycarbonyl) to obtain compound Z-13.

[0017] Step (3): Using N,N-dimethylformamide as the reaction solvent and N,N-diisopropylethylamine and O-benzotriazole-N,N,N-tetramethylhexafluorophosphate ureaium as condensing agents, compound Z-13 and compound H-5 undergo an amidation reaction to obtain compound Z-14;

[0018] Step (4): Using methanol as the reaction solvent, sodium methoxide was added to adjust the pH of the reaction system to 9-10. Compound Z-14 was reacted at room temperature to obtain the LYTAC compound with the structure shown in Formula I.

[0019] In step (1), the molar ratio of compound H-4 to carboxylated polyethylene glycol carboxyethyl is 1:1; the molar ratio of compound H-4 to 4-dimethylaminopyridine is 1:3; and the molar ratio of compound H-4 to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 1:3.

[0020] The amidation reaction was performed at room temperature.

[0021] After the reaction was completed, the reaction solution was washed with saturated sodium chloride aqueous solution, the organic layer was collected, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 20:1V / V) to purify and obtain compound H-5.

[0022] In step (2), the mass ratio of the 10% palladium on carbon to compound Z-12 is 1:10.

[0023] After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound Z-13.

[0024] In step (3), the molar ratio of compound Z-13 to compound H-5 is 1:1; the molar ratio of compound Z-13 to O-benzotriazole-N,N,N-tetramethylurea hexafluorophosphate is 1:1; and the molar ratio of compound Z-13 to N,N-diisopropylethylamine is 1:1.

[0025] The amidation reaction was performed at room temperature.

[0026] The LYTAC compound or its pharmaceutically acceptable salt described in this invention can significantly target and degrade the target protein 5-HT. 2A R degrades 5-HT via the clathrin-lysosome pathway. 2A R reduces the secretion of TGF-β, the LX-2 activating factor, in hepatic stellate cells, thereby preventing their activation. Simultaneously, it significantly inhibits the proliferation of already activated hepatic stellate cells, regulates apoptosis-related proteins, and thus induces apoptosis in activated hepatic stellate cells. Therefore, another object of this invention is to provide the aforementioned LYTAC compound or a pharmaceutically acceptable salt thereof for the preparation of a product targeting 5-HT. 2A Uses of R degradation agent.

[0027] Another object of the present invention is to provide the LYTAC compound or a pharmaceutically acceptable salt thereof in the preparation of a treatment for 5-HT 2A Uses of R protein in drugs for diseases.

[0028] The 5-HT 2A R protein-related diseases include liver fibrosis.

[0029] Another object of the present invention is to provide the use of the LYTAC compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating liver fibrosis.

[0030] Another object of the present invention is to provide a pharmaceutical composition which is formulated into a pharmaceutically acceptable dosage form using the LYTAC compound or a pharmaceutically acceptable salt thereof as the active ingredient or main active ingredient, with a pharmaceutically acceptable carrier.

[0031] The dosage form is any one of the following: tablets, capsules, powders, syrups, liquids, suspensions, lyophilized powder for injection, or injections.

[0032] The beneficial effects of this invention are:

[0033] Compared to the small molecule inhibitor risperidone, the LYTAC compound of this invention can significantly target and degrade the target protein 5-HT. 2A R can degrade 5-HT via the clathrin-lysosome pathway. 2A R reduces the secretion of TGF-β, the LX2 activator of hepatic stellate cells, thus preventing their activation. Simultaneously, it significantly inhibits the proliferation of already activated hepatic stellate cells, regulates apoptosis-related proteins, and thereby induces apoptosis in activated hepatic stellate cells. Attached Figure Description

[0034] Figure 1 Different concentrations of compound Z-15 were used to evaluate the effects of 5-HT on AML-12 cells. 2A The degradation effect of R; where A represents the effect of different concentrations of compound Z-15 on 5-HT. 2A The degradation effect of R, B represents the effect of different concentrations of risperidone on 5-HT. 2A The degradation effect of R.

[0035] Figure 2 Compound Z-15 exerts its degradation effect on the target protein 5-HT through a clathrin-lysosome mechanism. 2A Results of the investigation of the role of R; where A represents the degradation of 5-HT2AR protein by compound Z-15 dependent on the clathrin system, and B represents the degradation of 5-HT2AR protein by compound Z-15 dependent on the lysosomal system.

[0036] Figure 3 5-HT for different cell lines 2A The expression of R protein; where A represents 5-HT in different cell lines. 2A The expression level of R, and B represents the expression level of 5-HT in different cell lines. 2A Quantitative analysis results of R protein levels.

[0037] Figure 4 Compound Z-15 targets the protein 5-HT 2A The results of the investigation into the degradation effect of concentration gradient of compound Z-15 on 5-HT in normal human hepatocytes LO2 were studied; where A represents the effect of different concentrations of compound Z-15 on 5-HT in normal human hepatocytes LO2. 2A The degradation effect of R, B represents the effect of different concentrations of compound Z-15 on 5-HT in human hepatocellular carcinoma cells HepG2. 2A The effect of R on degradation, C represents the effect of different concentrations of compound Z-15 on 5-HT in normal rat liver cells BRL-3A. 2A The degradation effect of R, where D represents the effect of different concentrations of compound Z-15 on 5-HT in different cell lines. 2A Quantitative analysis results of R degradation.

[0038] Figure 5 Compound Z-15 targets the protein 5-HT 2A The results of the study on the time-dependent degradation effect of R; where A represents the effect of different treatment times of compound Z-15 on 5-HT. 2A The effect of different treatment times of compound Z-15 on 5-HT expression levels, B represents the effect of different treatment times of compound Z-15 on 5-HT. 2A Quantitative analysis results of R degradation.

[0039] Figure 6 For compound Z-15 and target protein 5-HT 2A Results of laser confocal fluorescence localization experiments for R.

[0040] Figure 7 The study investigated the effect of compound Z-15 on the release of the cytokine TGF-β from hepatocytes, which indirectly affected the activation of hepatic stellate cells. A represents the effect of compound Z-15 on the expression level of α-SMA protein in hepatic stellate cells, and B represents the quantitative analysis results of compound Z-15 on the expression level of α-SMA protein in hepatic stellate cells.

[0041] Figure 8 This study explores the optimal concentration and time for TGF-β activator to activate hepatic stellate cells. Specifically, A represents the effect of different concentrations of TGF-β activator on the expression level of α-SMA protein in hepatic stellate cells; B represents the quantitative analysis results of different concentrations of TGF-β activator on the expression level of α-SMA protein in hepatic stellate cells; C represents the effect of different treatment times of TGF-β activator on the expression level of α-SMA protein in hepatic stellate cells; and D represents the quantitative analysis results of different treatment times of TGF-β activator on the expression level of α-SMA protein in hepatic stellate cells.

[0042] Figure 9 The results of an immunofluorescence experiment were explored to investigate the activation of hepatic stellate cells by TGF-β.

[0043] Figure 10 5-HT in activated hepatic stellate cells 2A Results of the investigation into R expression; where A represents 5-HT in hepatic stellate cells in different states. 2A Immunofluorescence experiments were conducted to explore the expression of R, and B represented 5-HT in hepatic stellate cells at different states. 2A R protein expression level, C represents 5-HT in hepatic stellate cells in different states. 2A Quantitative analysis results of R protein expression level.

[0044] Figure 11 The results of this study explored the effects of different concentrations of compound Z-15 on the activity of LX-2 cells in different states.

[0045] Figure 12 To explore the inhibitory effect of compound Z-15 on the activation of hepatic stellate cells, A represents the effect of compound Z-15 on the expression level of α-SMA protein in hepatic stellate cells, and B represents the quantitative analysis results of the expression level of α-SMA protein in hepatic stellate cells.

[0046] Figure 13 The results of immunofluorescence experiments were used to explore the inhibitory effect of compound Z-15 on the activation of hepatic stellate cells.

[0047] Figure 14The results of the investigation on the apoptotic effect of compound Z-15 on hepatic stellate cells are as follows: A is the activator group (TGF-β group), B is the blank control group (DMSO group), C is the group treated with 1 μM compound Z-15, D is the group treated with 3 μM compound Z-15, E is the group treated with 5 μM compound Z-15, and F is the quantitative analysis result of the total number of apoptotic hepatic stellate cells.

[0048] Figure 15 This is the result of an investigation into the regulatory effect of compound Z-15 on apoptosis-related proteins in hepatic stellate cells. Detailed Implementation

[0049] To facilitate understanding of the technical solutions of the present invention, the following embodiments are provided to clearly and completely illustrate the technical solutions of the present invention. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to make the understanding of the technical solutions of the present invention more thorough and comprehensive.

[0050] The room temperature described in this invention is 25°C.

