An acly-targeting protac chimera with anti-mash activity, methods and uses

Abstract

This invention belongs to the field of biotechnology and pharmaceutical technology, and discloses an ACLY-targeting PROTAC chimera with anti-MASH activity using a glycol chain as the linking chain. Its general structural formula is Formula 1: R is -(CH2-O-CH2). n -, n is 2-6. The PROTAC compound synthesized in this invention is a novel compound. In a high-fat model established using L02 and HepG2 cells, the synthesized PROTAC compound exhibits activity in reducing TG content. Compared with the warhead, the synthesized PROTAC compound shows low toxicity activity in human hepatocellular carcinoma cells HepG2, normal human hepatocytes L02, and human umbilical vein endothelial cells HUVEC, making it a novel, low-toxicity, and highly effective drug for treating non-alcoholic steatohepatitis (NAH). This invention expands the application areas of PROTAC technology while developing a highly effective treatment for NHA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and pharmaceutical technology, and relates to the field of small molecule drug development, particularly an ACLY-targeting PROTAC chimera with anti-MASH activity using a glycol chain as the linking chain, its method, and its application. Background Technology

[0002] MASLD refers to excessive fat deposition in the liver caused by factors other than excessive alcohol consumption, viral infections, and other liver damage. If left untreated, it can develop into MASH. Untreated MASH can progress to cirrhosis and even liver cancer. Its incidence is extremely high, seriously threatening human life and health. However, currently only Resmetirom is available as a marketed drug for MASH. The pathogenesis of MASH is complex, and single-target therapies are insufficient to address the entire market. Developing a highly effective and safe drug for treating MASH is an urgent task and presents a significant challenge.

[0003] Studies have shown that excessive fat accumulation in most MASH patients originates from de novo fat synthesis. The raw material for de novo fat synthesis, acetyl-CoA, originates from pyruvate produced by glycolysis. Pyruvate in the cytoplasm is further glycolytically converted to acetyl-CoA. Acetyl-CoA enters the mitochondria and, through the tricarboxylic acid cycle, is converted to citrate. Citrate then enters the cytoplasm via the pyruvate-citrate shuttle mechanism, where it is converted to acetyl-CoA by adenosine triphosphate citrate lyase (ACLY). As a fundamental substrate for fatty acid and cholesterol biosynthesis, acetyl-CoA is an important target for treating MASH and dyslipidemia. ACLY can be selected as a target to inhibit acetyl-CoA production, thereby treating MASH.

[0004] Protein degradation-targeting chimeras (PROTACs) are a novel small-molecule protein-targeting degradation technology, consisting of three parts: a target protein (POI) ligand, an E3 ligase ligand (which recruits structures for the protein degradation system), and a linker connecting the two. When a PROTAC molecule enters the cell, the connecting strand in the middle folds and bends appropriately, allowing its two ends to penetrate into the hydrophobic cavities of the two receptors, bringing the E3 ubiquitin ligase closer to the target protein and forming a specific protein-protein interaction. When ATP is supplied, the E3 ligase ligand transfers the ubiquitin it carries to the substrate protein, causing ubiquitination. After the ternary complex dissociates, the ubiquitinated protein is recognized by the proteasome (UPS system), thus achieving the degradation of the target protein. PROTAC drugs have many advantages compared to traditional small-molecule drugs, such as lower cytotoxicity and the potential to overcome drug resistance. Currently, there are no reports of applying PROTAC technology to MASH.

[0005] Therefore, ACLY protein degraders based on PROTAC technology have a promising future as drugs for the treatment of MASH, bringing new hope to the treatment of MASH and expanding the application field of PROTAC technology. It is necessary to study them. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an ACLY-targeted PROTAC chimera with anti-MASH activity, a method, and an application, using a glycol chain as the linking chain.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] An ACLY-targeting PROTAC chimera with anti-MASH activity, using glycol chains as linking chains, has the general structural formula of Formula 1:

[0009]

[0010] In the formula, R stands for -(CH2-O-CH2). n -, where n is 2 to 6.

[0011] The specific structure of the PROTAC chimera is as follows:

[0012]

[0013] A method for preparing the PROTAC chimera as described above, comprising reacting a compound of formula 2 with a compound of formula 3:

[0014]

[0015] In Equation 2, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0016] Furthermore, Formula 2 is prepared by reacting with Formulas 4 and 5:

[0017]

[0018] In Equation 4, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0019] Formula 3 is prepared by reacting with Formulas 9 and 10:

[0020]

[0021] Furthermore, Formula 4 is prepared by reacting with Formulas 6 and 7:

[0022]

[0023] In Equation 6, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0024] Formula 9 is prepared by reacting with Formulas 11 and 12:

[0025]

[0026] Furthermore, Formula 6 is prepared by reacting Formulas 5 and 8:

[0027]

[0028] In Equation 8, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0029] Furthermore, Formula 10 is prepared by reactions of Formulas 13 and 14:

[0030]

[0031] Furthermore, the reaction formula is as follows:

[0032]

[0033] In the formula, R stands for -(CH2-O-CH2). n -, where n is 2 to 6.

