Synthesis of a protacs compound containing a pure carbon chain targeting acl y and its application in anti-nash

Abstract

The application belongs to the technical field of biology and medicine, and discloses a PROTAC compound with a carbon chain as a connecting chain, which is developed based on a PROTAC technology, and a structural general formula of the PROTAC compound is formula 1: wherein R is -(CH2) n -, and n is 3-8. The synthesized PROTAC compound is a new compound, has a TG content reducing activity in a high-fat model established by L02 and HepG2, and has a low-toxicity activity in human hepatoma cells HepG2, human normal liver cells L02 and human umbilical vein endothelial cells HUVEC compared with a warhead. The synthesized PROTAC compound is a new type of anti-non-alcoholic fatty liver disease drug with low toxicity and high efficiency. The application of the PROTAC technology is widened while developing an efficient treatment of anti-non-alcoholic fatty liver disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biology and medicine, relates to the field of small molecule drug development, and particularly relates to a PROTAC compound with anti-NASH activity based on PROTAC technology, a method and an application. BACKGROUND

[0002] NAFLD (non-alcoholic fatty liver disease, NAFLD) refers to excessive deposition of fat in the liver caused by factors other than excessive alcohol consumption, viruses and other liver damage factors, including non-alcoholic fatty liver (non-alcoholic fatty liver, NAFL) and non-alcoholic steatohepatitis (non-alcoholic steatohepatitis, NASH), wherein NASH treatment is not timely and can develop into cirrhosis and even liver cancer, causing death. NASH is closely related to metabolic disorders such as obesity, insulin resistance, type 2 diabetes and hyperlipidemia, and is prone to occur in middle-aged people, especially overweight and obese individuals, and its clinical manifestations are fatty hepatitis and fatty cirrhosis. NASH is the advanced stage of NAFLD and eventually develops into end-stage liver disease and hepatocellular carcinoma, and is considered one of the main causes of cirrhosis in adults in the United States and is the main cause of liver transplantation. However, there is currently only one marketed drug, Resmetirom, for NASH, and its target is THR-β, but the pathogenesis of NASH is complex and involves multiple targets, and a single target treatment drug is not sufficient to meet the entire market. Therefore, developing an efficient and safe drug for NASH is an urgent task and faces great challenges.

[0003] Excessive fat accumulation in most NASH patients is derived from the process of de novo lipogenesis, and the raw material for de novo lipogenesis is acetyl-CoA, which is derived from the cleavage of citrate by ATP citrate lyase (ACLY). Acetyl-CoA, as the basic substrate for fatty acid and cholesterol biosynthesis, is an important target for treating NASH and dyslipidemia, and ACLY can be selected as a target to inhibit the generation of acetyl-CoA and treat NASH.

[0004] Protein degradation-targeting chimeras (PROTACs) are a novel small-molecule protein-targeting degradation technology, consisting of a target protein ligand, an E3 ligase ligand, and a linker. When a PROTAC molecule enters the cell, the connecting chain in the middle bends, bringing the E3 ubiquitin ligase closer to the target protein. The ubiquitin carried by the E3 ligase ligand is then transferred to the substrate protein, causing ubiquitination. The ubiquitinated protein is then recognized by the proteasome, leading to the degradation of the target protein. PROTAC drugs have many advantages over traditional small-molecule drugs, such as lower cytotoxicity and the potential to overcome drug resistance. Currently, there are no reports of PROTAC technology being applied to NASH (Nephrotic Syndrome).

[0005] Therefore, among drugs targeting NASH, those developed based on PROTAC technology to treat NASH using ACLY protein degrading agents show great promise, bringing new hope to NASH treatment and broadening the application areas of PROTAC technology. It is necessary to conduct research on these drugs. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a PROTAC compound, method and application with anti-NASH activity based on PROTAC technology, which uses a carbon chain as the connecting chain.

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

[0008] A PROTAC compound developed based on PROTAC technology, with a carbon chain as the linking chain, has the general structural formula of Formula 1:

[0009]

[0010] In the formula, R stands for -(CH2). n -, where n is 3 to 8.

[0011] Furthermore, the structure of the PROTAC compound is specifically one of the following:

[0012]

[0013] The PROTAC compound described above is prepared by reacting compound 2 and compound 3.

[0014]

[0015] In Equation 2, R is -(CH2). n -, where n is 3 to 8.

