Synthesis of PROTACs compound containing pure carbon chain targeted ACLY and application of PROTACs compound in anti-NASH aspect

Abstract

The invention belongs to the technical field of biology and medicine, and discloses a PROTAC compound which is developed based on a PROTAC technology and takes a carbon chain as a connecting chain, the structural general formula of the PROTAC compound is 1: # imgabs0 #, in the formula, R is-(CH2) n-, and n is 3-8. 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 biology and pharmaceutical technology, and relates to the field of small molecule drug development, in particular to a PROTAC compound, method and application thereof developed based on PROTAC technology and having anti-NASH activity with a carbon chain as a connecting chain. Background Art

[0002] NAFLD (non-alcoholic fatty liver dis-ease, NAFLD) refers to excessive fat deposition in the liver caused by factors other than excessive drinking, viruses and other liver damage factors, including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). If NASH is not treated in time, it will develop into cirrhosis and even liver cancer and cause death. NASH is closely related to metabolic disorders such as obesity, insulin resistance, type 2 diabetes, and hyperlipidemia. It is more common in middle-aged people, especially overweight and obese individuals, and its clinical manifestations are fatty hepatitis and fatty liver cirrhosis. NASH is the progressive stage of NAFLD, and eventually develops into end-stage liver disease and hepatocellular carcinoma. It is considered to be one of the main causes of adult cirrhosis in the United States and the main cause of liver transplantation. However, there is only one marketed drug for NASH, Resmetirom, which targets THR-β. However, the disease mechanism of NASH is complex and involves multiple targets. Single-target therapeutic drugs are not enough to cope with the entire market. Therefore, developing an effective and safe drug for NASH is an urgent task and faces huge challenges.

[0003] The excessive fat accumulation of most NASH patients comes from the process of de novo fat synthesis. The raw material for de novo fat synthesis is acetyl-CoA, which is produced by the cleavage of citric acid under the action of 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 NASH and dyslipidemia. ACLY can be selected as a target to inhibit the production of acetyl-CoA and thus treat NASH.

[0004] Proteolysis-Targeting Chimeras (PROTACs) are a new type of small molecule protein targeted degradation technology, which consists of a target protein ligand, an E3 ligase ligand, and a linker. When the PROTAC molecule enters the cell, the intermediate connecting chain will bend, allowing the E3 ubiquitin ligase and the target protein to approach each other, and the ubiquitin carried by the E3 ligase ligand will be transferred to the substrate protein, causing the substrate protein to be ubiquitinated. The ubiquitinated protein will be recognized by the proteasome, 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 NASH.

[0005] Therefore, among the drugs targeting NASH, the development of ACLY protein degraders based on PROTAC technology to treat NASH has good prospects, bringing new hope for the treatment of NASH and broadening the application areas 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 a PROTAC compound, method and application developed based on PROTAC technology with anti-NASH activity and a carbon chain as a connecting chain.

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

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

[0009]

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

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

[0012]

[0013] The preparation method of the PROTAC compound as described above is prepared by reacting a compound of Formula 2 with a compound of Formula 3:

[0014]

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

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

[0017]

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

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

[0020]

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

[0022]

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

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

[0025]

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

[0027]

[0028] Further, Formula 6 is prepared by reacting Formula 5 and Formula 8:

[0029]

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

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

[0032]

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

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

[0035]

[0036] The use of the PROTAC compound as described above in the preparation of a drug 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] Furthermore, the PROTAC compound is combined with appropriate excipients to prepare a pharmaceutically acceptable corresponding 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 application relates to a PROTAC protein degrader targeting ACLY with a carbon chain as a connecting chain and its application. The problem to be solved by the present invention is to provide a new PROTAC drug for treating non-alcoholic fatty liver disease. The structural formula of the PROTAC molecule is shown in formula (I).

[0043]

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

[0045] 3. The purpose of the present invention is to solve the above technical problems and provide a class of ACLY protein targeting chimeras synthesized based on PROTAC technology and their application in anti-NASH activity. The present application discovered a class of PROTAC degraders targeting ACLY protein with a new structure and a structure as shown in Formula 1. Compared with the original drug BMS-303141, its cytotoxicity is greatly reduced. Among them, B7C has good lipid-lowering activity and ACLY protein degradation activity while alleviating liver inflammation. It is a new generation of low-toxic and high-efficiency new anti-NASH drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a graph showing the effects of compounds B3C to B8C of the present invention on lipid accumulation in L02 cells;

[0047] Figure 2 This is a graph showing the effects of compounds B3C to B8C of the present invention on the triglyceride content of L02 cells;

[0048] Figure 3 This is a graph showing the effects of compounds B3C to B8C of the present invention on the aspartate aminotransferase content of L02 cells;

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

[0050] Figure 5 This is a predicted map of the connection site between compound B7C and ACLY in the present invention. DETAILED DESCRIPTION

[0051] 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.