[0034] The preparation method is as follows:

[0035]

[0036] The application of PROTAC chimeras as described above in the preparation of drugs that target the degradation of ACLY.

[0037] Furthermore, the targeted ACLY degradation drug is an anti-nonalcoholic steatohepatitis drug;

[0038] Alternatively, the drug may be a drug with low toxicity in human hepatocellular carcinoma cells HepG2, normal human hepatocytes L02, and human umbilical vein endothelial cells HUVEC.

[0039] Alternatively, the PROTAC chimera can be combined with excipients to prepare a pharmaceutically acceptable drug formulation.

[0040] The advantages and positive effects of this invention are as follows:

[0041] 1. The PROTAC compound synthesized in this invention is a novel compound. In a high-fat model established using L02 and HepG2 cells, the synthesized PROTAC compound exhibits activity in reducing TG levels. Furthermore, compared to the target cell, the synthesized PROTAC compound shows low toxicity activity in human hepatocellular carcinoma cells (HepG2), normal human hepatocytes (L02), and human umbilical vein endothelial cells (HUVECs). This represents a novel, low-toxicity, and highly effective drug for treating non-alcoholic steatohepatitis (NAH). This invention expands the application areas of PROTAC technology while developing a highly effective treatment for NHA.

[0042] 2. This invention provides a class of ACLY protein-targeting chimeras synthesized using PROTAC technology, and discovers a new type of PROTAC degrader targeting ACLY protein with a structure as shown in Formula 1. It has good lipid-lowering activity and ACLY protein degradation activity, and is a new generation of low-toxicity and high-efficiency anti-MASH drugs.

[0043] 3. This invention utilizes protein hydrolysis-targeting chimeric compounds (PROTACs) to develop a class of drugs for treating non-alcoholic steatohepatitis (MASH). PROTACs compounds are rationally designed to link ACLY inhibitors with E3 ubiquitin ligases using a glycol chain as the linker. The PROTACs compounds of this invention can reduce warhead cytotoxicity, inhibit TG levels while alleviating liver inflammatory damage and reducing ACLY protein expression, thus exhibiting therapeutic or alleviating effects on MASH. Attached Figure Description

[0044] Figure 1 This is a diagram showing the effect of compounds B2OH-B6OH in this invention on lipid accumulation in L02 cells;

[0045] Figure 2The figure shows the effect of compounds B2OH-B6OH in this invention on triglyceride content in L02 cells;

[0046] Figure 3 The figure shows the effect of compounds B2OH-B6OH in this invention on the aspartate aminotransferase content in L02 cells;

[0047] Figure 4 This is a graph showing the degradation effect of compounds B3OH and B4OH on ACLY protein at a concentration of 20 μM in this invention;

[0048] Figure 5 This image shows the degradation effect of compound B3OH on ACLY protein at concentrations ranging from 10 to 100 μM. Detailed Implementation

[0049] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0050] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0051] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0052] An ACLY-targeting PROTAC chimera with anti-MASH activity, using glycol chains as linking chains, has the general structural formula of Formula 1:

[0053]

[0054] In the formula, R stands for -(CH2-O-CH2). n -, where n is 2 to 6.

[0055] Alternatively, the structure of the PROTAC chimera may be one or more of the following:

[0056]

[0057] A method for preparing the PROTAC chimera as described above, comprising reacting a compound of formula 2 with a compound of formula 3:

[0058]

[0059] In Equation 2, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0060] Formula 2 is prepared by reacting with Formulas 4 and 5:

[0061]

[0062] In Equation 4, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0063] Formula 3 is prepared by reacting with Formulas 9 and 10:

[0064]

[0065] Formula 4 is prepared by reacting with Formulas 6 and 7:

[0066]

[0067] In Equation 6, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0068] Formula 9 is prepared by reacting with Formulas 11 and 12:

[0069]

[0070] Formula 6 is prepared by reacting Formulas 5 and 8:

[0071]

[0072] In Equation 8, R is -(CH2-O-CH2) n -, where n is 2 to 6.

[0073] Formula 10 is prepared by reacting with Formulas 13 and 14:

[0074]

[0075] The reaction formula is as follows:

[0076]

[0077] In the formula, R stands for -(CH2-O-CH2). n -, where n is 2 to 6.

[0078] The preparation method is as follows:

[0079]

[0080] The application of PROTAC chimeras as described above in the preparation of drugs that target the degradation of ACLY.

[0081] Alternatively, the targeted ACLY degradation drug may be an anti-nonalcoholic steatohepatitis drug;

[0082] Alternatively, the drug may be a drug with low toxicity in human hepatocellular carcinoma cells HepG2, normal human hepatocytes L02, and human umbilical vein endothelial cells HUVEC.

[0083] Alternatively, the PROTAC chimera can be combined with excipients to prepare a pharmaceutically acceptable drug formulation.

[0084] Specifically, the relevant preparation and testing methods are as follows:

[0085] I. Compounds synthesized in this invention

[0086] 1. The synthetic route for preparing compound BMS-303141 of this invention is as follows:

[0087]

[0088] Furthermore, the specific synthesis steps for BMS-33141 are as follows:

[0089] (1) Synthesis of intermediate a: Under argon protection, 5-bromo-2-methoxyaniline (5g, 1eq) was added to a round-bottom flask, followed by the addition of phenylboronic acid (3.62g, 1.2eq), K2CO3 (10.26g, 3eq), Pd(PPh3)4 (700mg, 0.1eq) and a small amount of KF, dissolved in NN dimethylformamide, and reacted at 110℃ for 24h to obtain intermediate a.