[0016] Furthermore, Equation 2 is prepared by reactions of Equations 4 and 5:

[0017]

[0018] In Equation 4, R is -(CH2). n -, where n is 3 to 8.

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

[0020]

[0021] Furthermore, Equation 4 is prepared by reactions of Equations 6 and 7:

[0022]

[0023] In Equation 6, R is -(CH2). n -, where n is 3 to 8.

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

[0025]

[0026] Formula 10 is prepared by reacting with Formulas 13 and 14:

[0027]

[0028] Furthermore, formula 6 is prepared by reacting formulas 5 and 8 to obtain:

[0029]

[0030] In Equation 8, R is -(CH2). n -, where n is 3 to 8.

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

[0032]

[0033] In the formula, R stands for -(CH2). n -, where n is 3 to 8.

[0034] in, The preparation method is as follows:

[0035]

[0036] The application of PROTAC compounds 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] Furthermore, the PROTAC compound is combined with appropriate excipients to prepare a pharmaceutically acceptable 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 application relates to a PROTAC protein degrader targeting ACLY with a carbon chain as the linking chain and its application. The problem to be solved by this invention is to provide a novel PROTAC drug for treating non-alcoholic steatohepatitis. The structural formula of the PROTAC molecule is shown in formula (I).

[0043]

[0044] In the formula, R is -(CH2). n -, where n is 3 to 8.

[0045] 3. The purpose of this invention is to solve the above-mentioned technical problems by providing a class of ACLY protein-targeting chimeras synthesized using PROTAC technology and their application in anti-NASH activity. This application discovers a novel class of PROTAC degraders targeting ACLY protein with the structure shown in Formula 1. Compared with the original drug BMS-303141, its cytotoxicity is significantly reduced. Among them, B7C has good lipid-lowering activity and ACLY protein degradation activity while alleviating liver inflammation, making it a new generation of low-toxicity and highly effective anti-NASH drug. Attached Figure Description

[0046] Figure 1 The diagram shows the effect of compounds B3C to B8C in this invention on lipid accumulation in L02 cells.

[0047] Figure 2 The figure shows the effect of compounds B3C to B8C in this invention on triglyceride content in L02 cells.

[0048] Figure 3 The figure shows the effect of compounds B3C to B8C in this invention on the content of aspartate aminotransferase in L02 cells;

[0049] Figure 4 This is a diagram showing the degradation effect of compound B7C on ACLY protein in this invention;

[0050] Figure 5 This is a predicted diagram of the connection site between compound B7C and ACLY in this invention. Detailed Implementation

[0051] 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.

[0052] 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.

[0053] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0054] A PROTAC compound developed based on PROTAC technology, with a carbon chain as the linking chain, has the general structural formula of Formula 1:

[0055]

[0056] In the formula, R stands for -(CH2). n -, where n is 3 to 8.

[0057] Alternatively, the structure of the PROTAC compound may be one of the following:

[0058]

[0059] The PROTAC compound described above is prepared by reacting compound 2 and compound 3.

[0060]

[0061] In Equation 2, R is -(CH2). n -, where n is 3 to 8.

[0062] Furthermore, Equation 2 is prepared by reactions of Equations 4 and 5:

[0063]

[0064] In Equation 4, R is -(CH2).n -, where n is 3 to 8.

[0065] Formula 3 is prepared by reacting with Formulas 9 and 10:

[0066]

[0067] Furthermore, Equation 4 is prepared by reactions of Equations 6 and 7:

[0068]

[0069] In Equation 6, R is -(CH2). n -, where n is 3 to 8.

[0070] Formula 9 is prepared by reacting with Formulas 11 and 12:

[0071]

[0072] Formula 10 is prepared by reacting with Formulas 13 and 14:

[0073]

[0074] Furthermore, formula 6 is prepared by reacting formulas 5 and 8 to obtain:

[0075]

[0076] In Equation 8, R is -(CH2). n -, where n is 3 to 8.

[0077] The reaction formula is as follows:

[0078]

[0079] In the formula, R stands for -(CH2). n -, where n is 3 to 8.

[0080] in, The preparation method is as follows:

[0081]

[0082] The application of PROTAC compounds as described above in the preparation of drugs that target the degradation of ACLY.

[0083] Alternatively, the targeted ACLY degradation drug may be an anti-nonalcoholic steatohepatitis drug;

[0084] 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).

