ACLY targeted PROTAC chimera with anti-MASH activity, method and application

Abstract

The invention belongs to the technical field of biology and medicine, and discloses an ACLY-targeted PROTAC chimera which takes a glycol chain as a connecting chain and has anti-MASH activity, the structural general formula of the ACLY-targeted PROTAC chimera is as shown in formula 1: # imgabs0 #, R is-(CH2-O-CH2) n-, and n is 2-6. The synthesized PROTAC compound is a new compound, in a high-fat model established by L02 and HepG2, the synthesized PROTAC compound has the activity of reducing the TG content, and compared with a bullet, the synthesized PROTAC compound has low-toxicity activity in human hepatoma carcinoma cells HepG2, human normal hepatoma cells L02 and human umbilical vein endothelial cells HUVEC, and can be used for preparing a medicine for treating liver cancer. The medicine is a novel medicine for resisting non-alcoholic steatohepatitis with low toxicity and high efficiency. The application field of the PROTAC technology is widened while efficient treatment of the non-alcoholic steatohepatitis is developed.

Description

Technical Field

[0001] The present invention belongs to the field of biological and pharmaceutical technology, and relates to the field of small molecule drug development, in particular to an ACLY-targeted PROTAC chimera with anti-MASH activity using a glycol chain as a connecting chain, a method and an application thereof. Background Art

[0002] MASLD refers to excessive fat deposition in the liver caused by excessive drinking, excluding viral and other liver damage factors. If not treated in time, it will develop into MASH. If MASH is not treated in time, it will develop into cirrhosis and even liver cancer. Its incidence rate is extremely high, which seriously threatens human life and health. However, there is only one marketed drug for MASH, Resmetirom. The disease mechanism of MASH is complex, and a single target therapeutic drug is not enough to cope with the entire market. Developing an efficient and safe drug for the treatment of MASH is an urgent task and faces huge challenges.

[0003] Studies have shown that the excessive fat accumulation of most MASH patients comes from the process of de novo fat synthesis. The raw material of de novo fat synthesis, acetyl CoA, comes from pyruvate generated by glycolysis. Pyruvate generates acetyl CoA through glycolysis in the cytoplasm. Acetyl CoA enters the mitochondria and generates citric acid through the tricarboxylic acid cycle. Citric acid enters the cytoplasm through the pyruvate-citrate shuttle mechanism. After entering the cytoplasm, acetyl CoA is generated through adenosine triphosphate citrate lyase (ATP citrate lyase, ACLY). As the basic substrate for the biosynthesis of fatty acids and cholesterol, acetyl CoA is an important target for the treatment of MASH and dyslipidemia. ACLY can be selected as a target to inhibit the generation of acetyl CoA and thus treat MASH.

[0004] Proteolysis-Targeting Chimeras (PROTACs) is a new type of small molecule protein targeted degradation technology, consisting of three parts, including target protein (protein of interest, POI) ligand, E3 ligase ligand (structure that can recruit protein degradation system) and linker connecting the two. When the PROTAC molecule enters the cell, the middle connecting chain folds and bends appropriately, allowing its two ends to penetrate into the hydrophobic cavity of the two receptors, shortening the distance between the E3 ubiquitin ligase and the target protein, forming a specific protein-protein interaction, and when ATP is supplied, the E3 ligase ligand transfers the ubiquitin it carries to the substrate protein, making the substrate protein ubiquitinated. When the ternary complex dissociates, the ubiquitinated protein will be recognized by the proteasome, that is, the UPS system, thereby achieving the degradation of the target protein. PROTAC drugs have many advantages over traditional small molecule drugs, such as low cytotoxicity and theoretically overcoming drug resistance. At present, there are no reports on the application of PROTAC technology to MASH.

[0005] Therefore, ACLY protein degraders based on PROTAC technology have good prospects for drug development for the treatment of MASH, bringing new hope for the treatment of MASH and broadening the application field of PROTAC technology. It is necessary to study it. Summary of the invention

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

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

[0008] An ACLY-targeted PROTAC chimera with anti-MASH activity using a glycol chain as a connecting chain has a general structural formula of Formula 1:

[0009]

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

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

[0012]

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

[0014]

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

[0016] Further, the formula 2 is prepared by reacting formula 4 and formula 5:

[0017]

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

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

[0020]

[0021] Further, the formula 4 is prepared by reacting formula 6 and formula 7:

[0022]

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

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

[0025]

[0026] Further, the formula 6 is prepared by reacting formula 5 and formula 8:

[0027]

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

[0029] Further, formula 10 is prepared by reacting formula 13 and formula 14:

[0030]

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

[0032]

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

[0034] The preparation method is as follows:

[0035]

[0036] The use of the PROTAC chimera as described above in the preparation of drugs targeting the degradation of ACLY.

[0037] Furthermore, the targeted degradation of ACLY drug is an anti-non-alcoholic fatty hepatitis drug;

[0038] Alternatively, the drug is a drug with low toxicity in human liver cancer cells HepG2, human normal liver cells L02, and human umbilical vein endothelial cells HUVEC;

[0039] Alternatively, the PROTAC chimera is combined with an excipient to prepare a pharmaceutically acceptable corresponding drug preparation.