[0090] (2) Synthesis of intermediate b: 20.77 g (1 eq) of chlorosulfonic acid was weighed and 5 g (1 eq) of 2,4-dichlorophenol was added over 30 min. The mixture was reacted at 40 °C for 1.5 h. After cooling to room temperature, 8.52 g (1 eq) of thionyl chloride was added and the mixture was reacted at 50 °C for 40 min. The reaction solution was slowly poured into rapidly stirred ice water to obtain a white precipitate, which was dried to obtain intermediate b.

[0091] (3) Synthesis of BMS-303141: Under argon protection, intermediate b (4.255 g, 1 eq) was placed in a round-bottom flask, dissolved in 30 mL of pyridine, and intermediate a (5 g, 0.9 eq) was added. The reaction was carried out at 50 °C for 6 h. When the reaction solution turned into a transparent orange liquid, it was quenched with dilute hydrochloric acid, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound BMS-303141.

[0092] 2. The synthesis route of the compounds B2OH, B3OH, B4OH, B5OH, and B6OH of this invention is as follows:

[0093]

[0094] Furthermore, the specific synthesis steps are as follows:

[0095] (1) Synthesis of intermediate d (taking n=4 as an example): In a dry round-bottom flask, 10 g (1 eq) of tetraethylene glycol was dissolved in 30 mL of dichloromethane. At 0 °C, 8.837 g (0.9 eq) of p-toluenesulfonyl chloride and 10 mL of triethylamine were added. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 9d.

[0096] (2) Synthesis of intermediate e (taking n=4 as an example): In a round-bottom flask, 9d (1g, 1eq) was dissolved in 10mL of N,N-dimethylformamide. K2CO3 (991.68mg, 1.2eq) and pomalidomide (653.56mg, 1.1eq) were added to the reaction solution, and the reaction was carried out at 90℃ for 6h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, volume ratio) to obtain compound 9e.

[0097] (3) Synthesis of intermediate f (taking n=4 as an example): In a dry round-bottom flask, compound 9e (271 mg, 1 eq) was dissolved in 10 mL of dichloromethane. p-Toluenesulfonyl chloride (254.87 mg, 2 eq) and 0.3 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound 9f.

[0098] (4) Synthesis of the final product (taking B4OH as an example): In a round-bottom flask, 9f (70 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide. K2CO3 (121.3 mg, 1.2 eq) and BMS-303141 (282.4 mg, 1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, volume ratio) to obtain compound B4OH.

[0099] Example 1: Synthesis of compound B2OH

[0100] The structure is as follows:

[0101]

[0102] Synthesis of intermediate 7d

[0103]

[0104] In a dry round-bottom flask, diethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane. p-Toluenesulfonyl chloride (8.265 g, 0.9 eq) and 10 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1:1, v / v) to give compound 7d.

[0105] Synthesis of intermediate 7e

[0106]

[0107] In a round-bottom flask, 7d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (952.77 mg, 1.2 eq) and pomalidomide (668.91 mg, 1.1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound 7e.

[0108] Synthesis of intermediate 7f

[0109]

[0110] In a dry round-bottom flask, compound 7e (250 mg, 1 eq) was dissolved in 10 mL of dichloromethane. p-Toluenesulfonyl chloride (213.47 mg, 2 eq) and 0.3 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 3:1, v / v) to give compound 7f.

[0111] Synthesis of the final product B2OH

[0112]

[0113] In a round-bottom flask, 7f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (96.72 mg, 1.2 eq) and BMS-303141 (253.17 mg, 1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound B₂OH.

[0114] Yellow oily solid, yield 27.22%. 1 H NMR(400MHz,Chloroform-d)δ7.95(s,1H),7.46(d,J=9.1Hz,1H),7.20(m,5H),7.14-6.96(m,2H) ,6.92(s,1H),6.81(d,J=9.3Hz,2H),5.83-5.67(m,1H),4.96-4.82(m,1H),4.18-4.03(m,1H),3.9 8(dt,J=13.9,7.2Hz,2H),3.63-3.45(m,3H),3.42(s,1H),2.67(dd,J=35.1,9.9Hz,2H),2.52(t,J =8.0Hz,1H),2.40(d,J=13.6Hz,1H),2.32-2.22(m,1H),2.21-2.10(m,1H),1.97(q,J=7.3Hz,3H).

[0115] Example 2: Synthesis of compound B3OH

[0116] The structure is as follows:

[0117]

[0118] Synthesis of intermediate 8d

[0119]

[0120] In a dry round-bottom flask, triethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane. p-Toluenesulfonyl chloride (8.524 g, 0.9 eq) and 10 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 1:1, v / v) to give compound 8d.

[0121] Synthesis of intermediate 8e

[0122]

[0123] In a round-bottom flask, 8d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (973.25 mg, 1.2 eq) and pomalidomide (644 mg, 98 mg, 1.1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound 8e.