[0085] Alternatively, the PROTAC compound can be combined with appropriate excipients to prepare a pharmaceutically acceptable formulation.

[0086] Specifically, the relevant preparation and testing methods are as follows:

[0087] I. Compounds synthesized in this invention

[0088] 1. The synthetic route for preparing compound BMS-303141 of this invention is as follows:

[0089]

[0090] Furthermore, the specific synthesis steps for BMS-33141 are as follows:

[0091] (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.

[0092] (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.

[0093] (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) to obtain compound BMS-303141.

[0094] 2. The synthesis of compounds B3C, B4C, B5C, B6C, B7C, and B8C according to the present invention is as follows:

[0095]

[0096] Furthermore, the specific synthesis steps are as follows:

[0097] (1) Synthesis of intermediate d (taking n=3 as an example): In a dry round-bottom flask, compound 1,5-pentanediol (8 g, 1 eq) was dissolved in 10 mL of dichloromethane. p-Toluenesulfonyl chloride (13.6 g, 0.9 eq) and 8 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 = 1:1, volume ratio) to obtain compound 3d.

[0098] (2) Synthesis of intermediate e (taking n=3 as an example): In a round-bottom flask, 3d (300 mg, 1 eq) was dissolved in 5 mL of N,N-dimethylformamide. K2CO3 (156.2 mg, 1.2 eq) and pomalidomide (282 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 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 3e.

[0099] (3) Synthesis of intermediate f (taking n=3 as an example): In a dry round-bottom flask, compound 3e (50 mg, 1 eq) was dissolved in 5 mL of dichloromethane. p-Toluenesulfonyl chloride (53.05 mg, 2 eq) and 0.05 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 3f.

[0100] (4) Synthesis of the final product (taking B5C as an example): In a round-bottom flask, 3f (50 mg, 0.9 eq) was dissolved in 5 mL of N,N-dimethylformamide. K2CO3 (93.5 mg, 1.2 eq) and BMS-303141 (250 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 to obtain the crude product, which was purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, volume ratio) to obtain compound B5C.

[0101] Example 1: Synthesis of compound B3C

[0102] The structural formula is as follows:

[0103]

[0104] Synthesis of intermediate 1d

[0105]

[0106] In a dry round-bottom flask, compound 1,3-propanediol (8 g, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (18.04 g, 0.9 eq) and 8 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 1d.

[0107] Synthesis of intermediate 1e

[0108]

[0109] In a round-bottom flask, 1d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (342.2 mg, 1.2 eq) and pomalidomide (322 mg, 1.1 eq) were added to the reaction mixture, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction mixture 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 1e.

[0110] Synthesis of intermediate 1f

[0111]

[0112] In a dry round-bottom flask, compound 1e (50 mg, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (56.02 mg, 2 eq) and 0.05 mL 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 1f.

[0113] Synthesis of the final product B3C

[0114]

[0115] In a round-bottom flask, 1 part (50 mg, 0.9 eq) of N,N-dimethylformamide was dissolved. K₂CO₃ (106.5 mg, 1.2 eq) and BMS-303141 (230.1 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 B3C.

[0116] Yellow solid, yield 24.15%. 1 H NMR(400MHz,Chloroform-d)δ7.76-7.26(m,7H),7.21-7.11(m,1H),6.99(s,1H),6.87(dq,J=12.9,7. 0,5.2Hz,1H),5.82(ddt,J=17.0,10.2,6.8Hz,1H),5.41-5.31(m,1H),5.24(s,1H),4.93(d,J=9.0Hz, 1H),4.11-4.04(m,1H),3.89-3.79(m,1H),3.64(p,J=6.0Hz,1H),3.01-2.80(m,3H),2.80-2.66(m,2H ),2.66-2.52(m,1H),2.34(td,J=7.6,5.1Hz,1H),2.22(t,J=7.6Hz,1H),2.03(dt,J=10.0,5.9Hz,3H).

[0117] Example 2: Synthesis of compound B4C

[0118] The structural formula is as follows:

[0119]

[0120] Synthesis of intermediate 2d

[0121]

[0122] In a dry round-bottom flask, compound 1,4-butanediol (8 g, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (15.23 g, 0.9 eq) and 8 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 = 1:1, v / v) to give compound 2d.