[0040] The advantages and positive effects achieved by the present invention are:

[0041] 1. The PROTAC compound synthesized in the present invention is a new compound. In the high-fat model established with L02 and HepG2, the synthesized PROTAC compound has the activity of reducing TG content, and compared with the warhead, the synthesized PROTAC compound has low toxicity activity in human liver cancer cells HepG2, normal human liver cells L02, and human umbilical vein endothelial cells HUVEC, and is a new type of anti-non-alcoholic fatty hepatitis drug with low toxicity and high efficiency. While developing efficient treatments for non-alcoholic fatty hepatitis, the application field of PROTAC technology has been broadened.

[0042] 2. The present invention provides a class of ACLY protein targeting chimeras synthesized based on PROTAC technology, and discovers a class of PROTAC degraders targeting ACLY protein with a novel structure and a structure as shown in Formula 1, which have good lipid-lowering activity and ACLY protein degradation activity, and are a new generation of low-toxic and highly effective anti-MASH drugs.

[0043] 3. The present invention develops a class of drugs for treating non-alcoholic fatty liver disease (MASH) based on the technology of proteolysis targeting chimeras (PROTACs). PROTACs compounds are rationally designed by connecting ACLY inhibitors to E3 ubiquitin ligases using glycol chains as connecting chains. The PROTACs compounds of the present invention can reduce the cytotoxicity of the warhead, inhibit TG content, slow down liver inflammatory damage, and reduce ACLY protein expression, and have the effect of treating or alleviating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a graph showing the effects of compounds B2OH-B6OH in the present invention on lipid accumulation in L02 cells;

[0045] Figure 2This is a graph showing the effect of the compounds B2OH-B6OH in the present invention on the triglyceride content of L02 cells;

[0046] Figure 3 This is a graph showing the effect of the compounds B2OH-B6OH in the present invention on the aspartate aminotransferase content in L02 cells;

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

[0048] Figure 5 The graph shows the degradation effect of the compound B3OH in the present invention on ACLY protein in the concentration range of 10 to 100 μM. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0050] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

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

[0052] An ACLY-targeted PROTAC chimera with anti-MASH activity using a glycol chain as a connecting chain has a general structural formula of Formula 1:

[0053]

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

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

[0056]

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

[0058]

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

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

[0061]

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

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

[0064]

[0065]

[0066] Formula 4 is prepared by reacting Formula 6 and Formula 7:

[0067]

[0068] In formula 6, R is -(CH2-O-CH2) n -, n is 2 to 6.

[0069] Formula 9 is prepared by reacting formula 11 and formula 12:

[0070]

[0071] Formula 6 is prepared by reacting Formula 5 and Formula 8:

[0072]

[0073] In formula 8, R is -(CH2-O-CH2) n -, n is 2 to 6.

[0074] Formula 10 is prepared by reacting formula 13 and formula 14:

[0075]

[0076] The reaction formula is as follows:

[0077]

[0078] In the formula, R is -(CH2-O-CH2) n -, n is 2 to 6.

[0079] The preparation method is as follows:

[0080]

[0081] The use of the PROTAC chimera as described above in the preparation of drugs targeting the degradation of ACLY.

[0082] Alternatively, the targeted degradation of ACLY drug is an anti-nonalcoholic fatty hepatitis drug;

[0083] Alternatively, the drug is a drug with low toxicity in human liver cancer cells HepG2, human normal liver cells L02, and human umbilical vein endothelial cells HUVEC;

[0084] Alternatively, the PROTAC chimera is combined with an excipient to prepare a pharmaceutically acceptable corresponding drug preparation.

[0085] Specifically, the relevant preparation and detection are as follows:

[0086] 1. Compounds synthesized by the present invention

[0087] 1. Preparation of the compound BMS-303141 of the present invention, the synthetic route is as follows:

[0088]

[0089] Further, the specific synthesis steps of BMS-33141 are as follows:

[0090] (1) Synthesis of intermediate a: Under argon protection, 5-bromo-2-methoxyaniline (5 g, 1 eq) was placed in a round-bottom flask, and phenylboric acid (3.62 g, 1.2 eq), K2CO3 (10.26 g, 3 eq), Pd(PPh3)4 (700 mg, 0.1 eq) and a small amount of KF were added in sequence, dissolved in N-N-dimethylformamide, and reacted at 110°C for 24 h to obtain intermediate a.

[0091] (2) Synthesis of intermediate b: Weigh chlorosulfonic acid (20.77 g, 1 eq), add 2,4-dichlorophenol (5 g, 1 eq) within 30 min, and react at 40°C for 1.5 h. Cool to room temperature, add thionyl chloride (8.52 g, 1 eq), and react at 50°C for 40 min. Slowly pour the reaction solution into rapidly stirred ice water to obtain a white precipitate, which is then dried to obtain intermediate b.

[0092] (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 with ethyl acetate three times, and the organic phase was collected. The organic phase was washed with saturated NaCl aqueous solution three times 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.