[0051] Example 1

[0052] 1. Preparation of sugar chains

[0053]

[0054] Existing methods for synthesizing compound G-6 suffer from the problem of easily deteriorating intermediates during storage, the expensive use of benzyl 5-hydroxyvalerate, and lengthy subsequent synthetic routes. This invention optimizes the synthetic route, using more readily available raw materials and achieving higher yields. The specific method is as follows:

[0055] At room temperature, D-galactosamine pentaacetate (compound G-1, 389 g, 1.0 mol, 1.0 equivalent) was dissolved in 1,2-dichloroethane (500 mL), and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 70 mL, 1.5 mol, 1.5 equivalent) was added dropwise. After the addition was complete, the mixture was stirred at 50 °C for 90 minutes. Heating was stopped, and the mixture was stirred at room temperature for 4 hours. The reaction progress was monitored by TLC. After the reaction was completed, saturated sodium bicarbonate solution was added to the reaction solution to neutralize the remaining trimethylsilyl trifluoromethanesulfonate. The mixture was then extracted three times with dichloromethane. The dichloromethane layers were combined, dried with sodium sulfate, filtered, and concentrated under vacuum to obtain compound G-2 (263.02 g, yield 80%), a yellow oil.

[0056] Compound G-2 (329 g, 1.0 mol, 1.0 equivalent) was dissolved in anhydrous tetrahydrofuran. 100 g of type 4A molecular sieve was added as a desiccant, and the mixture was stirred at room temperature for 5 minutes. Then, 5-benzyloxy-1-pentanol (312 g, 1.5 mol, 1.5 equivalent) was added, and the mixture was stirred at room temperature for 30 minutes to remove water from the reagents. Subsequently, trimethylsilyl trifluoromethanesulfonate (111 g, 0.5 mol, 0.5 equivalent) was added dropwise over 10 minutes at room temperature with stirring. After the addition was complete, the mixture was stirred at room temperature for approximately 15 hours, and the reaction progress was monitored by TLC. The reaction mixture was washed with saturated brine, and the organic layer was collected. The organic layer was washed with saturated sodium bicarbonate solution, then extracted with dichloromethane, and the organic layer was collected. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain compound G-3 (444.55 g, yield 85%), a yellow oil.

[0057] Compound G-3 (523 g, 1 mol, 1.0 equivalent), 10% anhydrous palladium on carbon (53.7 g), and methanol (300 mL) were placed in a 500 mL round-bottom flask. The flask was evacuated and purged with hydrogen five times. The reaction was stirred for 3 hours at room temperature under a hydrogen atmosphere. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was filtered through diatomaceous earth. The filtrate was concentrated under vacuum and then subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 16:1 V / V) to obtain compound G-4 (290.11 g, yield 67%).

[0058] Compound G-4 (433 g, 1 mol, 1.0 equivalent) was dissolved in anhydrous dichloromethane (500 mL), and Dys-Martin oxidant (400 g, 1 mol, 1.0 equivalent) was added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was stirred with silica gel and subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 12:1 V / V) to purify compound G-5 (323.25 g, yield 75%).

[0059] Compound G-5 (431 g, 1 mol, 1.0 equivalent) was dissolved in 500 mL of acetonitrile to obtain a solution of compound G-5. Sodium dihydrogen phosphate (573 g, 1.2 equivalent) was dissolved in a mixed solvent of 30 mL of 30% hydrogen peroxide (3 equivalent) and 100 mL of distilled water to obtain a sodium dihydrogen phosphate solution. The two solutions were slowly mixed to obtain a mixed solution. Sodium chlorite (280 g, 5.34 equivalent) was dissolved in 230 mL of distilled water and slowly added dropwise to the mixed solution. The temperature was controlled at 10 °C, and the reaction was carried out for 4 hours. The reaction progress was monitored by TLC. After the reaction was completed, the sample was mixed with silica gel and subjected to silica gel column chromatography (eluent: dichloromethanol = 10:1 V / V) to purify compound G-6 (232 g, yield 52%), a white oily substance.

[0060] Data for compound G-6: 1 H NMR (500MHz, DMSO-d6, TMS) δH: 11.98 (1H, s), 7.84 (1H, d, J = 6.63Hz), 5.72, 5.21 (2H, s), 4.97 (1H, d, J = 6.67Hz), 4.49 (1 H,s),4.03,3.9,3.7(3H,m),3.42(2H,m),2.20(2H,m),2.10(3H,s),2.00(3H,s),1.89(3H,s),1.77(3H,s),1.43(4H,t). 13 C NMR(125MHz,DMSO-d6,TMS)δ:174.89,170.48,170.40,170.11,169.74,101.40,70.95,70.3 0,68.88,67.18,61.93,49.78,33.69,28.81,23.19,20.99,20.93,20.91,21.45.ESI:448.4.

[0061] 2. Preparation of branched chains

[0062]

[0063] Under a nitrogen atmosphere and at 15°C, 49.5 mL of 5.0 M sodium hydroxide solution (0.2475 mol, 0.1 equivalent) was added dropwise to a DMSO solution of 300 g (2.48 mol, 1.0 equivalent) of 2-amino-2-(hydroxymethyl)propane-1,3-diol (DMSO) in 500 mL of DMSO while stirring. After the addition was complete, the mixture was stirred at room temperature for 24 hours. The reaction mixture was extracted with ethyl acetate (4 × 3000 mL), and the organic layers were combined. The organic layers were washed successively with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 18:1 V / V) to obtain compound Z-7 (526 g, yield 42%) as a pale yellow oil. [M+H] + calculatedESI: 506.7.

[0064] At room temperature, while stirring, 365 mL of 25% Na₂CO₃ aqueous solution was added to a CH₂Cl₂ (750 mL) solution of compound Z-7 (50 g, 99.0 mmol, 1.0 equivalent). Benzyl chloroformate (50.5 g, 0.297 mol, 3.0 equivalent) was added dropwise at room temperature with stirring. After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was extracted with dichloromethane, and the organic layers were combined. The organic layers were washed sequentially with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 20:1 V / V) to purify compound Z-8 (42.4 g, 67% yield), a pale yellow oil.

[0065] Compound Z-8 (300 g, 468.9 mmol, 1.0 equivalent) was added to formic acid (3 L, 96% purity) and stirred at room temperature for 16 hours. After the reaction was completed, the pH of the reaction solution was adjusted to 12 with sodium hydroxide solution (1 mol / L), and the mixture was extracted with diethyl ether (3 × 6 L). The aqueous layers were combined, and the pH of the aqueous layer was adjusted to 4 with hydrochloric acid (2 mol / L). The mixture was then extracted with ethyl acetate, and the organic layers were combined. The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound Z-9 (198.96 g, 90%), a yellow oil.

[0066] Compound Z-9 (264 g, 560.51 mmol, 1.0 equivalent) was dissolved in a mixed solvent of dichloromethane (1400 mL) and acetonitrile (1400 mL). Then, N,N-diisopropylethylamine (578.4 g, 4.484 mol, 8.0 equivalent) and O-benzotriazole-N,N,N-tetramethylurea hexafluorophosphate (848 g, 2.231 mol, 4.0 equivalent) were added sequentially. The mixture was stirred at room temperature for 30 minutes. Then, N-(3-aminopropyl)carbamate tert-butyl ester (390 g, 2.238 mol, 4.0 equivalent) was added, and the mixture was stirred at room temperature for another 6 hours. The reaction solution was concentrated under reduced pressure to 1 / 20 of its volume. The residue was diluted with 3000 mL of dichloromethane and washed successively with saturated NaHCO3 solution, saturated NH4Cl solution, and saturated NaCl solution. After drying with anhydrous sodium sulfate and filtration, the residue was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 10:1 V / V) to purify and obtain compound Z-10 (336 g, yield 70%), which was white and foamy.

[0067] Data for compound Z-10: 1 H NMR (500MHz, DMSO-d6, TMS) δ H:8.17(s,1H),7.83(d,J=7.2Hz,3H),7.34(m,5H),6.82(m,3H)4.98(d,J=14.0Hz,2H),3.55(d,J=8.4Hz, 6H),3.48(m,6H),3.55(m,6H),3.02(m,6H),2.29(t,J=6.0Hz,6H),1.49(d,J=6.6Hz,6H),1.37(m,27H). 13 C NMR (125MHz, DMSO-d6, TMS)δ C :170.52(C×3),156.03(C×3),155.02,137.64,128.76(C×2),128.13,128.01(C×2),77.93(C×3),67.78(C×3 ),55.38(C×3),68.66,59.30,38.06(C×3),36.68(C×3),36.51(C×3),28.71(C×3),30.08(C×3),ESI:941.0.

[0068] Trifluoroacetic acid (350 mL) was added to a solution of compound Z-10 (350 g, 372.3 mmol, 1.0 equivalent) in dichloromethane (1.4 L), and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain 470 g of crude compound Z-11, a red oily substance, which was used directly in the next step of the reaction without purification.

[0069] Under nitrogen atmosphere and at room temperature, compound G-6 (66 g, 147.51 mmol, 3.50 equivalents) and N,N-diisopropylethylamine (37 g, 286.29 mmol, 6.78 equivalents) were dissolved in a mixed solvent of dichloromethane (200 mL) and acetonitrile (200 mL). Then, 1-hydroxybenzotriazole (24.5 g, 181.48 mmol, 4.30 equivalents) and O-benzotriazole-N,N,N-tetramethylhexafluorophosphate ureaium (56 g, 147.66 mmol, 3.50 equivalents) were added. The mixture was stirred at room temperature for 30 minutes. Then, compound Z-11 (41.4 g, 42.20 mmol, 1.00 equivalents) was added at room temperature, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with dichloromethane, and the organic layers were combined. The organic layers were washed with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 16:1 V / V). Then, it was subjected to rapid preparative HPLC (80% methanol / water) to purify compound Z-12 (41.2 g, yield 51%) as a pale yellow solid.