[0124] Synthesis of intermediate 8f

[0125]

[0126] In a dry round-bottom flask, compound 8e (250 mg, 1 eq) was dissolved in 10 mL of dichloromethane. p-Toluenesulfonyl chloride (233.83 mg, 2 eq) and 0.3 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 3:1, v / v) to give compound 8f.

[0127] Synthesis of the final product B3OH

[0128]

[0129] In a round-bottom flask, 8f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (99.61 mg, 1.2 eq) and BMS-303141 (266.7 mg, 1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound B₃OH.

[0130] Yellow oily solid, yield 24.68%. 1 H NMR(400MHz,Chloroform-d)δ7.58-7.42(m,3H),7.37(t,J=7.8Hz,2H),7.32-7.18(m,4H),7.13 -6.99(m,1H),6.93(d,J=6.5Hz,1H),6.80(d,J=8.4Hz,1H),5.75(dd,J=17.2,10.2Hz,1H),4.98 -4.82(m,1H),4.01(qd,J=13.5,7.3Hz,1H),3.67-3.48(m,8H),3.42(s,3H),2.85-2.70(m,1H), 2.59-2.47(m,1H),2.38(s,1H),2.27(t,J=7.6Hz,1H),2.15(t,J=7.7Hz,1H),2.03-1.87(m,3H).

[0131] Example 3: Synthesis of compound B4OH

[0132] The structure is as follows:

[0133]

[0134] Synthesis of intermediate 9d

[0135]

[0136] In a dry round-bottom flask, 10 g (1 eq) of tetraethylene glycol was dissolved in 30 mL of dichloromethane. Then, 8.837 g (0.9 eq) of p-toluenesulfonyl chloride and 10 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 1:1, v / v) to give compound 9d.

[0137] Synthesis of intermediate 9e

[0138]

[0139] In a round-bottom flask, 9d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (991.68 mg, 1.2 eq) and pomalidomide (653.56 mg, 1.1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound 9e.

[0140] Synthesis of intermediate 9f

[0141]

[0142] In a dry round-bottom flask, compound 9e (271 mg, 1 eq) was dissolved in 10 mL of dichloromethane. p-Toluenesulfonyl chloride (254.87 mg, 2 eq) and 0.3 mL of triethylamine were added at 0 °C, and the mixture was slowly heated to room temperature for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 3:1, v / v) to give compound 9f.

[0143] Synthesis of the final product B4OH

[0144]

[0145] In a round-bottom flask, 9f (70 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (121.3 mg, 1.2 eq) and BMS-303141 (282.4 mg, 1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound B₄OH.

[0146] Yellow oily solid, yield 26.05%. 1H NMR(400MHz,Chloroform-d)δ7.75-7.58(m,1H),7.53(d,J=7.0Hz,1H),7.46(d,J=8.5Hz,1H),7.41-7.26(m,3 H),7.13-7.03(m,1H),6.92(s,1H),6.87-6.74(m,1H),5.82-5.66(m,1H),5.28(d,J=5.5Hz,1H),5.18(d,J=9.2 Hz,2H),4.96-4.81(m,1H),4.15(t,J=4.9Hz,2H),4.07-3.92(m,2H),3.73-3.45(m,12H),2.89(dd,J=9.7,5.0 Hz,1H),2.80(t,J=8.1Hz,1H),2.76-2.66(m,2H),2.61-2.46(m,1H),2.15(t,J=7.5Hz,1H),1.99-1.91(m,2H).

[0147] Example 4: Synthesis of compound B5OH

[0148] The structure is as follows:

[0149]

[0150] Synthesis of intermediate 10d

[0151]

[0152] In a dry round-bottom flask, 10 g (1 eq) of pentaethylene glycol was dissolved in 30 mL of dichloromethane. Then, 8.962 g (0.9 eq) of p-toluenesulfonyl chloride and 10 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 1:1, v / v) to give compound 10d.

[0153] Synthesis of intermediate 10e

[0154]

[0155] In a round-bottom flask, 10d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (1.3 g, 1.2 eq) and pomalidomide (684 mg, 1.1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound 10e.

[0156] Synthesis of intermediate 10f

[0157]

[0158] In a dry round-bottom flask, compound 10e (250 mg, 1 eq) was dissolved in 10 mL of dichloromethane. p-Toluenesulfonyl chloride (273.09 mg, 2 eq) and 0.3 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 3:1, v / v) to give compound 10f.

[0159] Synthesis of the final product B5OH

[0160]

[0161] In a round-bottom flask, 10f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (126.7 mg, 1.2 eq) and BMS-303141 (273.05 mg, 1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound B₅OH.

[0162] Yellow oily solid, yield 25.33%. 1H NMR(400MHz,Chloroform-d)δ7.80-7.68(m,2H),7.67-7.59(m,1H),7.59-7.46(m,2H),7.47 -7.37(m,2H),7.37-7.22(m,4H),7.14(dd,J=7.0,2.6Hz,1H),6.95-6.80(m,1H),4.95(dt,J= 11.4,3.6Hz,1H),4.07-3.98(m,2H),3.69-3.42(m,18H),3.42-3.30(m,2H),3.22-3.09(m,1H ),3.03-2.89(m,1H),2.77(dt,J=11.7,4.5Hz,1H),2.45(d,J=2.5Hz,2H),2.26-2.16(m,1H).