[0093] 2. The synthesis of the compounds B2OH, B3OH, B4OH, B5OH and B6OH of the present invention is as follows:

[0094]

[0095] Further, the specific synthesis steps are as follows:

[0096] (1) Synthesis of intermediate d (taking n = 4 as an example): In a dry round-bottom flask, compound tetraethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane, p-toluenesulfonyl chloride (8.837 g, 0.9 eq) and 10 mL of triethylamine were added at 0°C, and the temperature was slowly raised to room temperature for 3 h. The reaction was monitored using TLC technology, and after the reaction was completed, it was purified using column chromatography (eluent system petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 9d.

[0097] (2) Synthesis of intermediate e (taking n = 4 as an example): In a round-bottom flask, 9d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (991.68 mg, 1.2 eq) and pomalidomide (653.56 mg, 1.1 eq) were added to the reaction solution. The reaction was allowed to react at 90°C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, water was added to quench the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic phase was collected, washed with a saturated aqueous solution of NaCl three times, and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography (eluent system of dichloromethane:methanol = 150:1, volume ratio) to obtain compound 9e.

[0098] (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, and the temperature was slowly raised to room temperature for 3 h. The reaction was monitored using TLC technology, and after the reaction was completed, it was purified using column chromatography (eluent system petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound 9f.

[0099] (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, and K2CO3 (121.3 mg, 1.2 eq) and BMS-303141 (282.4 mg, 1 eq) were added to the reaction solution. The reaction was allowed to react at 90°C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, water was added to quench the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic phase was collected, washed with a saturated aqueous solution of NaCl three times, and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography (eluent system of dichloromethane: methanol = 150:1, volume ratio) to obtain compound B4OH.

[0100] Example 1: Synthesis of Compound B2OH

[0101] The structural formula is as follows:

[0102]

[0103] Synthesis of intermediate 7d

[0104]

[0105] In a dry round-bottom flask, compound 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, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 7d.

[0106] Synthesis of intermediate 7e

[0107]

[0108] In a round-bottom flask, 7d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (952.77 mg, 1.2 eq) and pomalidomide (668.91 mg, 1.1 eq) were added to the reaction solution. The reaction was allowed to react at 90°C for 6 h. The reaction was monitored by TLC. The reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected. The organic phase was washed with saturated NaCl aqueous solution 3 times 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 7e.

[0109] Synthesis of intermediate 7f

[0110]

[0111] 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, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound 7f.

[0112] Synthesis of the final product B2OH

[0113]

[0114] In a round-bottom flask, 7f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (96.72 mg, 1.2 eq) and BMS-303141 (253.17 mg, 1 eq) were added to the reaction solution. The reaction was allowed to react at 90 °C for 6 h. The reaction was monitored by TLC. The reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected. The organic phase was washed with saturated NaCl aqueous solution 3 times 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 B2OH.

[0115] 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).

[0116] Example 2: Synthesis of Compound B3OH

[0117] The structural formula is as follows:

[0118]

[0119] Synthesis of intermediate 8d

[0120]

[0121] In a dry round-bottom flask, compound 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, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 8d.

[0122] Synthesis of intermediate 8e

[0123]

[0124] In a round-bottom flask, 8d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (973.25 mg, 1.2 eq) and pomalidomide (644, 98 mg, 1.1 eq) were added to the reaction solution. The reaction was allowed to react at 90°C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, water was added to quench the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic phase was collected, washed with saturated aqueous NaCl solution three times, 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 8e.

[0125] Synthesis of intermediate 8f

[0126]

[0127] 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, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound 8f.

[0128] Synthesis of the final product B3OH

[0129]

[0130] In a round-bottom flask, 8f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (99.61 mg, 1.2 eq) and BMS-303141 (266.7 mg, 1 eq) were added to the reaction solution. The reaction was allowed to react at 90 °C for 6 h. The reaction was monitored by TLC. The reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected. The organic phase was washed with saturated NaCl aqueous solution 3 times and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and vacuum, and purified by column chromatography (eluent system dichloromethane: methanol = 150: 1, volume ratio) to obtain compound B3OH.

[0131] 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).

[0132] Example 3: Synthesis of Compound B4OH

[0133] The structural formula is as follows:

[0134]

[0135] Synthesis of intermediate 9d

[0136]

[0137] In a dry round-bottom flask, tetraethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane, and p-toluenesulfonyl chloride (8.837 g, 0.9 eq) and 10 mL of triethylamine were added at 0°C, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 9d.

[0138] Synthesis of intermediate 9e

[0139]

[0140] In a round-bottom flask, 9d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (991.68 mg, 1.2 eq) and pomalidomide (653.56 mg, 1.1 eq) were added to the reaction solution. The reaction was allowed to react at 90°C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, water was added to quench the reaction solution, and the mixture was extracted with ethyl acetate three times. The organic phase was collected, washed with saturated aqueous NaCl solution three times, 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.

[0141] Synthesis of intermediate 9f

[0142]

[0143] 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 temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound 9f.