[0070] Data for compound Z-12:1 1H NMR (500 MHz, DMSO-d6, TMS) δ H : 8.17 (s, 1H), 8.09 (m, 3H), 7.97 - 7.91 (m, 6H), 7.35 - 7.30 (m, 5H), 6.02 (m, 3H), 5.98 (m, 3H), 5.33 (s, 3H), 5.07 (s, 1H), 5.02 (m, 3H), 4.56 (dd, J = 10.5, 4.2 Hz, 3H), 4.12 (d, J = 5.9 Hz, 6H), 3.86 - 3.66 (m, 15H), 3.53 - 3.20 (m, 15H), 2.41 (p, J = 6.2, 5.7 Hz, 6H), 2.19 (d, J = 11.0 Hz, 6H), 2.02 (t, J = 5.9 Hz, 18H), 2.04 (t, J = 7.4 Hz, 9H), 1.94 (s, 9H), 1.66 - 1.58 (m, 18H). 13 13C NMR (125 MHz, DMSO-d6, TMS) δ C : 176.02 (C×3), 174.04 (C×3), 173.64 (C×3), 172.14 (C×3), 171.78 (C×3), 157.14, 138.42, 129.57, 129.57, 129.07, 128.91, 128.91, 102.68×3, 72.20 (C×3), 71.80 (C×3), 70.47×3, 70.43 (C×3), 68.71 (C×3), 68.23 (C×3), 67.06, 62.75 (C×3), 51.65 (C×3), 37.80 (C×3), 37.89 (C×3), 36.7 (C×3), 30.38 (C×3), 30.08 (C×3), 23.69 (C×3), 22.97 (C×3), 20.66 (C×6), 20.63 (C×3). HRMS (ESI) calculated for C 87 H 134 N 10 O 38 [M + H] + , calculated 1928.9108, found 1928.9105 [M + H] + , calculated 2520.1836, found 2520.1830。

[0071] 3.5 - HT 2A Preparation of the R ligand

[0072]

[0073] In an ice bath, 2,3-dihydro-5H-oxazolo[2,3-B]quinazolin-5-one (1 g, 5.31 mM, 1.0 eq) was fully dissolved in 98% concentrated sulfuric acid (9 mL). The reaction vessel was placed in a low-temperature stirred reaction bath (0 °C), and a mixture of concentrated nitric acid (354 μL, 1.5 eq) and concentrated sulfuric acid (2:1 v / v) was slowly added dropwise. The reaction was allowed to proceed for 3 h, and the reaction was stopped by TLC. The reaction solution was poured into a beaker containing cold water, and 9.5 N sodium hydroxide was added dropwise to adjust the pH to neutral. A solid precipitated, which was filtered under reduced pressure and then purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 3:7 v / v) to obtain compound H-2 (yield 80%) as a white solid.

[0074] 6-fluoro-3-(4-piperidinyl)-1,2-benzisazole (1 g, 4.29 mM, 1.0 eq), toluene (30 mL), and compound H-2 (0.95 g, 4.31 mM, 1.0 eq) were added sequentially to a flask. The mixture was reacted at 110 °C for 48 h under sealed conditions. The reaction was stopped by TLC. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane:methanol = 32:1 V / V) to obtain compound H-3 (yield 56.4%) as a white solid.

[0075] In a flask, compound H-3 (1 g, 2.20 mM, 1.0 eq) was dissolved in a mixed solvent of ethanol and water (4:1 v / v). The pH was adjusted to 4.5 with concentrated hydrochloric acid, and iron powder (0.66 g, 11.79 mM, 5 eq) was added. The reaction was carried out at room temperature for 12 h, and the reaction was monitored by TLC. After the reaction was completed, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (eluent: dichloromethane:methanol = 30:1 v / v) to purify compound H-4 (yield 82%) as a white solid.

[0076] Under a nitrogen atmosphere, 4-dimethylaminopyridine (3.66 g, 30 mM, 3.0 eq) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (5.75 g, 30 mM, 3.0 eq) were added to a dichloromethane solution of carboxylated polyethylene glycol carboxyethyl (3.38 g, 10 mM, 1.0 eq, dissolved by sonication) at room temperature with stirring. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was washed with saturated sodium chloride aqueous solution, and the organic layer was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: dichloromethane:methanol = 20:1 V / V) to give compound H-5 in 80% yield.

[0077] Data for compound H-5: 1H NMR (500MHz, DMSO-d6, TMS) δ H :12.01(1H,s),11.48(1H,s),10.03(1H,s),8.27,7.93,7.87,7.41,7.12,6.97(6H,m),3.72,3.61( 2H,m),3.52(8H,m),3.12(1H,m),2.78(1H,m),2.53(1H,m),2.34(2H,m),2.40(2H,m),1.79(1H,m). 13 C NMR (125MHz, DMSO-d6, TMS)δ C :177.35,171.07,162.62,165.33,160.66,150.80,147.22,135.63,134.05,127.52,123.08,122.99,117.88,115.34,112.30,96.77,6 9.81(C×2),69.80(C×2),69.77(C×2),69.89,69.54,67.71,66.70,52.73,48.49,48.15,36.94,36.89,36.70,32.84,28.86.ESI:744.6.

[0078] 4. Preparation of LYTAC molecules

[0079]

[0080]

[0081] At room temperature, compound Z-12 (20 g, 10.37 mmol, 1.0 equivalent) and methanol (200 mL) were added to a flask to obtain a methanol solution of compound Z-12. Then, 10% palladium carbon (2 g) was added. The flask was evacuated and purged with hydrogen five times. The mixture was stirred for 4 hours at room temperature under hydrogen protection. The solid was filtered off, and the filtrate was concentrated under reduced pressure to obtain compound Z-13 (16.5 g, yield 89%), a white solid.

[0082] Compound H-5 (12.8 g, 24.57 mmol, 1.0 equivalent) was dissolved in N,N-dimethylformamide (500 mL) to obtain a solution of compound H-5. N,N-diisopropylethylamine (9.0 g, 69.64 mmol, 3.00 equivalent) and O-benzotriazole-N,N,N-tetramethylhexafluorophosphate ureaium (9.9 g, 27.03 mmol, 1.10 equivalent) were added to the H-5 solution, and the mixture was stirred at room temperature for 2 hours. Then, compound Z-13 (44 g, 24.57 mmol, 1.00 equivalent) was added, and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure and purified by rapid preparative HPLC (60% methanol / water) to obtain compound Z-14 (22 g, 41% yield), a pale yellow solid. HRMS (ESI): C 115 H 172 FN 15 O 45 for[M+H] + , calculated 2520.1836, found2520.1830.

[0083] Compound Z-14 was dissolved in methanol, and 0.5 M sodium methoxide solution was added until the pH reached 9. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, formic acid was added to quench the reaction, and the mixture was concentrated under vacuum to obtain the target compound Z-15 in 36% yield.

[0084] Data for compound Z-15: 1 H-NMR(500MHz,CD3OD,TMS):11.48(s,1H),10.03(s,1H),8.14(t,4H),7.70(m,6H),8.27,7.87,7.93,7.41,7.12,6.97(m,6H),6.02(m,3H) ),4.51(m,3H),4.37(m,3H),3.947(m,3H),4.32(d,J=4.0Hz,3H),4.32(m,3H),3.90(d,J=4.0Hz,1H),3.60(d,J=8.0Hz,6H),3.51(m,6H),3 35(m,6H),3.42(m,12H),3.62(m,6H),3.51(m,6H),3.12(m,2H),2.78-2.53(m,4H ),2.40(m,6H),2.35-2,13(m,18H),1.99(s,9H),1.88(m,6H),1.53-1.44(m,12H). 13C NMR(125MHz,CD3OD,TMS)δ173.95(C×3),171.99(C×3),171.06(C×3),172.22,171.06,166.31,164.67,163.26,153.61 ,147.78,140.50,128.47,125.64,119.11,118.48,114.43,109.50,103.27(C×3),99.29,77.14(C×3),73.39(C×3),71 .61-71.32(C×3),70.09-68.47(C×3),61.49,63.44(C×3),62.32(C×3),53.94(C×3),50.47(C×3),38.24(C×3),38.14( C×3),37.89(C×3),36.96(C×3),30.50(C×3),32.05(C×3),31.19(C×3),27.50,24.91(C×3),23.83(C×3).HRMS(ESI):C 97 H 154 FN 15 O 37 for[M+H] + , calculated 2142.0943, found 2142.0940.

[0085] Example 2

[0086] In vitro anti-liver fibrosis study

[0087] Instruments: Clean bench (SW-CJ-1FD, AIRTECH, Suzhou Jingantai), constant temperature CO2 incubator (3111, Thermo, USA), inverted biological microscope (IX71, Olympus, Japan), enzyme-linked immunosorbent assay (ELISA) system (Model680, Bio-rad, USA), plate shaker (Kylin-bell lab Instruments), autoclave (YXO.SG41.280, Shanghai Huaxian), centrifuge (Sigma), Tianneng gel imaging system (Shanghai Tianneng).