[0163] Example 5: Synthesis of compound B6OH

[0164] The structure is as follows:

[0165]

[0166] Synthesis of intermediate 11d

[0167]

[0168] In a dry round-bottom flask, hexaethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane. p-Toluenesulfonyl chloride (9.112 g, 0.9 eq) and 10 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 1:1, v / v) to give compound 11d.

[0169] Synthesis of intermediate 11e

[0170]

[0171] In a round-bottom flask, 11d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (1.42 mg, 1.2 eq) and pomalidomide (683.2 mg, 1.1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound 11e.

[0172] Synthesis of intermediate 11f

[0173]

[0174] In a dry round-bottom flask, compound 11e (250 mg, 1 eq) was dissolved in 10 mL of dichloromethane. p-Toluenesulfonyl chloride (290.35 mg, 2 eq) and 0.3 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After completion, the mixture was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 3:1, v / v) to give compound 11f.

[0175] Synthesis of the final product B6OH

[0176]

[0177] In a round-bottom flask, 11f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide. K₂CO₃ (145.8 mg, 1.2 eq) and BMS-303141 (288.02 mg, 1 eq) were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, and purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound B₆OH.

[0178] Yellow oily solid, yield 23.71%. 1 HNMR(400MHz,Chloroform-d)δ7.77(d,J=2.4Hz,1H),7.69-7.52(m,3H),7.52-7.40(m, 3H),7.36(dd,J=12.1,7.0Hz,4H),7.24-7.14(m,1H),6.87(d,J=9.4Hz,1H),4.97(dd,J =13.6,5.9Hz,1H),3.96(t,J=7.0Hz,2H),3.66-3.49(m,8H),3.42(s,1H),3.20(s,1H), 2.04(d,J=3.5Hz,1H),1.53(t,J=6.8Hz,2H),1.45(t,J=7.3Hz,3H),1.42-1.23(m,14H).

[0179] II. Study on the anti-NASH effect of the compounds of this invention

[0180] Unless otherwise specified, the implementation methods in the following examples are conventional methods. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available products.

[0181] Experimental Example 1: Cytotoxicity Test of PROTAC Compound

[0182] The drug concentration was set in vitro, and the cytotoxicity of the PROTAC compound was tested using a cell proliferation (MTT) assay. The cells used for the cytotoxicity test in this invention were: human hepatocellular carcinoma cells HepG2, normal human hepatocytes L02, and human umbilical vein endothelial cells HUVEC.

[0183] Cells were loaded at a rate of 1×10 5 Cells were seeded per empty cell in 96-well plates and incubated at 37°C with 5% CO2 for 24 h to allow adherence. The desired PROTAC compound was dissolved in dimethyl sulfoxide (DMSO) and added to the 96-well plates at concentration gradients of 100, 80, 40, 20, and 10 μM. The plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, 5 mg / mL MTT was added to each well, and the plates were incubated for another 4 h. The culture medium was aspirated, and 100 μL of DMSO was added to each well. The plates were then shaken at 37°C for 10 min. The OD values ​​of each well were measured at 492 and 630 nm using a microplate reader, and the results were recorded. Cell viability was plotted against quantitation.

[0184] Results calculation: Cell viability = (OD value of experimental group cells - OD value of blank group cells) / (OD value of control group cells - OD value of blank group cells) × 100%.

[0185] The experimental group consisted of the drug-treated group; the blank group consisted of the blank culture medium group to detect the effect of the blank culture medium on cell viability; and the control group consisted of the cell culture medium with added DMSO to detect the effect of the drug solvent on cell viability.

[0186] The results, as shown in Tables 1 and 2, indicate that the five newly synthesized PROTAC compounds exhibited lower cytotoxicity than the BMS-303141 warhead at concentrations below 100 μM, achieving a reduction in toxicity. Among them, the PROTAC compound B4OH, with four glycol chains as linkers, showed weak toxicity in HepG2 cells, with an IC50 value of [missing information]. 50 The value was 88.38 μM, but the toxicity was still significantly reduced compared to BMS-303141.

[0187] Table 1. Cytotoxicity of PROTAC compounds on HepG2, L02, and HUVEC cells.

[0188]

[0189]

[0190] Table 2. IC50 of PROTAC compounds in HepG2, LO2, and HUVEC cells. 50

[0191]

[0192] Experimental Example 2: Test of the lipid-lowering activity of PROTAC compounds

[0193] Solution preparation:

[0194] ① Preparation of Oil Red O staining solution

[0195] Preparation of Oil Red O stock solution: Add 0.45 g of Oil Red O powder to a 50 mL centrifuge tube, add 45 mL of isopropanol, and prepare a saturated isopropanol solution of Oil Red O. Store at room temperature away from light and use after 24 hours.

[0196] Preparation of Oil Red O working solution: Centrifuge the Oil Red O stock solution, take the supernatant, mix the supernatant with double-distilled water at a volume ratio of 3:2, filter through a 0.45μM filter membrane, and use immediately after preparation.