[0144] Synthesis of the final product B4OH

[0145]

[0146] In a round-bottom flask, 9f (70 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (121.3 mg, 1.2 eq) and BMS-303141 (282.4 mg, 1 eq) were added to the reaction solution. The reaction was allowed to react at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, water was added to quench it, and ethyl acetate was extracted 3 times. The organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and vacuum, and purified by column chromatography (eluent system dichloromethane: methanol = 150: 1, volume ratio) to obtain compound B4OH.

[0147] 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).

[0148] Example 4: Synthesis of Compound B5OH

[0149] The structural formula is as follows:

[0150]

[0151] Synthesis of intermediate 10d

[0152]

[0153] In a dry round-bottom flask, compound pentaethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane, p-toluenesulfonyl chloride (8.962 g, 0.9 eq) and 10 mL of triethylamine were added at 0°C, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 10d.

[0154] Synthesis of intermediate 10e

[0155]

[0156] In a round-bottom flask, 10d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (1.3 g, 1.2 eq) and pomalidomide (684 mg, 1.1 eq) were added to the reaction solution. The reaction was allowed to react at 90°C for 6 h. The reaction was monitored by TLC. The reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected. The organic phase was washed with saturated aqueous NaCl solution 3 times 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 10e.

[0157] Synthesis of intermediate 10f

[0158]

[0159] 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, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound 10f.

[0160] Synthesis of the final product B5OH

[0161]

[0162] In a round-bottom flask, 10f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (126.7 mg, 1.2 eq) and BMS-303141 (273.05 mg, 1 eq) were added to the reaction solution. The reaction was allowed to react at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction solution was cooled to room temperature, water was added to quench it, and ethyl acetate was extracted 3 times. The organic phase was collected, washed 3 times with saturated NaCl aqueous solution, and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and vacuum, and purified by column chromatography (eluent system dichloromethane: methanol = 150: 1, volume ratio) to obtain compound B5OH.

[0163] 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).

[0164] Example 5: Synthesis of Compound B6OH

[0165] The structural formula is as follows:

[0166]

[0167] Synthesis of intermediate 11d

[0168]

[0169] In a dry round-bottom flask, compound 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, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 11d.

[0170] Synthesis of intermediate 11e

[0171]

[0172] In a round-bottom flask, 11d (1 g, 1 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (1.42 mg, 1.2 eq) and pomalidomide (683.2 mg, 1.1 eq) were added to the reaction solution. The reaction was allowed to react at 90°C for 6 h. The reaction was monitored by TLC. The reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected. The organic phase was washed with saturated NaCl aqueous solution 3 times 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 11e.

[0173] Synthesis of intermediate 11f

[0174]

[0175] 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, and the temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, column chromatography was used for purification (eluent system petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound 11f.

[0176] Synthesis of the final product B6OH

[0177]

[0178] In a round-bottom flask, 11f (50 mg, 0.9 eq) was dissolved in 10 mL of N,N-dimethylformamide, and K2CO3 (145.8 mg, 1.2 eq) and BMS-303141 (288.02 mg, 1 eq) were added to the reaction solution. The reaction was allowed to react at 90 °C for 6 h. The reaction was monitored by TLC. The reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected. The organic phase was washed with saturated NaCl aqueous solution 3 times 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 B6OH.

[0179] 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).

[0180] II. Study on the Anti-NASH Effect of the Compounds of the Invention

[0181] The implementation methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0182] Experimental Example 1: Cytotoxicity test of PROTAC compounds

[0183] The drug concentration was set in vitro, and the cytotoxicity of the PROTAC compound was tested by cell proliferation (MTT) assay. The cells used in the cytotoxicity test of the present invention were: human hepatoma cell HepG2, human normal hepatocyte L02, and human umbilical vein endothelial cell HUVEC.

[0184] The cells were cultured at 1×10 5 Cells / empty were inoculated into 96-well plates and cultured in an incubator at 37°C and 5% CO2 for 24 hours to adhere to the wall. The PROTAC compound to be tested was dissolved in dimethyl sulfoxide and added to the above 96-well plate according to a concentration gradient of 100, 80, 40, 20, and 10 μM. It was cultured in an incubator at 37°C and 5% CO2 for 24 hours. After the incubation, MTT at a concentration of 5 mg / mL was added to each well and continued to be incubated in the incubator for 4 hours. The culture medium was aspirated, 100 μL of dimethyl sulfoxide was added to each well, and the plate was shaken at 37°C for 10 minutes. The OD value of each well was measured at 492 and 630 nm using an enzyme reader, and the results were recorded. The cell survival rate was plotted against the quantity.

[0185] Result calculation: Cell survival rate = (OD value of cells in the experimental group - OD value of cells in the blank group) / (OD value of cells in the control group - OD value of cells in the blank group) × 100%.

[0186] The experimental group was the drug-added group; the blank group was the blank culture medium group, in order to detect the effect of the blank culture medium on cell survival rate; the control group was the cell culture medium group with DMSO added, in order to detect the effect of the drug solvent on cell survival rate.