[0088] Reagents: 1640 medium (Gibco), fetal bovine serum (Gibco), Western and IP cell lysate (Sigma), trypsin (Sigma), DMSO (Sigma), BCA protein assay kit (Sigma), New-Surper-ECL assay kit (Sigma), α-Smooth Muscle Actin (ACTA2) specific primary antibody (ABclonal), HTR2A Rabbit pAb specific primary antibody (ABclonal), SR-2A specific primary antibody (Santa Cruz), LAMP2 specific primary antibody (ABclonal), Bad specific primary antibody (ABclonal), Cleaved PARP (Asp214) specific primary antibody (ABclonal), Bax specific primary antibody (Beyotime), PARP specific primary antibody (Beyotime), GAPDH specific primary antibody (Proteintech), TGF-β1 Protein specific primary antibody (ABclonal), Cy3 Goat Anti-Rabbit IgG (H+L) (ABclonal), Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) (Beyotime), DAPI staining solution (Beyotime), horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (Proteintech), horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (Beyotime), proteasome inhibitor (MG132) (Abcam), 2-deoxy-D-glucose (Sigma), chloroquine (Sigma), nystatin (Sigma), calpain inhibitor I (ALLN) (Sigma), leupeptin (Sigma), 5-(N-methyl-N-isobutyl)amiloride (EIPA) (Sigma), Lyso-Tracker Red (Beyotime), Annexin V-FITC / PI double staining apoptosis detection kit (KGI Biotechnology), Oil Red staining solution (for cultured cells) (Nanjing Senbeijia Biotechnology Co., Ltd.), total cholesterol (T-CHO) test kit (Nanjing Jiancheng Bioengineering Institute), triglyceride (TG) test kit (Nanjing Jiancheng Bioengineering Institute), High Gene plus Transfection reagent (ABclonal), BSA (Beyotime), anti-fluorescence quenching mounting solution (containing DAPI) (Beyotime), immunostaining permeabilization solution (Triton X-100) (Beyotime), 4% paraformaldehyde fixative (Beyotime), cell spreader (Biosharp).

[0089] Cell lines: human normal hepatocytes LO2, human hepatocellular carcinoma cells HepG2, mouse normal hepatocytes AML-12, rat normal hepatocytes BRL-3A, and human hepatic stellate cells LX-2 (Jiangsu Kaiji Biotechnology Co., Ltd.); BRL-3A and HepG2 were cultured in DMEM medium, and the remaining cells were cultured in 1640 medium.

[0090] Cytotoxicity test

[0091] The cytotoxicity of compound Z-15 was tested using the MTT assay (tetramethylazozolium blue colorimetric method).

[0092] method:

[0093] Preparation of 1640 complete culture medium: Add 50 mL of fetal bovine serum to 450 mL of 1640 culture medium to prepare 1640 complete culture medium, store it in a 4℃ refrigerator, and wait for use.

[0094] Preparation of DMEM complete medium: Add 50 mL of fetal bovine serum to 450 mL of DMEM medium to prepare DMEM complete medium, and store it in a refrigerator at 4°C for later use.

[0095] The above-mentioned cryopreserved cell lines were revived and inoculated into complete culture medium. They were then cultured in a CO2 incubator at 37°C, with the medium changed daily. When the cells were in the exponential growth phase and in good condition, 1 mL of 0.25% trypsin was added for digestion for 1–2 minutes. Digestion was stopped when adherent cells became rounded and wrinkled under a microscope. Cells were collected, and culture medium was added to prepare a single-cell suspension. Cells were counted at a density of 5 × 10⁶ cells per well. 4 Calculate the required cell suspension volume based on the number of cells and the total number of wells. Seed the cell suspension at 100 μL / well in a 96-well cell culture plate. Seal the 96-well cell culture plate with PBS and incubate at 37°C in a CO2 incubator for 24 h. Set up a blank control group and a drug-treated group. The blank control group was replaced with complete culture medium without compound Z-15, and the drug-treated group was replaced with drug-containing culture medium (the final concentration of compound Z-15 was prepared using complete culture medium at 0.005, 0.05, 0.5, 5, 10, 25, and 50 μM). Each treatment was performed in triplicate, and the plates were cultured for another 48 h. After the culture was completed, 10 μL / well of 5 mg / mL MTT reagent was added to the 96-well plate, and the plates were incubated for another 4 h. The culture medium was removed from the wells, and 100 μL of DMSO was added to each well. The plates were shaken on a plate shaker for 10 min. The absorbance of each well was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated.

[0096]

[0097] The cell viability of compound Z-15 is shown in Table 1. Compound Z-15 did not show significant cytotoxicity to hepatocytes, indicating that the compound has good safety.

[0098] Table 1. Effects of compound Z-15 on the viability of different hepatocytes

[0099]

[0100] Western blot analysis detected the effect of compound Z-15 on 5-HT 2A Degradation activity of R

[0101] AML-12 cells were cultured in 1640 medium. Once the cells were in good growth condition, cells in the logarithmic growth phase were harvested and seeded into 6-well plates (6 × 10⁻⁶ cells / well). 5 In the cell / well array, blank control group, drug group, and risperidone group were set up. Blank control group: cultured in a complete culture set containing 0.1% DMSO. Drug group: cultured in drug-containing medium (compound Z-15 final concentrations were 0.02, 0.05, 0.1, 0.5, and 1 μM). Risperidone group: cultured in drug-containing medium (5-HT... 2A The final concentrations of the R antagonist risperidone were 0.02, 0.05, 0.1, 0.5, and 1 μM. Cells in each group were incubated for 48 h. The supernatant was collected in centrifuge tubes, and the cells were washed twice with PBS and transferred to centrifuge tubes. 1 mL of EDTA trypsin was added to the 6-well plate for digestion, and the cells were transferred to centrifuge tubes. The cells were centrifuged (1000 rpm for 5 min), and the pellet (i.e., cells) was collected in a 1.5 mL centrifuge tube. RIPA cell lysis buffer was added, and the cells were centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected as the protein solution, which was stored at -78 °C for later use. Protein quantification was performed using a BCA assay kit to determine the protein concentration of each sample. Samples were diluted to the same concentration with 5× protein loading buffer and separated by electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE, 10% gel). The separated samples were then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder in TBST buffer. The membrane was washed three times with TBST and incubated overnight at 4°C with Prdx2 specific primary antibody and β-actin specific primary antibody. The membrane was then washed three times with TBST and treated with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) secondary antibody at room temperature for 2 hours. The membrane was then washed three times with TBST. Finally, protein bands were visualized using a chemiluminescence imaging system. The corresponding band optical density values ​​were determined using image processing software (Image Lab Plus 6.0). Each treatment was repeated three times, and the results are expressed as averages.

[0102] Compound Z-15 against 5-HT 2A The degradation activity results of R are as follows: Figure 1 As shown, it can be observed that 48 hours after administration, 5-HT levels in risperidone-treated AML-12 cells... 2A R (i.e.) Figure 1 The expression level of SR-2A (hereinafter the same) did not change significantly. Compared with the same concentration of risperidone, compound Z-15 showed no significant effect on the target protein 5-HT. 2A R exhibits significant degradation activity, and can significantly reduce 5-HT. 2A R protein levels; Compound Z-15 can significantly degrade 5-HT at a concentration of 500 nM. 2A R, and this effect shows concentration dependence.

[0103] Western blot analysis detected the effect of compound Z-15 on 5-HT 2A Degradation mechanism of R

[0104] (1) BRL-3A cell lines were seeded in DMEM complete medium and cultured in a CO2 incubator at 37℃. The medium was changed once a day. When the cells were in the exponential growth phase and in good condition, 1 mL of 0.25% trypsin digestion solution was added for 1-2 min. Digestion was stopped when the adherent cells became rounded and wrinkled under a microscope. The cells were collected, and a single-cell suspension was prepared by adding medium. The cells were counted at 5 × 10⁶ cells per dish. 4The required volume of cell suspension was calculated based on the number of cells and the total number of culture dishes. The cell suspension was inoculated into 6 cm culture dishes and cultured for 24 h. The following groups were established: a blank control group (DMSO group), a compound Z-15 treatment group (LYTAC group), a chloroquine treatment group (Chloroquine+LYTAC group), a 2-deoxy-D-glucose treatment group (2-deoxyglucose+LYTAC group), a nystatin treatment group (Nystatin+LYTAC group), and a 5-(N-methyl-N-isobutyl)amilolide treatment group (EIPA+LYTAC group). The blank control group was replaced with a complete culture set containing 0.1% DMSO. The compound Z-15 treatment group was replaced with a complete culture set containing 0.1% DMSO. The chloroquine treatment group used a 0.1% DMSO-containing culture set. The following groups were cultured: DMSO complete medium (chloroquine dissolved in the medium to a final concentration of 50.0 μM); 2-deoxy-D-glucose dosing group: replaced with 0.1% DMSO complete medium (2-DOG dissolved in the medium to a final concentration of 50.0 mM); nystatin dosing group: replaced with 0.1% DMSO complete medium (nystatin dissolved in the medium to a final concentration of 30.0 μM); 5-(N-methyl-N-isobutyl)amiloride dosing group: replaced with 0.1% DMSO complete medium (5-(N-methyl-N-isopropyl)amiloride (EIPA). BRL-3A cells in each group were pre-incubated for 2 hours in complete DMSO medium (EIPA dissolved in the medium to a final concentration of 100 mM). The medium was then discarded. The blank control group was replaced with fresh complete medium, while the other treatment groups were replaced with complete medium containing 500 nM compound Z-15. Cells were incubated for 12 hours. The supernatant was collected in centrifuge tubes, and the culture dishes were washed twice with PBS and transferred to centrifuge tubes. 1 mL of EDTA trypsin was added to the culture dishes to digest the cells. When most cells were observed to be rounded under a microscope, complete medium was added to stop the digestion. The culture media were combined and centrifuged (1000 rpm, 5 min). The pellet (cells) was collected and placed in a 1.5 mL centrifuge tube. The protein extraction and quantification, SDS-PAGE electrophoresis and transfer, development and analysis methods used in the "Western blot assay for the degradation activity of compound Z-15 on 5-HT2AR" were employed to analyze the 5-HT2AR degradation activity of compound Z-15 in each group of BRL-3A cells. 2A The level of R protein was quantitatively analyzed.