[0052] 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.

[0053] 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.

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

[0055]

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

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

[0058]

[0059] The preparation method of the PROTAC compound as described above is prepared by reacting a compound of Formula 2 with a compound of Formula 3:

[0060]

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

[0062] Further, Formula 2 is prepared by reacting Formula 4 and Formula 5:

[0063]

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

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

[0066]

[0067] Further, Formula 4 is prepared by reacting Formula 6 and Formula 7:

[0068]

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

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

[0071]

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

[0073]

[0074] Further, Formula 6 is prepared by reacting Formula 5 and Formula 8:

[0075]

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

[0077] The reaction formula is as follows:

[0078]

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

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

[0081]

[0082] The use of the PROTAC compound as described above in the preparation of a drug targeting the degradation of ACLY.

[0083] Alternatively, the targeted degradation of ACLY drug is an anti-nonalcoholic fatty hepatitis drug;

[0084] 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.

[0085] Alternatively, the PROTAC compound is combined with appropriate excipients to prepare a pharmaceutically acceptable preparation.

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

[0087] 1. Compounds synthesized by the present invention

[0088] 1. Preparation of the compound BMS-303141 of the present invention, the synthetic route is as follows:

[0089]

[0090] Further, the specific synthesis steps of BMS-33141 are as follows:

[0091] (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.

[0092] (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.

[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 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 petroleum ether: ethyl acetate = 1:1) to obtain compound BMS-303141.

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

[0095]

[0096] Further, 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, 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 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, and K2CO3 (156.2 mg, 1.2 eq) and pomalidomide (282 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 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, 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 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, and K2CO3 (93.5 mg, 1.2 eq) and BMS-303141 (250 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 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, 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 1d.

[0107] Synthesis of intermediate 1e

[0108]

[0109] In a round-bottom flask, 1d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide, and K2CO3 (342.2 mg, 1.2 eq) and pomalidomide (322 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 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 temperature was slowly raised to room temperature for 3 hours. 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 1f.

[0113] Synthesis of the final product B3C

[0114]

[0115] In a round-bottom flask, 1f (50 mg, 0.9 eq) was dissolved in N,N-dimethylformamide, 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, and 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 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 temperature was slowly raised to room temperature for 3 h. The reaction was detected using TLC technology, and after the reaction was completed, it was purified by column chromatography (eluent system petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain 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, and K2CO3 (274.5 mg, 1.2 eq) and pomalidomide (309 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 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 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 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, and 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 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 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 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 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, water was added to quench the reaction solution, 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 purified by column chromatography (eluent system dichloromethane: methanol = 150: 1, volume ratio) to obtain 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, 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 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, and 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 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 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 4d.

[0155] Synthesis of intermediate 4e

[0156]

[0157] In a round-bottom flask, 4d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide, and K2CO3 (382.2 mg, 1.2 eq) and pomalidomide (402 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 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 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 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, and 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 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-heptanediol (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 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 5d.

[0171] Synthesis of intermediate 5e

[0172]

[0173] In a round-bottom flask, 5d (300 mg, 1 eq) was dissolved in N,N-dimethylformamide, and K2CO3 (156.2 mg, 1.2 eq) and pomalidomide (282 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 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 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 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, and 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 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 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 6d.

[0187] Synthesis of intermediate 6e

[0188]

[0189] In a round-bottom flask, 6d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide, and K2CO3 (173.2 mg, 1.2 eq) and pomalidomide (363.2 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. 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 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 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 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, and 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 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 the Invention

[0198] 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.

[0199] Experimental Example 1: Cytotoxicity test of PROTAC compounds

[0200] 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.

[0201] 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.

[0202] 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%.

[0203] 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.

[0204] The results are shown in Tables 1 and 2. Compared with the warhead BMS-303141, the six newly synthesized PROTAC compounds synthesized in the present invention have less cytotoxicity than the warhead BMS-303141 at a concentration below 100 μM. BMS-303141 has an IC of 2.0 in L02 cells. 50 The value is 29.25 μM, while the synthesized PROTACs compounds have no cytotoxicity in the range of 10 to 100 μM, achieving the function of attenuation.

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

[0206]

[0207] Table 2 Cytotoxicity of PROTAC compounds on HepG2, L02, and HUVEC cells

[0208]

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

[0210] Solution preparation:

[0211] ① Preparation of Oil Red O dye solution

[0212] 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.