[0197] ② Preparation of 4% CHAPS lysis buffer

[0198] Accurately weigh 1.8018 g of urea, 0.7613 g of thiourea, 0.05 g of dithiothreitol, 0.2 g of CHAPS (an amphoteric surfactant), and 24.25 mg of Tris-base into a 50 mL centrifuge tube, and add 5 mL of the solution to dissolve. Store at 4°C.

[0199] ③ Preparation of culture medium containing FFAs and 1% BSA

[0200] Accurately weigh 5g of bovine serum albumin (BSA) and dissolve it in a preheated (55°C) complete culture medium to prepare a culture medium containing 1% bovine serum albumin.

[0201] Sodium oleate and sodium palmitate were separately prepared into solutions with a final concentration of 200 mmol / L using 0.1 mol / L sodium hydroxide aqueous solution and dissolved by heating at 95 °C. The sodium oleate and sodium palmitate solutions were then mixed thoroughly at a volume ratio of 2:1 and added to an appropriate amount of preheated (55 °C) complete medium containing 1% BSA to prepare a medium with a final concentration of 2 mmol / L containing FFAs and 1% BSA. This medium was then rapidly filtered through a 0.45 μM filter membrane in a clean bench while still hot. The required experimental concentration was diluted with medium containing 1% BSA.

[0202] Specific experimental steps:

[0203] L02 cells were administered at a dose of 1×10⁻⁶. 5 Cells were seeded at a density of 10 mL / mL in 100 mm cell culture dishes and 1 mL / well in 6-well cell culture plates. The seeded culture plates were incubated at 37°C with 5% CO2 for 24 h. The DMEM medium in the culture dishes was discarded, and the cells were washed three times with 1×PBS. Then, an equal volume of serum-free DMEM medium was added, and the cells were cultured for another 12 h under the same conditions to starve them. After 12 hours, the serum-free medium in the culture dishes was discarded. DMSO plus DMEM medium was used as the blank group, and DMSO plus FFA medium was used as the model group. Fenofibrate acid (20 μmol / L) was used as the positive drug, BMS-303141 concentration was 20 μmol / L, and the concentration of the PROTAC compound to be tested was 10, 20, 40, 80, and 100 μM. DMSO, fenofibrate acid and the drug to be tested were mixed with complete medium containing 1% BSA beforehand. The mixed medium was added to cell culture dishes and culture plates and cultured for another 24 hours.

[0204] Discard the culture medium in the 6-well cell culture plate, add an appropriate amount of 1×PBS and wash three times, discard the 1×PBS, add 4% paraformaldehyde fixative (M / M, mass percentage) and fix for 40 min, then discard the 4% paraformaldehyde fixative, add an appropriate amount of distilled water and wash, discard the distilled water, then add 1 mL of Oil Red O working solution and stain in the dark for 40 min, then discard the stain, add an appropriate amount of distilled water and wash, discard the distilled water, add 1 mL of 75% ethanol solution (V / V, volume percentage) and differentiate for 10 s, quickly discard the 75% ethanol solution (V / V, volume percentage), add an appropriate amount of distilled water and wash, discard the distilled water, add 1 mL of hematoxylin staining solution and stain for 10 min, recover the hematoxylin staining solution, place the 6-well cell culture plate in tap water, wash with running water and differentiate for 5 min, add 50% glycerol aqueous solution (V / V, volume percentage) and mount, and photograph and examine under a 400x trinocular inverted biological microscope. Discard the culture medium in a 100mm cell culture dish, add an appropriate amount of 1×PBS and wash three times. Discard the 1×PBS, shake using a cross-shaped motion for 1 min. After washing, aspirate the PBS solution, add 1 mL of 0.25% trypsin (M / M, mass percentage) for cell digestion, add 1 mL of trypsin solution, and digest for 2 min. A layer of mist will form at the bottom of the culture dish. Add 2 mL of 1640 medium to rinse off the cells adhering to the bottom of the culture dish, transfer to a centrifuge tube, and centrifuge at 800 rpm for 5 min. Aspirate the supernatant from the centrifuge tube, add 100 μL of 4% CHAPS lysis buffer (V / V, volume percentage), and lyse on ice for 1 h.

[0205] TG content detection was performed according to the kit instructions, as shown in Table 3.

[0206] Table 3. Test methods for TG content

[0207]

[0208] The standard is the calibrator included in the kit.

[0209] The TG content is calculated according to formula (3-1).

[0210]

[0211] Where Cpr represents the protein content of the PROTAC compound to be tested.

[0212] AST content detection should be performed according to the kit instructions, as shown in Table 4.

[0213] Table 4. Test methods for AST content

[0214]

[0215]

[0216] The effects of BMS-303141 and PROTAC compounds on morphological changes in the FFAs-induced MASH cell model are as follows: Figure 1 As shown in the figure, after co-culturing with 20 μmol / L BMS-303141 and PROTAC compounds for 24 h, morphological observation revealed that compounds BMS-303141, B3OH, and B4OH reduced intracellular lipid accumulation. Furthermore, B3OH and B4OH reduced lipid content more significantly than BMS-303141, showing a marked improvement over the model group. In contrast, the lipid content of other compounds showed almost no significant change, remaining comparable to the model group. Based on these data and morphological observation, it can be concluded that the PROTACs synthesized in this invention have a superior ability to reduce lipid accumulation compared to the original drug BMS-303141.