[0105] (2) As above, BRL-3A cells were seeded in 6cm culture dishes and cultured for 24h (50×10⁻⁶ cells). 4 / plate), and separate groups were set up for the following: blank control group (DMSO group), compound Z-15 treatment group (LYTAC group), leupetin blank group (Leupetin group), leupetin treatment group (Leupetin+LYTAC group), calpain inhibitor I (ALLN) blank group (ALLN group), and calpain inhibitor I treatment group (ALLN+LYTAC group). The blank control group and compound Z-15 treatment group were replaced with complete medium containing 0.1% DMSO, while the leupetin blank group and leupetin treatment group were replaced with complete medium containing leupetin (leupetin dissolved in the medium to a final concentration of 80 μM). The calpain inhibitor I blank group and the calpain inhibitor I dosing group were replaced with complete culture medium containing calpain inhibitor I (calpain inhibitor I was dissolved in the culture medium to a final concentration of 1.0 mM) and cultured. BRL-3A cells in each group were pre-incubated for 2 h. Then the old culture medium was discarded. The compound Z-15 dosing group, the leupetin dosing group, and the calpain inhibitor I dosing group were replaced with complete culture medium containing 500 nM compound Z-15. The blank control group, the leupetin blank group, and the calpain inhibitor I blank group were replaced with fresh complete culture medium. The cells in each group were incubated for 12 h. The supernatant was collected in centrifuge tubes, the culture dishes were washed twice with PBS, and the cells were transferred to centrifuge tubes. 1 mL of EDTA trypsin was added for digestion. When most cells were observed to be rounded under a microscope, complete culture medium was added to stop digestion. The culture media were combined in centrifuge tubes and centrifuged (1000 rpm, 5 min). The cell pellet was collected in 1.5 mL small centrifuge tubes. The protein samples were extracted and quantified, and subjected to SDS-PAGE electrophoresis and transfer, development and analysis were analyzed using the method described in "Western blot assay to detect the degradation activity of compound Z-15 on 5-HT2AR". This method was employed to detect the degradation activity of 5-HT2AR in BRL-3A cells from each group. 2A Quantitative analysis of R protein levels was performed.

[0106] The results are as follows Figure 2 As shown. By Figure 2 A. It can be observed that the cell membrane protein degradation was significant in the compound Z-15 treatment group; in the 2-deoxy-D-glucose treatment group and the chloroquine treatment group, the degradation of target proteins in cells was inhibited, and the target protein 5-HT was reduced. 2A R expression was not reduced; however, nystatin and 5-(N-methyl-N-isopropyl)amiloride, being inhibitors of endocytosis via other pathways, showed no inhibition of target protein degradation and reduced target protein expression in the nystatin and 5-(N-methyl-N-isopropyl)amiloride groups, demonstrating that target protein degradation depends on clathrin endocytosis. Figure 2 As shown in Figure B, pretreatment of cells with two lysosomal inhibitors, Leupeptin and ALLN, reduced 5-HT levels in the cells. 2AThe expression of the R protein was not reduced, and even with the addition of compound Z-15, it could no longer exert its effect on 5-HT. 2A The degradation effect of R protein. These results indicate that compound Z-15 degrades 5-HT through lysosomes. 2A R function.

[0107] Western blot experiments investigated the effect of compound Z-15 on 5-HT 2A Effect of R concentration on degradation dependence

[0108] (1) Resuscitate BRL-3A, HepG2, LO2, and LX-2 cells, and seed them into culture flasks using the corresponding culture media. Place the flasks in a CO2 incubator and culture at 37°C with a 5% CO2 concentration. Change the culture medium daily and observe the cell status under a microscope. Once the cells are in good growth condition, under aseptic conditions, take cells in the exponential growth phase and seed them into 6cm culture dishes (50×10⁻⁶ cells / cm²). 4 The cells were cultured in culture dishes for 24 hours. The supernatant was collected in centrifuge tubes, and the culture dishes were washed twice with PBS. The supernatant was then transferred to centrifuge tubes, and 1 mL of EDTA trypsin was added to the culture dishes for digestion. The culture media were then combined in centrifuge tubes and centrifuged (1000 rpm, 5 min). The pellet (i.e., cells) was collected in 1.5 mL centrifuge tubes. The protein extraction and quantification, SDS-PAGE electrophoresis and transfer, development and analysis methods used in the "Western blot experiment to detect the degradation activity of compound Z-15 on 5-HT2AR" were employed to analyze the intracellular 5-HT2AR in different cell lines. 2A The level of R protein was quantitatively analyzed.

[0109] 5-HT in different cell lines 2A The expression of R protein is as follows Figure 3 As shown, 5-HT 2A 5-HT is almost not expressed in human hepatic stellate cells (LX-2), but is expressed more abundantly in normal human hepatocytes (LO2) and normal rat hepatocytes (BRL-3A). It is also expressed in HepG2 liver cancer cells. 2A R is the most frequently expressed.

[0110] (2) HepG2, BRL-3A and LO2 cells were seeded into 6cm culture dishes (50×10⁻⁶ cells per 100cm inch). 4 In petri dishes, compound Z-15 was prepared into drug-containing media (concentrations of 20, 50, 100, 500, and 1000 nM using the corresponding media) and cultured in media containing 0.1% DMSO. Figure 4After DMSO treatment for 24 hours, the supernatant was collected in centrifuge tubes. The culture dishes were washed twice with PBS and transferred to centrifuge tubes. EDTA trypsin was added for digestion. Once most cells were observed to be round under a microscope, complete culture medium was added to stop digestion. The culture media were combined in centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The pellet (i.e., cells) was collected and placed in a 1.5 mL centrifuge tube. The protein sample extraction and quantification, SDS-PAGE electrophoresis, membrane transfer, development, and analysis methods described in "Western blot assay for the degradation activity of compound Z-15 on 5-HT2AR" were used to analyze intracellular 5-HT2AR in different cell lines. 2A The level of R protein was quantitatively analyzed. The corresponding band optical density values ​​were determined using image processing software (Image Lab Plus 6.0). Each concentration was tested three times, and the results are expressed as averages.

[0111] In normal rat hepatocytes (BRL-3A), the expression of 5-HT2AR protein decreased at a concentration of 100 nM, demonstrating the degradation effect of compound Z-12. At concentrations of 500 nM and 1.0 μM, 5-HT2AR protein expression was very low, indicating relatively complete degradation. In human hepatocellular carcinoma cells (HepG2), a degradation trend was observed at a concentration of 100 nM, with a significantly enhanced degradation effect at 500 nM. The same pattern was observed in normal human hepatocytes (LO2), where a concentration of 500 nM reduced 5-HT2AR protein expression, indicating a significant degradation effect. These results suggest that the degradation effect of compound Z-15 on 5-HT2AR protein becomes increasingly significant with increasing concentration.

[0112] Western blot experiments investigated the effect of compound Z-15 on 5-HT 2A Effect of R time-degradation dependent

[0113] BRL-3A cells were revived and seeded into culture flasks using DMEM complete medium. These flasks were then placed in a CO2 incubator at 37°C and a 5% CO2 concentration. The medium was changed daily, and cell status was observed under a microscope. Once the cells were in good growth condition, under aseptic conditions, cells in the exponential growth phase were harvested and seeded into 6cm culture dishes (50×10⁶ cells / cm²). 4 The culture was carried out in a petri dish for 24 hours, then replaced with a medium containing 500 nM compound Z-15 and cultured for 3, 6, 12, 24 and 48 hours, respectively. A blank control group was also set up. Figure 5DMSO treatment: Cells were cultured in complete medium containing 0.1% DMSO. The supernatant was collected in centrifuge tubes, the culture dishes were washed twice with PBS, and the cells were transferred to centrifuge tubes. EDTA trypsin was added to digest the cells until most cells became round. Complete medium was added to stop digestion, and the cultures were combined in centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes, and the pellet (cells) was collected in 1.5 mL centrifuge tubes. The protein extraction and quantification, SDS-PAGE electrophoresis, membrane transfer, development, and analysis techniques used in the "Western blot assay for the degradation activity of compound Z-15 on 5-HT2AR" were employed to analyze the degradation activity of 5-HT2AR in the BRL-3A cell line. 2A The level of R protein was quantitatively examined. The corresponding band density values ​​were determined using image processing software (Image Lab Plus 6.0). Each concentration was tested three times, and the results are expressed as averages.