[0123] Synthesis of intermediate 2e

[0124]

[0125] In a round-bottom flask, 2d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (274.5 mg, 1.2 eq) and pomalidomide (309 mg, 1.1 eq) were added to the reaction mixture, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction mixture 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 then purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound 2e.

[0126] Synthesis of intermediate 2f

[0127]

[0128] In a dry round-bottom flask, compound 2e (50 mg, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (60.23 mg, 2 eq) and 0.05 mL 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 2f.

[0129] Synthesis of the final product B4C

[0130]

[0131] In a round-bottom flask, 2f (50 mg, 0.9 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (103.3 mg, 1.2 eq) and BMS-303141 (212.4 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 B4C.

[0132] Yellow solid, yield 24.02%. 1H NMR(400MHz,Chloroform-d)δ7.65(d,J=2.6Hz,1H),7.60-7.47(m,3H),7.43(t,J=7.7Hz,2H),7.33(q ,J=7.5,6.2Hz,1H),7.19-7.05(m,1H),6.99(s,1H),6.84(d,J=8.3Hz,1H),5.86-5.71(m,1H),5.45-5. 19(m,2H),5.00-4.90(m,1H),4.05(dt,J=12.8,6.7Hz,2H),3.60(q,J=6.6Hz,3H),3.47(d,J=21.2Hz,3 H),2.95-2.73(m,2H),2.65-2.52(m,1H),2.40-2.31(m,1H),2.22(t,J=7.6Hz,1H),2.08-1.95(m,3H).

[0133] Example 3: Synthesis of compound B5C

[0134] The structural formula is as follows:

[0135]

[0136] Synthesis of intermediate 3D

[0137]

[0138] In a dry round-bottom flask, compound 1,5-pentanediol (8 g, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (13.6 g, 0.9 eq) and 8 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 = 1:1, v / v) to give compound 3d.

[0139] Synthesis of intermediate 3e

[0140]

[0141] In a round-bottom flask, 3d (300 mg, 1 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (156.2 mg, 1.2 eq) and pomalidomide (282 mg, 1.1 eq) were added to the reaction mixture, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction mixture 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 3e.

[0142] Synthesis of intermediate 3f

[0143]

[0144] In a dry round-bottom flask, compound 3e (50 mg, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (53.05 mg, 2 eq) and 0.05 mL 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 3f.

[0145] Synthesis of the final product B5C

[0146]

[0147] In a round-bottom flask, 3f (50 mg, 0.9 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (93.5 mg, 1.2 eq) and BMS-303141 (250 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 B5C.

[0148] Yellow solid, yield 26.67%. 1H NMR(400MHz,Chloroform-d)δ7.45(m,J=7.8Hz,6H),7.17(d,J=7.1Hz,1H),6.99(s,1H),6.88(d,J=8.4Hz,1H),5.35(s,1H),5.24(s,2H),4. 90(s,1H),3.85(dd,J=14.6,7.3Hz,2H),3.65(dt,J=12.1,6.7Hz,6H),2.73(d,J=10.6Hz,2H),2.62(s,1H),2.49-2.17(m,5H),2.02(m,3H).

[0149] Example 4: Synthesis of compound B6C

[0150] The structural formula is as follows:

[0151]

[0152] Synthesis of intermediate 4d

[0153]

[0154] In a dry round-bottom flask, compound 1,5-hexanediol (8 g, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (11.62 g, 0.9 eq) and 8 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 = 1:1, v / v) to give compound 4d.

[0155] Synthesis of intermediate 4e

[0156]

[0157] In a round-bottom flask, 4d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (382.2 mg, 1.2 eq) and pomalidomide (402 mg, 1.1 eq) were added to the reaction mixture, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction mixture 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 4e.

[0158] Synthesis of intermediate 4f

[0159]

[0160] In a dry round-bottom flask, compound 4e (50 mg, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (56.02 mg, 2 eq) and 0.05 mL 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 4f.

[0161] Synthesis of the final product B6C

[0162]

[0163] In a round-bottom flask, 4f (50 mg, 0.9 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (111.2 mg, 1.2 eq) and BMS-303141 (273.4 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 B6C.