[0187] The results are shown in Tables 1 and 2. Compared with the warhead BMS-303141, the five newly synthesized PROTAC compounds synthesized in the present invention have less cytotoxicity than the warhead BMS-303141 at a concentration below 100 μM, achieving the function of attenuation. Among them, the PROTAC compound B4OH with four-length glycol chains as connecting chains has weak toxicity in HepG2 cells, and its IC 50 The value was 88.38 μM, but the toxicity was still significantly reduced compared with BMS-303141.

[0188] Table 1 Cytotoxicity of PROTAC compounds on HepG2, L02, and HUVEC cells

[0189]

[0190]

[0191] Table 2 IC of PROTAC compounds in HepG2, L02 and HUVEC cells 50

[0192]

[0193] Experimental Example 2: Test of lipid-lowering activity of PROTAC compounds

[0194] Solution preparation:

[0195] ① Preparation of Oil Red O dye solution

[0196] Preparation of Oil Red O stock solution: Add 0.45g of Oil Red O powder to a 50mL centrifuge tube and add 45mL of isopropanol to make a saturated solution of Oil Red O in isopropanol. Store at room temperature away from light and use after 24 hours.

[0197] Preparation of Oil Red O working solution: Centrifuge the Oil Red O stock solution, take the supernatant, mix the supernatant with double distilled water in a volume ratio of 3:2, filter through a 0.45 μM filter membrane, and use it immediately after preparation.

[0198] ② Preparation of 4% CHAPS lysis buffer

[0199] Accurately weigh 1.8018 g of urea, 0.7613 g of thiourea, 0.05 g of dithiothreitol, 0.2 g of CHAPS (amphoteric surfactant), and 24.25 mg of Tris-base into a 50 mL centrifuge tube, add 5 mL to dissolve, and store at 4°C.

[0200] ③Preparation of culture medium containing FFAs and 1% BSA

[0201] Accurately weigh 5 g of bovine serum albumin (BSA), add it into a complete culture medium preheated at 55°C to dissolve it, and prepare a culture medium containing 1% bovine serum albumin.

[0202] Sodium oleate and sodium palmitate were prepared into solutions with a final concentration of 200mmol / L using 0.1mol / L sodium hydroxide aqueous solution, heated at 95℃ to dissolve, and then the sodium oleate and sodium palmitate solutions were mixed evenly in a volume ratio of 2:1, and added to an appropriate amount of complete medium containing 1% BSA preheated at 55℃ to prepare a medium containing FFAs and 1% BSA with a final concentration of 2mmol / L. The medium was quickly filtered using a 0.45μM filter membrane on a clean bench while hot. The concentration required for the experiment was diluted with a medium containing 1% BSA.

[0203] Specific experimental steps:

[0204] L02 cells were collected at 1×10 5 The density of cells / mL was inoculated in 100mm cell culture dishes and 6-well cell culture plates, where 10mL was inoculated in 100mm cell culture dishes and 1mL was inoculated in each well of 6-well cell culture plates. The culture plates inoculated with cells were placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours, the DMEM medium in the culture dishes was discarded, and an appropriate amount of 1×PBS was added for washing three times, and then an equal amount of serum-free DMEM medium was added to continue culturing for 12 hours in the same environment to starve the cells. After 12 hours, the serum-free medium in the culture dish was discarded, and DMSO plus DMEM medium was used as the blank group, and DMSO plus FFAs medium was used as the model group. Fenofibric acid (20 μmol / L) was used as the positive drug, the concentration of BMS-303141 was 20 μmol / L, and the concentration of the PROTAC compound to be tested was 10, 20, 40, 80, and 100 μM. DMSO, fenofibric acid and the drug to be tested were pre-mixed with complete medium containing 1% BSA, and the mixed medium was added to the cell culture dish and culture plate and continued to be cultured for 24 hours.

[0205] The culture medium in the 6-well cell culture plate was discarded, and an appropriate amount of 1×PBS was added to wash three times. The 1×PBS was discarded, and 4% paraformaldehyde fixative (M / M, mass percentage) was added to fix for 40 minutes, then the 4% paraformaldehyde fixative was discarded, and an appropriate amount of distilled water was added to wash, and the distilled water was discarded. Then 1 mL of Oil Red O working solution was added and stained for 40 minutes in the dark, and then the staining solution was discarded, and an appropriate amount of distilled water was added to wash, and the distilled water was discarded. 1 mL of 75% ethanol solution (V / V, volume percentage) was added to differentiate for 10 seconds, and the 75% ethanol solution (V / V, volume percentage) was quickly discarded, and an appropriate amount of distilled water was added to wash, and the distilled water was discarded. 1 mL of hematoxylin staining solution was added and stained for 10 minutes. The hematoxylin staining solution was recovered, and the 6-well cell culture plate was placed in tap water, washed and differentiated with running water for 5 minutes, and 50% glycerol aqueous solution (V / V, volume percentage) was added to seal the slides, and photographed and detected under a trinocular inverted biological microscope at 400 times. Discard the culture medium in the 100mm cell culture dish, add an appropriate amount of 1×PBS to wash three times, discard the 1×PBS, shake with the cross method for 1min, and after washing, aspirate the PBS solution, add 1mL0.25% trypsin (M / M, mass percentage) to digest the cells, add 1mL trypsin solution, and digest for 2min. A layer of aerosol appears at the bottom of the culture dish, add 2mL 1640 culture medium, wash off the cells attached to the bottom of the culture dish, aspirate into a centrifuge tube, and centrifuge at 800r / min for 5min. Aspirate the supernatant in the centrifuge tube, add 100μL 4% CHAPS lysis solution (V / V, volume percentage), and lyse on ice for 1h.