[0114] The results are as follows Figure 5 As shown, after 24 hours of treatment with compound Z-15, BRL-3A cells exhibited clear degradation of 5-HT2AR protein. Quantitative analysis revealed that compound Z-15 degraded approximately 50% of the 5-HT2AR protein 12 hours after administration, and almost completely degraded the intracellular 5-HT2AR protein 24 hours after administration. This indicates that the degradation effect of compound Z-15 on 5-HT2AR becomes increasingly significant with increasing administration time.

[0115] Laser confocal fluorescence localization experiment

[0116] BRL-3A cell lines were inoculated into complete culture medium and cultured in a CO2 incubator at 37°C. The culture medium was changed daily. When the cells were in the exponential growth phase and in good condition, 1 mL of 0.25% trypsin was added for 1–2 minutes for digestion. Digestion was stopped when the adherent cells became rounded and wrinkled under a microscope. The cells were collected, added to culture medium to prepare a single-cell suspension, and the cells were counted at a ratio of 1 × 10⁻⁶. 5Add 1 mL / mL of the solution to a 15 mm laser confocal dish and incubate for 24 hours. For the drug-treated group, replace the medium with drug-containing medium (compound Z-15 final concentration 1 μM). A blank control group is set up using drug-free complete medium containing 0.1% DMSO. Incubate for another 12 hours, then add 100 nM lysosome targeting reagent (Lyso Tracker Red) and incubate for a total of 40 minutes. Remove the culture dishes and let them stand at room temperature for 5 minutes. Wash three times with PBS for 3 minutes each time, then permeate with 0.5% Triton X-100 (prepared in PBS) at room temperature for 20 minutes. Wash three times with PBS for 3 minutes each time, then blot dry with absorbent paper. Add 5% BSA to a slide and block at room temperature for 40 minutes. Blot off the blocking solution with absorbent paper, do not wash, add sufficient diluted primary antibody to each dish, and incubate overnight at 4°C. Remove the confocal dishes from the refrigerator, warm them at 37°C for 30 minutes, and wash three times with PBS for 5 minutes each time. Wash the slides three times with PBS for 5 minutes each time. After blotting off excess liquid with absorbent paper, add diluted fluorescent secondary antibody and incubate in a humidified chamber at 37°C for 40 minutes. Wash three more times with PBS for 5 minutes each time. Take pictures using a laser confocal microscope.

[0117] The results are as follows Figure 6 As shown, the top row represents the blank control group, and the bottom row represents the drug-treated group. From left to right, each row shows blue-stained cell nuclei, green-stained 5-HT2AR, red-stained lysosomes, and a Merge plot. The target protein and lysosomes exhibit significant co-localization, appearing orange, indicating that the target protein 5-HT2AR... 2A R is transported to the lysosome by compound Z-15 and degraded.

[0118] Western blot experiments were conducted to investigate the effects of compound Z-15 on the release of the cytokine TGF-β from hepatocytes and its indirect effect on the activation of hepatic stellate cells.

[0119] BRL-3A cells were seeded into 6-well plates, with the cell density controlled at 70%-90%; the next day, 4 μg of 5HT ordered from Genscript Biotech was taken. 2AThe plasmid _pcDNA3.1(+)-myc-His A (optimized host Human, N-terminal lent with the Kozak sequence GCCACC, cloned into the expression vector pcDNA3.1(+) / myc-His A using KpnI / ApaI; after construction, the target protein is fused with the vector backbone Myc and 6xHis at the C-terminus) was added to a centrifuge tube containing 200 μL of serum-free DMEM basal medium and mixed thoroughly by pipetting. Then, 8 μL of High Gene plus transfection reagent was added and mixed by pipetting. 200 μL of the plasmid / High Geneplus transfection reagent complex was evenly added to the wells of a 6-well cell culture plate, and the plate was gently shaken to distribute it evenly. Cells were transfected for 4-6 hours, and half of the medium was replaced with fresh complete medium. Stable transfection with high expression of 5-HT was achieved after 24-48 hours of transfection. 2A R's BRL-3A cell line.

[0120] The Normal group, Transfection group, blank control group, and drug-treated group were set up. Unactivated LX-2 cells were seeded into 6cm culture dishes (50×10⁶ cells / cm²). 4 LX-2 cells were cultured in wells for 24 hours. Normal group: Unactivated LX-2 cells were incubated in medium containing 0.1% DMSO for 12 hours. Transfection group: Cells stably transfected with high expression of 5-HT... 2A R's BRL-3A cells were incubated in culture medium for 24 hours, centrifuged at 8000 rpm for 10 minutes, and 8 mL of the supernatant was collected and co-incubated with unactivated LX-2 cells for 12 hours. Blank control group: stably transfected with high expression of 5-HT 2A R's BRL-3A cells were incubated in medium containing 0.1% DMSO for 24 hours, centrifuged at 8000 rpm for 10 minutes, and 8 mL of the supernatant was collected and co-incubated with unactivated LX-2 cells for 12 hours. The drug-treated group was stably transfected with high expression of 5-HT. 2ABRL-3A cells were incubated for 24 hours in drug-containing medium (compound Z-15 final concentrations of 100 nM, 500 nM, and 1000 nM) containing 0.1% DMSO. After centrifugation at 8000 rpm for 10 minutes, 8 mL of supernatant was collected and co-incubated with LX-2 cells for 12 hours. The supernatant was collected in centrifuge tubes, and the culture dishes were washed twice with PBS and transferred to centrifuge tubes. EDTA trypsin was added to the culture dishes for digestion. When most cells became round, complete medium was added to stop the digestion process. The medium was combined with the culture dishes and centrifuged at 1000 rpm for 5 minutes. The cell pellet was collected in 1.5 mL centrifuge tubes. The levels of α-SMA protein inside LX-2 cells were quantitatively analyzed using the methods described in "Western blot assay for the degradation activity of compound Z-15 on 5-HT2AR", including protein extraction and quantification, SDS-PAGE electrophoresis, membrane transfer, imaging, and analysis. The corresponding strip optical density values ​​were determined using image processing software (Image Lab Plus 6.0). Each treatment was repeated three times, and the results are expressed as average values.

[0121] The results are as follows Figure 7 As shown, stable transfection with high expression of 5-HT was used. 2A Treatment of LX-2 cells with the supernatant of BRL-3A cells increased the expression of α-SMA, a marker of liver fibrosis, in LX-2 cells, indicating that LX-2 cells were activated. Treatment of BRL-3A cells with compound Z-15 resulted in high expression of 5-HT. 2A R is degraded, and then stably transfected with high expression of 5-HT 2A Co-incubation of BRL-3A cell supernatant with LX-2 cells showed that α-SMA protein expression in LX-2 cells gradually decreased with increasing dosage of compound Z-15. This demonstrates that compound Z-15 degrades 5-HT 2A R induces changes in the inflammatory pathway signaling of BRL-3A in hepatocytes, indirectly affecting the activation of LX-2 hepatic stellate cells.

[0122] Western blot experiments were conducted to investigate the optimal concentration and time for TGF-β activation of LX-2 cells.

[0123] Unactivated LX2 cells were seeded in 6cm culture dishes (50×10⁶ cells / cm²). 4Cells were cultured in 1640 complete medium for 24 h, then replaced with drug-containing medium containing different concentrations of TGF-β activator (0, 1, 2.5, 5, 10, 20 ng / mL) for another 24 h. The supernatant was collected in centrifuge tubes, and the culture dishes were washed twice with PBS and transferred to centrifuge tubes. 1 mL of EDTA trypsin was added to the culture dishes for digestion. When most cells were observed to be rounded under a microscope, complete medium was added to stop digestion. The culture dishes were combined and centrifuged (1000 rpm, 5 min). The cell pellet was collected in 1.5 mL centrifuge tubes. The level of intracellular α-SMA protein was quantitatively analyzed using the methods described in "Western blot assay for the degradation activity of compound Z-15 on 5-HT2AR", including protein extraction and quantification, SDS-PAGE electrophoresis and transfer, development and analysis.

[0124] The results are as follows Figure 8 As shown in A and B, the optimal activation concentration of the activator for LX-2 cells is 5 ng / mL, and this concentration will be used as the baseline for subsequent experiments.

[0125] Unactivated LX2 cells were seeded into 6cm culture dishes (50×10⁶ cells / cm²). 4 The cells were cultured in a medium containing 5 ng / mL TGF-β for 24 h, and then cultured for 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h, respectively. The supernatant was collected in centrifuge tubes, and the culture dishes were washed twice with PBS and transferred to centrifuge tubes. 1 mL of EDTA trypsin was added to the culture dishes for digestion. When most cells were observed to be rounded under a microscope, complete culture medium was added to stop the digestion. The culture media were combined and centrifuged (1000 rpm, 5 min). The cell pellet was collected in a 1.5 mL centrifuge tube. The level of α-SMA protein in the cells was quantitatively analyzed using the methods described in "Western blot assay for detecting the degradation activity of compound Z-15 on 5-HT2AR", including protein extraction and quantification, SDS-PAGE electrophoresis and transfer, development and analysis.