[0164] Yellow solid, yield 25.32%. 1 H NMR (400MHz, Chloroform-d) δ7.73(d,J=8.2Hz,1H),7.58(d,J=2.6Hz,1H),7.46(dq,J=6.5,3.6,2.8Hz,2H),7.36(dq,J=7.5,4.0 Hz,1H),7.29(s,2H),7.15-7.00(m,1H),6.92(s,1H),6.85-6.74(m,1H),5.82-5.68(m,1H),5.28(t,J=4.8Hz,1H),5.17(s,1H),4 .91-4.81(m,1H),4.27-4.06(m,1H),4.06-3.93(m,2H),3.85(s,1H),3.74(dd,J=8.7,5.7Hz,1H),3.58(dtd,J=24.2,8.9,4.2Hz, 6H),2.87(s,1H),2.84-2.75(m,1H),2.75-2.64(m,1H),2.56-2.48(m,1H),2.38(s,1H),2.15(t,J=7.6Hz,1H),2.03-1.90(m,3H).

[0165] Example 5: Synthesis of compound B7C

[0166] The structural formula is as follows:

[0167]

[0168] Synthesis of intermediate 5d

[0169]

[0170] In a dry round-bottom flask, compound 1,5-heptadecyl glycol (8 g, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (10.38 g, 0.9 eq) and 8 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 = 1:1, v / v) to give compound 5d.

[0171] Synthesis of intermediate 5e

[0172]

[0173] In a round-bottom flask, 5d (300 mg, 1 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (156.2 mg, 1.2 eq) and pomalidomide (282 mg, 1.1 eq) were added to the reaction mixture, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction mixture 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 then purified by column chromatography (eluent system: dichloromethane:methanol = 150:1, v / v) to give compound 5e.

[0174] Synthesis of intermediate 5f

[0175]

[0176] In a dry round-bottom flask, compound 5e (50 mg, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (53.05 mg, 2 eq) and 0.05 mL 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 5f.

[0177] Synthesis of the final product B7C

[0178]

[0179] In a round-bottom flask, 5f (50 mg, 0.9 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (110.8 mg, 1.2 eq) and BMS-303141 (251.3 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 B7C.

[0180] Yellow solid, yield 26.81%. 1 H NMR(400MHz,Chloroform-d)δ7.74(d,J=7.8Hz,2H),7.62-7.46(m,2H),7.44-7 .32(m,2H),7.18(d,J=7.8Hz,2H),7.07(d,J=7.2Hz,2H),5.98(d,J=11.6Hz,3H) ,4.93(dd,J=12.3,5.4Hz,1H),4.05(d,J=40.9Hz,2H),3.82-3.70(m,3H),3.65 -3.43(m,4H),3.33(q,J=7.2Hz,2H),2.82-2.67(m,7H),2.06(d,J=19.5Hz,4H).

[0181] Example 6: Synthesis of compound B8C

[0182] The structural formula is as follows:

[0183]

[0184] Synthesis of intermediate 6d

[0185]

[0186] In a dry round-bottom flask, compound 1,5-octanediol (8 g, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (9.39 g, 0.9 eq) and 8 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 = 1:1, v / v) to give compound 6d.

[0187] Synthesis of intermediate 6e

[0188]

[0189] In a round-bottom flask, 6d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (173.2 mg, 1.2 eq) and pomalidomide (363.2 mg, 1.1 eq) were added to the reaction mixture, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC. After the reaction mixture 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 6e.

[0190] Synthesis of intermediate 6f

[0191]

[0192] In a dry round-bottom flask, compound 6e (50 mg, 1 eq) was dissolved in dichloromethane. p-Toluenesulfonyl chloride (60.3 mg, 2 eq) and 0.05 mL 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 6f.

[0193] Synthesis of the final product B8C

[0194]

[0195] In a round-bottom flask, 6f (50 mg, 0.9 eq) was dissolved in N,N-dimethylformamide. K₂CO₃ (121 mg, 1.2 eq) and BMS-303141 (284.3 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 B8C.

[0196] Yellow solid, yield 25.77%. 1H NMR(400MHz,Chloroform-d)δ7.84-7.59(m,2H),7.53(s,2H),7.42(t,J=7.7Hz,2H),7.35(d,J=7.2H z,2H),7.16(d,J=7.1Hz,1H),7.00(d,J=11.2Hz,1H),6.88(d,J=8.4Hz,2H),5.53(s,1H),4.98-4.85( m,1H),4.06-3.91(m,2H),3.79(t,J=7.5Hz,3H),3.69-3.57(m,1H),3.49(d,J=8.2Hz,3H),3.21-3.02 (m,2H),2.83(d,J=6.4Hz,2H),2.81-2.65(m,6H),2.65-2.52(m,1H),2.45(s,2H),2.30-2.15(m,2H).