[0206] The TG content was detected according to the instructions of the kit, as shown in Table 3.

[0207] Table 3 Test method for TG content

[0208]

[0209] The standard substance is the calibration substance included in the kit.

[0210] The TG content is calculated according to formula (3-1).

[0211]

[0212] Wherein, Cpr is the corresponding protein content of the PROTAC compound to be tested.

[0213] The AST content detection is carried out according to the instructions of the kit, and can be specifically carried out as shown in Table 4.

[0214] Table 4 Test method for AST content

[0215]

[0216]

[0217] The effects of BMS-303141 and PROTAC compounds on the morphological changes of FFAs-induced MASH cell models are shown in the following table. Figure 1 As shown in the figure, after adding 20 μmol / L of BMS-303141 and PROTAC compounds for co-culture for 24 hours, it can be seen from morphological observation that compounds BMS-303141, B3OH and B4OH reduce intracellular lipid accumulation, and the lipid content reduced by B3OH and B4OH is more than that of BMS-303141, which is significantly better than the model group, while the intracellular lipid content of other compounds has almost no significant change, which is equivalent to the model group. Combined with the above data, it can be concluded through morphological observation that the ability of the PROTACs compounds synthesized in the present invention to reduce lipid accumulation is better than that of the original drug BMS-303141.

[0218] The effects of BMS-303141 and PROTAC compounds on the TG content of FFAs-induced MASH cell models were investigated. Figure 2As shown in the figure, it can be seen that the TG content of the blank group is very low, with a TG content of 0.02mmol / gprot. After stimulation with FFAs, the TG content of the model group increased significantly, with a TG content of 0.24mmol / gprot. The positive control fenofibric acid can reverse the increase in TG content induced by FFAs and reduce the TG content to 0.12mmol / gprot. After adding BMS-303141 or PROTAC compounds at a concentration of 20μmol / L, BMS-303141 and B3OH and B4OH have obvious lipid-lowering activities, and B3OH and B4OH have stronger lipid-lowering activities than the warhead BMS-313141. Among them, the TG content of BMS-303141 is 0.16mmol / gprot, the TG content of B3OH is 0.13mmol / gprot, and the TG content of B4OH is 0.11mmol / gprot. Combined with the above data, it is further verified that the lipid-lowering activity of the PROTACs compounds synthesized in the present invention is stronger than that of the original drug BMS-303141.

[0219] The effects of BMS-303141 and PROTAC compounds on AST content in FFAs-induced MASH cell models were investigated. Figure 3 As shown, it can be seen from the figure that the AST content of the blank group is very low, with an AST value of 47.16U / gprot. After stimulation by FFAs, the AST content of the model group increased significantly, with an AST value of 74.79U / gprot. The positive drug fenofibric acid and the original drug BMS-303141 can reduce the AST content, of which the fenofibric acid AST value is 64.15U / gprot and the BMS-303141 AST value is 62.06U / gprot, but the effect is not significant. After adding B3OH and B4OH at a concentration of 20μmol / L, the B3OH AST value is 45.18U / gprot, the B4OH AST value is 52.82U / gprot, and liver damage is significantly reduced, of which the B3OH reduction effect is significant, which is equivalent to the blank group. From the above data, we can see that the lipid-lowering activity of the PROTACs compound synthesized by the present invention is stronger than that of the original drug BMS-303141, and can reduce liver damage to a certain extent.

[0220] Experimental Example 3: ACLY degradation ability test of PROTAC compounds

[0221] ① Protein sampling

[0222] L02 cells were cultured at 1×10 5Cells / well were inoculated into 100mm cell culture dishes and cultured in an incubator at 37°C and 5% CO2 for 24 hours to adhere to the wall. The DMEM medium in the culture dish was discarded, and an appropriate amount of 1×PBS was added to wash three times, and then an equal amount of serum-free DMEM medium was added, and the cells were continued to be cultured for 12 hours in the same environment to starve the cells. After 12 hours, the serum-free medium was aspirated, and DMSO plus DMEM medium was used as a blank group, and DMSO plus FFAs medium was used as a model group. Fenofibric acid (20μmol / L) was used as a positive drug, the concentration of BMS-303141 was 20μmol / L, and the concentrations of B3OH and B4OH were 20μM. DMSO, fenofibrate, B3OH and B4OH were pre-mixed with complete medium containing 1% BSA, and the mixed medium was added to the cell culture dish and culture plate and continued to be cultured for 24 hours. Aspirate the culture medium in the 100mm cell culture dish into a centrifuge tube, add 2mL 1×PBS to wash the culture dish, shake it with the cross method for 1min, aspirate PBS into the above centrifuge tube, add 1mL 0.25% trypsin to digest the cells, digest for 2min, a layer of aerosol will appear at the bottom of the culture dish, add 2mL of the culture medium in the above centrifuge tube to stop digestion, wash off the cells attached to the bottom of the culture dish, aspirate into a centrifuge tube, centrifuge at 2500r / min for 5min, quickly pour out the supernatant, add 1mL PBS and blow evenly, transfer to a 2mL EP tube, centrifuge at 2500r / min for 5min, quickly pour out the supernatant, add 100μL protein lysis buffer, lyse on ice for 1h, blow to suspend, centrifuge at 13500r / min in a 4℃ centrifuge for 20min, retain the supernatant, and perform protein quantification.