[0217] The effects of BMS-303141 and PROTAC compounds on TG content in a FFA-induced MASH cell model were investigated, and the results are as follows: Figure 2As shown in the figure, the TG content in the blank group was very low, at 0.02 mmol / gprot. In the model group, after stimulation with FFAs, the TG content increased significantly, reaching 0.24 mmol / gprot. The positive control, fenofibrate acid, reversed the FFA-induced increase in TG content, reducing it to 0.12 mmol / gprot. After adding 20 μmol / L of BMS-303141 or PROTAC compounds, BMS-303141 showed significant lipid-lowering activity compared to B3OH and B4OH, with B3OH and B4OH exhibiting stronger lipid-lowering activity than the precursor BMS-313141. Specifically, BMS-303141 had a TG content of 0.16 mmol / gprot, B3OH had a TG content of 0.13 mmol / gprot, and B4OH had a TG content of 0.11 mmol / gprot. Based on the above data, it is further verified that the lipid-lowering activity of the PROTACs compound synthesized in this invention is stronger than that of the original drug BMS-303141.

[0218] The effects of BMS-303141 and PROTAC compounds on AST content in a FFA-induced MASH cell model were investigated, and the results are as follows: Figure 3 As shown in the figure, the AST content in the blank group was very low, with an AST value of 47.16 U / gprot. In the model group, after stimulation with FFAs, the AST content significantly increased, reaching 74.79 U / gprot. The positive control drug fenofibrate and the original drug BMS-303141 could reduce the AST content, with fenofibrate at 64.15 U / gprot and BMS-303141 at 62.06 U / gprot, but the effect was not significant. After adding B3OH and B4OH at a concentration of 20 μmol / L, the AST value of B3OH was 45.18 U / gprot, and that of B4OH was 52.82 U / gprot, significantly reducing liver damage. The reduction effect of B3OH was particularly significant, comparable to that of the blank group. Based on the above data, we can see that the PROTACs compounds synthesized in this invention have stronger lipid-lowering activity than the original drug BMS-303141 and can reduce liver damage to a certain extent.

[0219] Experiment Example 3: Degradation ability test of PROTAC compound ACLY

[0220] ① Protein sample collection

[0221] L02 cells were fed at a rate of 1×10 5Cells were seeded per well into 100 mm cell culture dishes and cultured at 37°C with 5% CO2 for 24 h to allow adherence. The DMEM medium in the culture dishes was discarded, and the cells were washed three times with 1×PBS. Then, an equal volume of serum-free DMEM medium was added, and the cells were cultured for another 12 h under the same conditions to starve them. After 12 h, the serum-free medium was aspirated. DMSO plus DMEM medium was used as the control group, and DMSO plus FFAs medium was used as the model group. Fenofibrate (20 μmol / L) was used as the positive control, BMS-303141 was at a concentration of 20 μmol / L, and B3OH and B4OH were at concentrations of 20 μM. DMSO, fenofibrate, B3OH, and B4OH were pre-mixed with complete medium containing 1% BSA. The mixed medium was then added to cell culture dishes and culture plates and cultured for another 24 h. Transfer the culture medium from a 100mm cell culture dish to a centrifuge tube. Add 2mL of 1×PBS to wash the dish and shake using a cross-shaking method for 1 minute. Transfer the PBS to the centrifuge tube and add 1mL of 0.25% trypsin to digest the cells for 2 minutes. When a misty layer appears at the bottom of the dish, add 2mL of the culture medium from the centrifuge tube to stop digestion. Rinse off any cells adhering to the bottom of the dish and transfer the mixture to a centrifuge tube. Centrifuge at 2500 rpm for 5 minutes. Quickly discard the supernatant, add 1mL of PBS, mix well, transfer to a 2mL EP tube, centrifuge at 2500 rpm for 5 minutes, quickly discard the supernatant, add 100μL of protein lysis buffer, lyse on ice for 1 hour, resuspend, and centrifuge at 13500 rpm for 20 minutes at 4°C. Retain the supernatant for protein quantification.

[0222] ② Protein quantification

[0223] Protein standard curves were constructed, and protein concentrations were determined using the BSA method. CHAPS protein lysis buffer was mixed with bovine serum albumin to prepare seven concentrations: 0, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL, which were used as protein standard solutions. Protein concentrations were determined using 96-well plates. 100 μL / well of Coomassie Brilliant Blue was added, followed by 2 μL / well of protein standard solution, ensuring thorough mixing to prevent air bubbles. Three replicates were performed. Absorbance was measured at 600 nm using a microplate reader, and the mean values ​​were calculated to plot a standard curve (see Table 5).