[0197] II. Study on the anti-NASH effect of the compounds of this invention

[0198] 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.

[0199] Experimental Example 1: Cytotoxicity Test of PROTAC Compound

[0200] 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.

[0201] 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.

[0202] 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%.

[0203] 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.

[0204] The results, as shown in Tables 1 and 2, indicate that the six newly synthesized PROTAC compounds exhibited lower cytotoxicity than the warhead BMS-303141 at concentrations below 100 μM. BMS-303141 showed lower IC50 values ​​in L02 cells. 50 The value was 29.25 μM, while the synthesized PROTACs compounds showed no cytotoxicity in the range of 10–100 μM, thus achieving the function of attenuation.

[0205] Table 1. Cytotoxicity of PROTAC compounds on HepG2, L02, and HUVEC cells.

[0206]

[0207] Table 2. Cytotoxic effects of PROTAC compounds on HepG2, L02, and HUVEC cells.

[0208]

[0209] Experimental Example 2: Test of the lipid-lowering activity of PROTAC compounds

[0210] Solution preparation:

[0211] ① Preparation of Oil Red O staining solution

[0212] 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.

[0213] Preparation of Oil Red O working solution: Centrifuge the Oil Red O storage 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.

[0214] ② Preparation of 4% CHAPS lysis buffer

[0215] 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.

[0216] ③ Preparation of culture medium containing FFAs and 1% BSA

[0217] 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.

[0218] 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 using medium containing 1% BSA.

[0219] Specific experimental steps:

[0220] 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 then discarded, and the cells were washed three times with 1×PBS. An equal volume of serum-free DMEM medium was then added, and the cells were cultured for another 12 h under the same conditions to starve them. After 12 hours, the serum-free culture medium in the culture dishes was discarded. DMSO and 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 concentrations of the PROTAC compound to be tested were 10, 20, 40, 80, and 100 μM. DMSO, fenofibrate acid and the drug to be tested were mixed with complete culture medium containing 1% BSA beforehand. The mixed culture medium was added to cell culture dishes and culture plates and cultured for another 24 hours.

[0221] 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 (MM, 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.

[0222] TG content detection was performed according to the kit instructions, as shown in Table 3.

[0223] Table 3. Test methods for TG content

[0224]

[0225] The standard is the calibrator included in the kit.

[0226] The TG content is calculated according to formula (3-1).

[0227]

[0228] Where Cpr represents the protein content of the PROTAC compound to be tested.

[0229] AST content detection should be performed according to the kit instructions, as shown in Table 4.

[0230] Table 4. Test methods for AST content

[0231]

[0232] The effects of BMS-303141 and PROTAC compounds on morphological changes in a FFA-induced NASH 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, B6C, and B7C reduced intracellular lipid accumulation. Furthermore, the reduction in lipid accumulation by B6C and B7C was less than that by BMS-303141, significantly better than the model group. Other compounds showed almost no significant change in intracellular lipid content, comparable to the model group. Based on the above 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.

[0233] Preliminary screening identified B6C and B7C as effective compounds.

[0234] The effects of BMS-303141 and PROTAC compounds on TG levels in a FFA-induced NASH cell model were investigated, and the results are as follows: Figure 2 As shown in the figure, the TG content in the blank group was very low, while the TG content in the model group increased significantly after stimulation with FFAs. The positive control, fenofibrate acid, reversed the FFA-induced increase in TG content. After adding 20 μmol / L of BMS-303141 or PROTAC compounds, the TG content of BMS-303141 was 0.26 mmol / gprot, B6C 0.19 mmol / gprot, and B7C 0.13 mmol / gprot. BMS-303141, B6C, and B7C showed significant lipid-lowering activity, with B6C and B7C exhibiting stronger lipid-lowering activity than the precursor BMS-313141. Based on these data, it is further verified that the lipid-lowering activity of the synthesized PROTACs in this invention is stronger than that of the original drug BMS-303141.