[0223] ② Protein quantification

[0224] The protein standard curve was drawn and the protein concentration was determined using the BSA method. According to the corresponding proportions, CHAPS protein lysate was prepared with bovine serum albumin into 7 concentrations of 0, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL for use as protein standard solutions. A 96-well plate was used to determine the protein concentration. 100 μL / well Coomassie brilliant blue was added in a dark place, and then 2 μL / well protein standard solution was added. No bubbles should appear during the mixing process, and 3 replicate wells were set. Using an ELISA reader, the absorbance was tested at a wavelength of 600 nm, and the mean was calculated to draw a standard curve, as shown in Table 5.

[0225] Table 5 BSA standard dilution standard

[0226] 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

[0227] Dilute the protein sample by CHARPS, and use the same operation method as above to calculate the protein concentration of each group according to the protein curve. After calculation and multiple dilutions, when the protein concentration of each group is basically consistent at 1.2-1.5 mg / mL, subsequent experiments can be carried out, and the samples can be stored at -80℃ first.

[0228] The effective compounds B3OH and B4OH in the present invention are compared, and the experimental results are as follows Figure 4 As shown in the figure, compared with the model group, BMS-303141, B3OH, and B4OH all have degradation effects on ACLY, and the degradation effect of B3OH is stronger than that of B4OH. The positive drug fenofibric acid has no degradation ability on ACLY. In the follow-up, the degradation ability of B3OH on ACLY was further explored, and 10, 20, 40, 80, and 100 μM were selected for concentration-dependent testing. The results are shown in Figure 5 As shown, the results show that with the increase of concentration, the ability of B3OH to degrade ACLY gradually increases, which is concentration-dependent. Combined with the above data, it can be seen that compared with the warhead BMS-303141, the ability of B3OH to degrade ACLY is significantly improved, and the degradation ability of ACLY is concentration-dependent.

[0229] Combined with the above results, it can be seen that PROTAC modification of the warhead BMS-303141 can indeed improve the activity of the original drug. Based on this result, it is hoped that compounds with better activity can be obtained by modifying the original drug, trying more types of connection chains, and increasing the types of E3 ubiquitin ligases.

[0230] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. An ACLY-targeted PROTAC chimera with anti-MASH activity using a glycol chain as a connecting chain, characterized in that: Its general structural formula is Formula 1: In the formula, R-(CH2-O-CH2) n -, n is 2 to 6.

2. The PROTAC chimera according to claim 1, characterized in that: The specific structure of the chimera is:

3. The method for preparing a PROTAC chimera according to claim 1 or 2, characterized in that: The synthetic route of the PROTACs compound is shown below.