[0126] The results are as follows Figure 8 As shown in C and D, the optimal activation time for LX-2 cells by the activator is 24 hours, and this time will be used as the baseline for subsequent experiments.

[0127] Immunofluorescence staining experiment

[0128] Use sterile forceps to place the cell smear into a 6-well plate; digest the unactivated LX-2 cells with trypsin, and thoroughly pipette them to form a single-cell suspension. Remove the 6-well plate containing the cell smear and drop 1 mL of the single-cell suspension onto the smear one drop at a time into each well. Then incubate the plate at 37°C and 5% CO2 for 12 hours. Set up a blank control group, a co-incubation group (Culture), and a TGF-β group (TGF-β + LX-2). Discard the old culture medium. For the blank control group, the co-incubation group (Culture), and the TGF-β group, add 2 mL of blank culture medium, 1000 nM compound Z-15 group BRL-3A supernatant collected according to the experimental method of "Western blot experiment to investigate the effect of compound Z-15 on the release of cytokine TGF-β from hepatocytes and its indirect effect on the activation of hepatic stellate cells", and fresh culture medium containing 5 ng / mL activator TGF-β to each well. Each sample is set up in triplicate. Co-incubate for 12 h, remove the culture medium, wash three times with PBS, fix in 4% paraformaldehyde for 15 min, and then wash three times with PBS for 3 min each time; 0.5% Triton X-100 (prepared with PBS) was used for permeation at room temperature for 20 min. The slides were then washed three times with PBS for 3 min each time. The PBS was blotted dry with absorbent paper. 5% BSA was added to the slides, and the slides were blocked at room temperature for 40 min. The blocking solution was blotted off with absorbent paper, but the slides were not washed. Sufficient diluted primary antibody was added to each slide, and the slides were placed in a humidified chamber and incubated overnight at 4°C. The slides were then removed from the refrigerator and warmed to 37°C for 30 min. The slides were washed three times with PBS for 5 min each time, and the slides were rinsed three times with PBS for 5 min each time. Excess liquid was blotted dry with absorbent paper. Diluted secondary fluorescent antibody was added, and the slides were incubated at 37°C for 40 min in a humidified chamber. The slides were then washed three times with PBS for 5 min each time. DAPI was added and incubated in the dark for 5-7 min to stain the nuclei. Excess DAPI was then washed away with PBS. The liquid on the slides was blotted dry with absorbent paper. The slides were then mounted with mounting medium containing an anti-fluorescence quencher, and the images were observed and acquired under a fluorescence microscope.

[0129] See results Figure 9 The expression of α-SMA in the co-incubation group (Culture) and the TGF-β group (TGF-β+LX-2) was significantly increased, indicating that LX-2 cells were significantly activated. The TGF-β group with activator was more effective than the co-incubation group in the activated state.

[0130] Immunofluorescence staining and Western blotting assays were used to detect 5-HT after activation of LX-2 cells. 2A R expression

[0131] Unactivated LX-2 cells were seeded into 6 cm diameter culture dishes, with 50 × 10⁶ cells per dish. 4Cells were cultured for 24 hours. A blank control group, a BRL-3A co-incubation group (Culture), and a TGF-β group (TGF-β+LX-2) were set up. The old culture medium was discarded. For the blank control group, the BRL-3A co-incubation group, and the TGF-β group, 2 mL of blank culture medium, 1000 nM supernatant of BRL-3A cells from the compound Z-15 group (collected using the experimental method "Western blot experiment to investigate the effect of compound Z-15 on the release of cytokine TGF-β from hepatocytes and its indirect effect on the activation of hepatic stellate cells"), and fresh culture medium containing 5 ng / mL activator TGF-β were added to each well. Each sample was tested in triplicate. All cells were incubated for 12 hours. The supernatant was collected into centrifuge tubes, and the culture dishes were washed twice with PBS and transferred to centrifuge tubes. EDTA trypsin was added to the culture dishes for digestion. The culture media were combined and transferred to centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes and collected into 1.5 mL centrifuge tubes. The intracellular SR-2A protein was extracted, quantified, subjected to SDS-PAGE electrophoresis, transferred to a membrane, developed, and analyzed using a Western blot assay to detect the degradation activity of compound Z-15 on 5-HT2AR.

[0132] like Figure 10 The results showed that LX-2 cells were activated in both the BRL-3A co-incubation group (Culture) and the TGF-β activation group (TGF-β), and LX-2 cells' 5-HT... 2A R expression was significantly increased. This further demonstrates that LX-2 cells highly express 5-HT after activation. 2A R. Compound Z-15 primarily targets 5-HT. 2A R plays a role.

[0133] The effect of different concentrations of compound Z-15 on LX-2 cell viability was detected by CCK8 assay.

[0134] The test compound Z-15 was dissolved in DMSO to prepare a 1 mM stock solution, which was then diluted with 1640 complete medium to obtain drug-containing mediums of different concentrations (0.02, 0.05, 0.5, 5, 10, 25, 50, 75, 100 μM).

[0135] When the LX-2 cell density reached 80%–90%, the cells were digested with trypsin and collected for counting. 3 × 10⁶ cells were collected per well. 3Cells were seeded at a density of 1,000 cells / well in 96-well cell culture plates, with 6 replicates per group, and incubated. The next day, the old culture medium in the 96-well plates was discarded, and fresh drug-containing culture medium was added. Each sample was seeded in 3 replicates, and a control group (culture medium containing 0.1% DMSO) was also included. Incubation continued for 48 hours. When administering the drug to a parallel 96-well plate of LX2 cells, 5 ng / mL of the activator TGF-β (i.e., 0.1% DMSO) was added simultaneously. Figure 11 Add TGF-β+LX-2 and incubate for another 48 hours. After the culture is complete, add 10 μL of CCK-8 reagent to each well, return the culture plate to the incubator, and incubate for 1 hour. Measure the absorbance (OD value) of each well using a multi-mode microplate reader at a wavelength of 450 nm to calculate the cell viability.

[0136] Cell viability (%) = [OD(experimental group) - OD(blank group)] / [OD(control group) - OD(blank group)] × 100%

[0137] The blank group consisted of cell-free blank culture medium.

[0138] From Table 2 and Figure 11 It can be seen that when the concentration of compound Z-15 was reduced to 5 μM, the survival rate of activated hepatic stellate cells increased to 74.43%, while the survival rate of normal human hepatic stellate cells at the same concentration was 103.76%. This indicates that compound Z-15 at 5 μM can inhibit the proliferation of activated LX-2 cells, while having little inhibitory effect on the proliferation of inactivated LX-2 cells. Therefore, this concentration was chosen for subsequent experiments.

[0139] Table 2. Effect of compound Z-15 on LX-2 cell viability

[0140]

[0141] Western blotting assay was used to detect changes in α-SMA expression in activated LX-2 cells after drug administration.

[0142] Unactivated LX-2 cells were seeded in 6cm culture dishes (50×10⁶ cells / cm²). 4The cells were cultured in 1000-300-300 medium dishes for 24 hours. Four control groups were established: a blank control group (LX-2 group), an LX-2+TGF-β group, a solvent control group (DMSO group), and a LYTAC group. The old medium was discarded and replaced with fresh medium, fresh medium containing 5 ng / mL TGF-β activator, fresh medium containing 5 ng / mL TGF-β activator and 0.1% DMSO, and fresh medium containing 5 ng / mL TGF-β activator and 5 μM compound Z-15, respectively. Each treatment had three replicates, and the cells were incubated for a total of 12 hours. The supernatant was collected in centrifuge tubes, and the culture dishes were washed twice with PBS and transferred to centrifuge tubes. 1 mL of EDTA trypsin was added to the culture dishes for digestion. When most cells were observed to be rounded under a microscope, complete medium was added to stop digestion. The cultures were combined and centrifuged (1000 rpm, 5 min). The cells were collected in 1.5 mL centrifuge tubes. The level of intracellular α-SMA protein was quantitatively analyzed using the methods described in "Western blot experiment to detect the degradation activity of compound Z-15 on 5-HT2AR", including protein sample extraction and quantification, SDS-PAGE electrophoresis and membrane transfer, development and analysis.

[0143] like Figure 12 As shown, after adding the activator TGF-β (i.e., LX-2+TGF-β), the expression level of α-SMA protein in LX-2 cells significantly increased, compared with that in unactivated LX-2 cells (i.e., Figure 12 The difference in expression levels between LX-2 cells and the solvent control group (DMSO group) was statistically significant (P<0.001). Compared with the solvent control group (DMSO group), the addition of 5 μM compound Z-15 significantly reduced the expression of α-SMA protein in cells (P<0.001). The results indicate that compound Z-15 can not only inhibit the proliferation of activated LX-2 cells, but also reduce the expression level of α-SMA protein.