[0235] The effects of BMS-303141 and PROTAC compounds on AST content in a FFA-induced NASH cell model were investigated, and the results are as follows: Figure 3As shown in the figure, the AST content in the blank group was very low, with an AST value of 27.88 U / gprot. In the model group, after stimulation with FFAs, the AST content significantly increased, reaching 76.25 U / gprot. The positive control drug fenofibrate and the original drug BMS-303141 could reduce the AST content, with fenofibrate at 58.7 U / gprot and BMS-303141 at 59.12 U / gprot, but the effect was not significant. After adding B7C at a concentration of 20 μmol / L, the B7C AST value was 31.93 U / gprot, comparable to the blank group, significantly reducing liver damage. These data indicate that the synthesized PROTACs compounds have stronger lipid-lowering activity than the original drug BMS-303141 and can reduce liver damage to a certain extent.

[0236] Experiment Example 3: Degradation ability test of PROTAC compound ACLY

[0237] ① Protein sample collection

[0238] L02 cells were fed at a rate of 1×10 5 Cells 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 in the same environment to starve them. After 12 h, serum-free medium was aspirated, and fenofibrate (20 μmol / L) was used as a positive control. BMS-303141 concentration was 20 μmol / L, and B7C concentrations were 10, 20, 40, 80, and 100 μM. DMSO, fenofibrate, and B7C 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 and mix well. Transfer the mixture to a 2mL EP tube and centrifuge at 2500 rpm for 5 minutes. Quickly discard the supernatant, add 100μL of protein lysis buffer, and lyse on ice for 1 hour. Resuspend the mixture and centrifuge at 13500 rpm for 20 minutes at 4°C. Retain the supernatant for protein quantification.

[0239] ② Protein quantification

[0240] Protein standard curves were constructed, and protein concentrations were determined using the BSA method. 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) of CHAPS protein lysis buffer and bovine serum albumin were prepared according to appropriate ratios and used as protein standard solutions. Protein concentrations were measured 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 avoid air bubbles. Three replicates were performed. The absorbance was measured at 600 nm using a microplate reader, and the mean values ​​were calculated to plot a standard curve.

[0241] Table 5. Dilution Standards for BSA Standards

[0242] Sample 1 2 3 4 5 6 7 BSA concentration (pg / pL) 0 0.125 0.25 0.5 1 1.5 2 Sample (pL) 2 2 2 2 2 2 2 Coomassie blue (pL) 100 100 100 100 100 100 100 Protein content (pg) 0 0.25 0.5 1 2 3 4

[0243] Protein samples were diluted with CHAPS, and the same procedure described above was used to calculate the protein concentration for each group based on the protein curve. After calculation and multiple dilutions, when the protein concentration for each group was approximately consistent between 1.2-1.5 mg / mL, subsequent experiments could be carried out. Samples could be stored at -80℃ initially.

[0244] Experimental results are as follows Figure 4 As shown, compared with the model group, both BMS-303141 and B7C degraded ACLY, while the positive control drug fenofibrate acid showed no ability to degrade ACLY. Compared with the warhead BMS-303141, B7C showed a significantly improved ACLY degradation ability at a concentration of 20 μM, and the degradation ability of B7C on ACLY was concentration-dependent.

[0245] Experimental Example 4: Prediction of the linking site between PROTAC compound and ACLY

[0246] By simulating the docking of B7C and ACLY (PDB code: 3WME) using Autodock software, it was found that, Figure 5 As shown, the warhead of the B7C is in the active pocket of the ACLY, while the connecting chain and pomalidomide end are exposed to the solvent so that the other end can be connected to pomalidomide, which is consistent with the design concept of RPOTAC.

[0247] The above results demonstrate that modifying the BMS-303141 warhead with PROTAC can indeed improve the activity of the original drug and enhance its targeting. 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.

[0248] 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 PROTAC compound developed based on PROTAC technology with a carbon chain as a connecting chain, characterized in that: the structure of the compound is one of the following: The synthesis route of the PROTAC compound is as follows:

2. The method for preparing the PROTAC compound according to claim 1, characterized in that: The synthesis steps of B6C are as follows:

3. The method of claim 2, wherein: Synthesis of intermediate 4d: In a dry round-bottom flask, compound 1,5-hexanediol 8 g was dissolved in dichloromethane, and p-toluenesulfonyl chloride 11.62 g and 8 mL of triethylamine were added at 0°C, and slowly warmed to room temperature, and reacted for 3 h; TLC technology was used to detect the reaction, 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 4d; Synthesis of intermediate 4e: In a round-bottom flask, 4d 500 mg was dissolved in N,N-dimethylformamide, K2CO3 382.2 mg, pomalidomide 402 mg were added to the reaction solution, and reacted at 90°C for 6 h; TLC method was used to monitor the reaction, and after the reaction solution was cooled to room temperature, water was added to quench, extracted with ethyl acetate for 3 times, collected the organic phase, washed with saturated NaCl aqueous solution for 3 times, dried with anhydrous Na2SO4; The crude product was concentrated under reduced pressure and vacuum, and purified by column chromatography, and the eluent system was dichloromethane:methanol = 150:1, volume ratio, to obtain compound 4e; Synthesis of intermediate 4f: In a dry round-bottom flask, compound 4e 50 mg was dissolved in dichloromethane, p-toluenesulfonyl chloride 56.02 mg and 0.05 mL of triethylamine were added at 0°C, and slowly warmed to room temperature, and reacted for 3 h; TLC technology was used to detect the reaction, and 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 4f; Synthesis of final product B6C: In a round-bottom flask, 4f 50 mg was dissolved in N,N-dimethylformamide, K2CO3 111.2 mg, BMS-303141 273.4 mg were added to the reaction solution, and reacted at 90°C for 6 h; TLC method was used to monitor the reaction, and after the reaction solution was cooled to room temperature, water was added to quench, extracted with ethyl acetate for 3 times, collected the organic phase, washed with saturated NaCl aqueous solution for 3 times, dried with anhydrous Na2SO4; The crude product was concentrated under reduced pressure and vacuum, and purified by column chromatography, and the eluent system was dichloromethane:methanol = 150:1, volume ratio, to obtain compound B6C; Alternatively, the synthesis steps of B7C are as follows: Synthesis of intermediate 5d: In a dry round-bottom flask, compound 1,5-heptanediol 8 g was dissolved in dichloromethane, and p-toluenesulfonyl chloride 10.38 g and 8 mL of triethylamine were added at 0°C, and slowly warmed to room temperature, and reacted for 3 h; TLC technology was used to detect the reaction, 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 5d; Synthesis of intermediate 5e: ​ In a round-bottom flask, 5d 300 mg was dissolved in N,N-dimethylformamide, K2CO3 156.2 mg, pomalidomide 282 mg were added to the reaction solution, and the reaction was carried out at 90°C for 6 h; the reaction was monitored by TLC method, and after the reaction solution was cooled to room temperature, water was added for quenching, and ethyl acetate was extracted for 3 times, the organic phase was collected, and the organic phase was washed with saturated NaCl aqueous solution for 3 times, and dried with anhydrous Na2SO4; the crude product was concentrated under reduced pressure and vacuum, and purified by column chromatography, and the eluent system was dichloromethane:methanol = 150:1, volume ratio, to obtain compound 5e; Synthesis of intermediate 5f In a dry round-bottom flask, compound 5e 50 mg was dissolved in dichloromethane, and p-toluenesulfonyl chloride 53.05 mg and 0.05 mL of triethylamine were added at 0°C, and slowly warmed to room temperature, and reacted for 3 h; the reaction was detected by TLC technology, and after the reaction was completed, the product was purified by column chromatography, and the eluent system was petroleum ether:ethyl acetate = 3:1, volume ratio, to obtain compound 5f; Synthesis of final product B7C: In a round-bottom flask, 5f 50 mg was dissolved in N,N-dimethylformamide, K2CO3 110.8 mg, BMS-303141 251.3 mg were added to the reaction solution, and the reaction was carried out at 90°C for 6 h; the reaction was monitored by TLC method, and after the reaction solution was cooled to room temperature, water was added for quenching, and ethyl acetate was extracted for 3 times, the organic phase was collected, and the organic phase was washed with saturated NaCl aqueous solution for 3 times, and dried with anhydrous Na2SO4; the crude product was concentrated under reduced pressure and vacuum, and purified by column chromatography, and the eluent system was dichloromethane:methanol = 150:1, volume ratio, to obtain compound B7C.

4. Use of the PROTAC compound of claim 1 in the preparation of a drug for targeted degradation of ACLY.

5. Use of the PROTAC compound of claim 1 in the preparation of a drug for resisting non-alcoholic fatty liver disease.

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