4. The preparation method according to claim 3, characterized in that: The steps for synthesizing B2OH are as follows: Synthesis of intermediate 7d: In a dry round-bottom flask, compound 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, and the temperature was slowly raised to room temperature, and the reaction was carried out for 3 h. The reaction was detected by TLC technology, and after the reaction was completed, column chromatography was used for purification, and the eluent system was petroleum ether: ethyl acetate = 1:1, volume ratio, to obtain compound 7d; Synthesis of intermediate 7e: In a round-bottom flask, 7d (1g, 1eq) was dissolved in 10mL N,N-dimethylformamide, K2CO3 (952.77mg, 1.2eq) and pomalidomide (668.91mg, 1.1eq) were added to the reaction solution, and the reaction was carried out at 90°C for 6h; the reaction was monitored by TLC, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound 7e; Synthesis of intermediate 7f: 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, and the temperature was slowly raised to room temperature. The reaction was reacted for 3 h. The reaction was detected by TLC technology. After the reaction was completed, the compound was purified by column chromatography, and the eluent system was petroleum ether: ethyl acetate = 3:1, volume ratio, to obtain compound 7f. Synthesis of the final product B2OH: 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, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150:1, volume ratio, to obtain compound B2OH; Alternatively, the B3OH synthesis steps are as follows: Synthesis of intermediate 8d: In a dry round-bottom flask, compound 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, and the temperature was slowly raised to room temperature, and the reaction was carried out for 3 h. The reaction was detected by TLC technology, and after the reaction was completed, column chromatography was used for purification, and the eluent system was petroleum ether: ethyl acetate = 1:1, volume ratio, to obtain compound 8d; Synthesis of intermediate 8e: In a round-bottom flask, 8d (1g, 1eq) was dissolved in 10mL N,N-dimethylformamide, K2CO3 (973.25mg, 1.2eq) and pomalidomide (644, 98mg, 1.1eq) were added to the reaction solution, and the reaction was carried out at 90°C for 6h; the reaction was monitored by TLC, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound 8e; Synthesis of intermediate 8f: 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, and the temperature was slowly raised to room temperature. The reaction was reacted for 3 h. The reaction was detected by TLC technology. After the reaction was completed, the compound was purified by column chromatography, and the eluent system was petroleum ether: ethyl acetate = 3:1, volume ratio, to obtain compound 8f. Synthesis of the final product B3OH: 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, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound B3OH; Alternatively, the B4OH synthesis steps are as follows: Synthesis of intermediate 9d: In a dry round-bottom flask, compound tetraethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane, p-toluenesulfonyl chloride (8.837 g, 0.9 eq) and 10 mL of triethylamine were added at 0°C, and the temperature was slowly raised to room temperature, and the reaction was carried out for 3 h. The reaction was detected by TLC technology, and after the reaction was completed, column chromatography was used for purification, and the eluent system was petroleum ether: ethyl acetate = 1:1, volume ratio, to obtain compound 9d; Synthesis of intermediate 9e: 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, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated aqueous NaCl solution 3 times, and dried over anhydrous Na2SO4; the crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane:methanol=150:1, volume ratio, to obtain compound 9e; Synthesis of intermediate 9f: 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 temperature was slowly raised to room temperature. The reaction was reacted for 3 h. The reaction was detected by TLC technology. After the reaction was completed, the compound was purified by column chromatography, and the eluent system was petroleum ether: ethyl acetate = 3:1, volume ratio, to obtain compound 9f. Synthesis of the final product B4OH: 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, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound B4OH; Alternatively, the B5OH synthesis steps are as follows: Synthesis of intermediate 10d: In a dry round-bottom flask, compound pentaethylene glycol (10 g, 1 eq) was dissolved in 30 mL of dichloromethane, p-toluenesulfonyl chloride (8.962 g, 0.9 eq) and 10 mL of triethylamine were added at 0°C, and the temperature was slowly raised to room temperature. The reaction was reacted for 3 h. The reaction was detected by TLC technology. After the reaction was completed, column chromatography was used for purification, and the eluent system was petroleum ether: ethyl acetate = 1:1, volume ratio, to obtain compound 10d. Synthesis of intermediate 10e: In a round-bottom flask, 10d (1g, 1eq) was dissolved in 10mL N,N-dimethylformamide, K2CO3 (1.3g, 1.2eq) and pomalidomide (684mg, 1.1eq) were added to the reaction solution, and the reaction was carried out at 90°C for 6h; the reaction was monitored by TLC, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound 10e; Synthesis of intermediate 10f: 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, and the temperature was slowly raised to room temperature. The reaction was reacted for 3 h. The reaction was detected by TLC technology. After the reaction was completed, column chromatography was used for purification, and the eluent system was petroleum ether: ethyl acetate = 3:1, volume ratio, to obtain compound 10f. Synthesis of the final product B5OH: 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, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150:1, volume ratio, to obtain compound B5OH; Alternatively, the B6OH synthesis steps are as follows: Synthesis of intermediate 11d In a dry round-bottom flask, compound 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, and the temperature was slowly raised to room temperature. The reaction was reacted for 3 h. The reaction was detected by TLC technology. After the reaction was completed, column chromatography was used for purification, and the eluent system was petroleum ether: ethyl acetate = 1:1, volume ratio, to obtain compound 11d. Synthesis of intermediate 11e: In a round-bottom flask, 11d (1g, 1eq) was dissolved in 10mL N,N-dimethylformamide, K2CO3 (1.42mg, 1.2eq) and pomalidomide (683.2mg, 1.1eq) were added to the reaction solution, and the reaction was carried out at 90°C for 6h; the reaction was monitored by TLC, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried with anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound 11e; Synthesis of intermediate 11f: 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, and the temperature was slowly raised to room temperature. The reaction was reacted for 3 h. The reaction was detected by TLC technology. After the reaction was completed, column chromatography was used for purification, and the eluent system was petroleum ether: ethyl acetate = 3:1, volume ratio, to obtain compound 11f. Synthesis of the final product B6OH: In a round-bottom flask, 11f (50 mg, 0.9 eq) was dissolved in 10 mL 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, and the reaction solution was cooled to room temperature, quenched with water, extracted with ethyl acetate 3 times, and the organic phase was collected, washed with saturated NaCl aqueous solution 3 times, and dried over anhydrous Na2SO4. The crude product was concentrated under reduced pressure and purified by column chromatography, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound B6OH.

5. Use of the PROTAC chimera as described in claim 1 or 2 in the preparation of a drug targeting the degradation of ACLY.

6. Use of the PROTAC chimera as described in claim 1 or 2 in the preparation of anti-MASH drugs.

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