[0144] Immunofluorescence staining experiment

[0145] Use sterile forceps to place the cell smears into a 6-well plate; digest the unactivated LX-2 cells with trypsin, thoroughly pipette them to form a single-cell suspension, remove the 6-well plate containing the cell smears, add 1 ml of the single-cell suspension to each well, and drop it drop by drop onto the glass slide, then incubate in a 37°C, 5% CO2 incubator. Then, the old medium was replaced with medium containing 5 ng / mL activator TGF-β. After incubation for 24 hours, LX-2 cells were activated. Then, DMSO control group, 5 μM compound Z-15 treatment group, and 10 μM compound Z-15 treatment group were set up. The activated human astrocytes LX-2 were treated with medium containing 0.1% DMSO, medium containing 5 μM compound Z-15, and medium containing 10 μM compound Z-15, respectively, and incubated for a total of 12 h. The medium was aspirated, washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, and then washed three times with PBS for 3 min each time. The slides were permeated with 0.5% Triton X-100 (prepared with PBS) at room temperature for 20 min, and then washed with PBS three times for 3 min each time. The PBS was blotted dry with absorbent paper, 5% BSA was added to the slides, and the slides were blocked at room temperature for 40 min. The blocking solution was blotted off with absorbent paper, and the slides were not washed. Sufficient amount of diluted primary antibody was added to each slide and placed in a humidified chamber for overnight incubation at 4°C. Remove the cell slides from the refrigerator and incubate them at 37°C for 30 minutes. Rinse three times with PBS, blot dry with absorbent paper, add diluted fluorescent secondary antibody, and incubate at 37°C for 40 minutes in a humidified chamber. Rinse the slides three times with PBS for 5 minutes each time. Add DAPI and incubate in the dark for 5-7 minutes to stain the nuclei. Wash away excess DAPI with PBS, blot dry with absorbent paper, and mount the cell slides with mounting medium containing anti-fluorescence quencher. Then observe and acquire images under a fluorescence microscope.

[0146] The experimental results are shown in Figure 13 The results showed that, compared with the DMSO control group, different concentrations of compound Z-15 reduced the expression level of α-SMA protein in activated LX-2 cells, indicating that compound Z-15 can effectively inhibit the activation effect of TGF-β on LX-2 cells, which is consistent with the results of the experiment "Western blotting experiment to detect changes in α-SMA expression in activated LX-2 cells after drug administration". This further demonstrates that compound Z-15 can reduce the expression of the fibrosis marker protein α-SMA in activated LX-2 cells, proving that compound Z-15 can inhibit the activation of stellate cells.

[0147] Annexin V / PI double staining flow cytometry cell proliferation assay

[0148] LX-2 cells were seeded into culture flasks and incubated at 37°C with 5% CO2 for 24 hours. Then, the old medium was replaced with medium containing 5 ng / mL TGF-β activator. After 24 hours of incubation, all LX-2 cells were activated. The activated LX-2 cells were washed three times with PBS, then digested with trypsin, and the cell pellets were collected by centrifugation. The cell pellets were diluted to 4 × 10⁻⁶ cells / mL with 1640 complete medium. 5 Single-cell suspensions of 1 cell / mL were seeded into 6cm culture dishes for culture. The culture medium was replaced the following day with fresh medium (activator group, TGF-β group), medium containing 0.1% DMSO (blank control group, DMSO group), and drug-containing medium containing compound Z-15 (1μM, 3μM, 5μM, respectively) (administered groups of 1μM, 3μM, and 5μM compound Z-15). After 24 hours of culture, all hepatic stellate cells were collected, centrifuged at 2000 rpm for 5 min, and cell clusters were collected. The cells were then washed twice with pre-cooled 0℃ PBS. Under light-protected conditions, 500μL of 1× Binding Buffer was added to the collected cell clusters to fully resuspend the cells, followed by 5μL of Annexin V-FITC reagent and finally 5μL of PI reagent. The cells were incubated at room temperature in the dark for 15 minutes, and cell proliferation was detected by flow cytometry.

[0149] The results are as follows Figure 14 As shown, TGF-β can promote the activation of hepatic stellate cells LX-2 and reduce the apoptosis rate. There was no statistically significant difference in the apoptosis rate between the activator group and the DMSO group. After administration of compound Z-15, the cell apoptosis rate increased significantly (P<0.001), and the number of dead cells increased significantly at high doses, indicating that compound Z-15 has a significant pro-apoptotic effect on activated LX-2 cells.

[0150] Western blotting assay was used to detect changes in the expression of apoptosis-related proteins in activated LX-2 cells after drug administration.

[0151] Unactivated LX-2 cells were seeded into 12 6cm culture dishes (50×10⁶ cells / cm²). 4LX-2 cells were cultured in fresh medium for 24 hours in 3 replicates per dish. Three dishes were used as unactivated LX-2 cells and replaced with fresh medium. The remaining 9 dishes were replaced with medium containing 5 ng / mL of activator TGF-β and incubated for 24 hours. LX-2 cells were activated. The next day, the LX-2+TGF-β group, the solvent control group, the TGF-β group (TGF-β+LX-2), and the drug-treated group (LYTAC) replaced their old medium with fresh medium, fresh medium containing 0.1% DMSO, and fresh medium containing 5 μM compound Z-15, respectively. The LX-2 group was replaced with fresh medium. The incubation was carried out for a total of 12 hours. Collect the supernatant into centrifuge tubes, wash the culture dishes twice with PBS, and transfer the washed cells to centrifuge tubes. Add EDTA trypsin to the culture dishes for digestion, combine the culture media into 15 mL centrifuge tubes, centrifuge at 1000 rpm for 5 minutes, and collect the cells into 1.5 mL centrifuge tubes. The degradation activity of compound Z-15 on 5-HT2AR was detected using Western blot. Intracellular protein samples were extracted, quantified, subjected to SDS-PAGE electrophoresis, transferred to a membrane, developed, and analyzed to obtain the levels of Bax, Bcl-2, PARP, and Cleaved PARP proteins.

[0152] The results are as follows Figure 15 The results showed that, compared with normal LX-2 cells, activated LX-2 cells (LX-2+TGF-β) increased the expression of the apoptosis-inhibiting protein Bcl-2 and decreased the expression of the apoptosis-promoting protein Bax. In addition, the expression of Cleaved-PARP (a marker of apoptosis) was significantly reduced, indicating that activated LX-2 cells inhibited apoptosis. The opposite was true in the drug-treated group, indicating that compound Z-15 promotes apoptosis of human hepatic stellate cells LX-2 by regulating the expression of apoptosis-related proteins (Bcl-2, Bax, PARP, and Cleaved-PARP).

Claims

1. A LYTAC compound with the structure shown in Formula I, or a pharmaceutically acceptable salt thereof:

2. A method for preparing the LYTAC compound according to claim 1, characterized in that: The synthesis route is as follows:

3. The method for preparing the LYTAC compound according to claim 2, characterized in that: include: Step (1): Under a nitrogen atmosphere, using dichloromethane as the reaction solvent, 4-dimethylaminopyridine as the catalyst, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride as the condensing agent, compound H-4 undergoes an amidation reaction with carboxylated polyethylene glycol carboxyethyl to obtain compound H-5. Step (2): Under a hydrogen atmosphere, with methanol as the reaction solvent and 10% palladium carbon as the catalyst, compound Z-12 was deprotected at room temperature to obtain compound Z-13. Step (3): Using N,N-dimethylformamide as the reaction solvent and N,N-diisopropylethylamine and O-benzotriazole-N,N,N-tetramethylhexafluorophosphate ureaium as condensing agents, compound Z-13 and compound H-5 undergo an amidation reaction to obtain compound Z-14; Step (4): Using methanol as the reaction solvent, sodium methoxide was added to adjust the pH of the reaction system to 9-10. Compound Z-14 was reacted at room temperature to obtain the LYTAC compound with the structure shown in Formula I.

4. The method for preparing the LYTAC compound according to claim 3, characterized in that: In step (1), the molar ratio of compound H-4 to carboxylated polyethylene glycol carboxyethyl is 1:1; the molar ratio of compound H-4 to 4-dimethylaminopyridine is 1:3; and the molar ratio of compound H-4 to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 1:

3.

5. The method for preparing the LYTAC compound according to claim 3, characterized in that: In step (3), the molar ratio of compound Z-13 to compound H-5 is 1:1; the molar ratio of compound Z-13 to O-benzotriazole-N,N,N-tetramethylurea hexafluorophosphate is 1:1; and the molar ratio of compound Z-13 to N,N-diisopropylethylamine is 1:

1.

6. The LYTAC compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a product targeting 5-HT 2A Uses of R degradation agent.

7. The LYTAC compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a treatment for 5-HT 2A Uses of R protein in drugs for diseases.

8. The use according to claim 7, characterized in that: The 5-HT 2A R protein-related diseases include liver fibrosis.

9. A pharmaceutical composition, characterized in that: It is formulated into a pharmaceutically acceptable dosage form using the LYTAC compound of claim 1 or its pharmaceutically acceptable salt as the active ingredient or main active ingredient, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that: The dosage form is tablet, capsule, powder, syrup, liquid, suspension, lyophilized powder for injection or injection.

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