[0224] Table 5. Dilution Standards for BSA Standards

[0225] sample 1 2 3 4 5 6 7 BSA concentration (μg / μL) 0 0.125 0.25 0.5 1 1.5 2 Sample (μL) 2 2 2 2 2 2 2 Coomassie Brilliant Blue (μL) 100 100 100 100 100 100 100 Protein content (μg) 0 0.25 0.5 1 2 3 4

[0226] Protein samples were diluted using CHARPS, and the same procedure described above was followed to calculate the protein concentration for each group based on the protein curve. After calculation and multiple dilutions, when the protein concentrations for each group were approximately consistent between 1.2 and 1.5 mg / mL, subsequent experiments could be carried out. Samples could be stored at -80°C initially.

[0227] The experimental results are as follows: The effective compounds B3OH and B4OH from this invention are compared. Figure 4 As shown, compared with the model group, BMS-303141, B3OH, and B4OH all exhibited degradation activity against ACLY, with B3OH showing stronger degradation activity than B4OH. The positive control drug fenofibrate acid showed no degradation ability against ACLY. Further investigation into the ACLY degradation ability of B3OH was conducted, using concentration-dependent tests at 10, 20, 40, 80, and 100 μM. The results are as follows. Figure 5 As shown, the results indicate that the ability of B3OH to degrade ACLY gradually increases with increasing concentration, exhibiting a concentration-dependent effect. Combined with the above data, this demonstrates that compared to the BMS-303141 warhead, B3OH significantly enhances its ACLY degradation ability, and this degradation ability is concentration-dependent.

[0228] The above results demonstrate that modifying the BMS-303141 warhead with PROTAC can indeed improve the activity of the original drug. Based on this result, further modifications to the original drug, exploring more types of linker chains, and increasing the types of E3 ubiquitin ligases may yield compounds with even better activity.

[0229] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A glycol-linked ACLY-targeting PROTAC chimera with anti-MASH activity, characterized in that: The specific structure of the chimera is as follows: or .

2. The method for preparing the PROTAC chimera as described in claim 1, characterized in that: The synthesis steps for B3OH are as follows: Synthesis of intermediate 8d: ; In a dry round-bottom flask, 10 g of triethylene glycol was dissolved in 30 mL of dichloromethane. 8.524 g of p-toluenesulfonyl chloride and 10 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography with a petroleum ether:ethyl acetate ratio of 1:1 (v / v) to obtain compound 8d. Synthesis of intermediate 8e: ; In a round-bottom flask, 1 g of 8d was dissolved in 10 mL of N,N-dimethylformamide. 973.25 mg of K₂CO₃ and 644.98 mg of pomalidomide were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, purified by column chromatography with a dichloromethane:methanol eluent ratio of 150:1 (v / v) to obtain compound 8e. Synthesis of intermediate 8f: ; In a dry round-bottom flask, 250 mg of compound 8e was dissolved in 10 mL of dichloromethane. 233.83 mg of p-toluenesulfonyl chloride and 0.3 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After the reaction was complete, the compound 8f was purified by column chromatography with a petroleum ether:ethyl acetate ratio of 3:1 (v / v). Synthesis of the final product B3OH: ; In a round-bottom flask, 50 mg of 8f was dissolved in 10 mL of N,N-dimethylformamide. 99.61 mg of K₂CO₃ and 266.7 mg of BMS-303141 were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, purified by column chromatography with a dichloromethane:methanol eluent ratio of 150:1 (v / v) to obtain compound B₃OH. Alternatively, the synthesis steps for B4OH are as follows: Synthesis of intermediate 9d: ; In a dry round-bottom flask, 10 g of tetraethylene glycol was dissolved in 30 mL of dichloromethane. 8.837 g of p-toluenesulfonyl chloride and 10 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography with a petroleum ether:ethyl acetate ratio of 1:1 (v / v) to obtain compound 9d. Synthesis of intermediate 9e: ; In a round-bottom flask, 1 g of 9d was dissolved in 10 mL of N,N-dimethylformamide. 991.68 mg of K₂CO₃ and 653.56 mg of pomalidomide were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The crude product was concentrated under reduced pressure, purified by column chromatography with a dichloromethane:methanol eluent ratio of 150:1 (v / v) to obtain compound 9e. Synthesis of intermediate 9f: ; In a dry round-bottom flask, 271 mg of compound 9e was dissolved in 10 mL of dichloromethane. 254.87 mg of p-toluenesulfonyl chloride and 0.3 mL of triethylamine were added at 0 °C. The mixture was slowly heated to room temperature and reacted for 3 h. The reaction was monitored by TLC. After the reaction was complete, the compound 9f was purified by column chromatography with a petroleum ether:ethyl acetate ratio of 3:1 (v / v). Synthesis of the final product B4OH: ; In a round-bottom flask, 70 mg of 9f was dissolved in 10 mL of N,N-dimethylformamide. 121.3 mg of K2CO3 and 282.4 mg of BMS-303141 were added to the reaction solution, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, it was quenched with water, extracted three times with ethyl acetate, and the organic phase was collected. The organic phase was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography with dichloromethane:methanol in a volume ratio of 150:1 to obtain compound B4OH.

3. The application of the PROTAC chimera as described in claim 1 in the preparation of drugs targeting the degradation of ACLY.

4. The use of the PROTAC chimera as described in claim 1 in the preparation of anti-MASH drugs.

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