[0213] 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.

[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 (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.

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

[0217] Accurately weigh 5 g of bovine serum albumin (BSA) and add it to a complete culture medium preheated at 55° C. to dissolve it, thereby preparing a culture medium containing 1% bovine serum albumin.

[0218] 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 at 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.

[0219] Specific experimental steps:

[0220] 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, 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 the 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 concentrations of the PROTAC compounds to be tested were 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.

[0221] 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 (MM, mass percentage) was added to fix for 40 minutes, and 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. 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.

[0222] The TG content was detected according to the instructions of the kit, as shown in Table 3.

[0223] Table 3 Test method for TG content

[0224]

[0225] The standard substance is the calibration substance included in the kit.

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

[0227]

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

[0229] 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.

[0230] Table 4 Test method for AST content

[0231]

[0232] The effects of BMS-303141 and PROTAC compounds on the morphological changes of FFAs-induced NASH 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, B6C and B7C reduce intracellular lipid accumulation, and the amount of lipid accumulation reduced by B6C and B7C is less 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.

[0233] B6C and B7C were initially screened out as effective compounds.

[0234] The effects of BMS-303141 and PROTAC compounds on the TG content of FFAs-induced NASH cell models were investigated. Figure 2 As shown in the figure, it can be seen that the TG content of the blank group is very low. After stimulation with FFAs, the TG content of the model group increased significantly, and the positive control fenofibric acid can reverse the increase in TG content induced by FFAs. After adding BMS-303141 or PROTAC compound at a concentration of 20 μmol / L, the TG content of BMS-303141 was 0.26mmol / gprot, the TG content of B6C was 0.19mmol / gprot, and the TG content of B7C was 0.13mmol / gprot. BMS-303141, B6C and B7C had obvious lipid-lowering activity, and the lipid-lowering activity of B6C and B7C was stronger than that of the warhead BMS-313141. Combined with the above data, it is further verified that the lipid-lowering activity of the PROTACs compound synthesized by the present invention is stronger than that of the original drug BMS-303141.

[0235] The effects of BMS-303141 and PROTAC compounds on AST content in FFAs-induced NASH cell models were investigated. Figure 3As shown in the figure, it can be seen that the AST content of the blank group is very low, with an AST value of 27.88U / gprot. After stimulation with FFAs, the AST content of the model group increased significantly, with an AST value of 76.25U / gprot. The positive drug fenofibric acid and the original drug BMS-303141 can reduce the AST content, among which the AST value of fenofibric acid is 58.7U / gprot and the AST value of BMS-303141 is 59.12U / gprot, but the effect is not significant. After adding B7C at a concentration of 20μmol / L, the AST value of B7C was 31.93U / gprot, which is equivalent to the blank group, significantly reducing liver damage. From the above data, it can be seen that the lipid-lowering activity of the PROTACs compound we synthesized is stronger than that of the original drug BMS-303141, and can reduce liver damage to a certain extent.

[0236] Experimental Example 3: ACLY degradation ability test of PROTAC compounds

[0237] ① Protein sampling

[0238] L02 cells were cultured at 1×10 5 Cells / 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 to continue culturing for 12 hours in the same environment to starve the cells. After 12 hours, the serum-free medium was taken, fenofibric acid (20μmol / L) was used as the positive drug, the concentration of BMS-303141 was 20μmol / L, and the concentration of B7C was 10, 20, 40, 80, 100μM. DMSO, fenofibrate and B7C 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, and 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.

[0239] ② Protein quantification

[0240] The protein standard curve was drawn and the protein concentration was determined using the BSA method. CHAPS protein lysate and bovine serum albumin were prepared 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 according to the corresponding proportions and used 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 were allowed to form 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.

[0241] Table 5 BSA standard dilution standard

[0242] 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

[0243] Dilute the protein sample with CHAPS 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℃.

[0244] The experimental results are as follows Figure 4 As shown in the figure, compared with the model group, BMS-303141 and B7C both had degradation effects on ACLY, while the positive drug fenofibric acid had no degradation ability on ACLY. Compared with the warhead BMS-303141, B7C had a significantly improved ability to degrade ACLY at a concentration of 20 μM, and the degradation ability of B7C on ACLY was concentration-dependent.

[0245] Experimental Example 4: Prediction of the binding site between PROTAC compounds and ACLY

[0246] The Autodock software was used to simulate the docking of B7C and ACLY (PDB code: 3WME). Figure 5 As shown, the warhead end of B7C is in the active pocket of ACLY, while the linker chain and pomalidomide end are exposed to the solvent to facilitate the connection of the other end with pomalidomide, which is consistent with the design concept of RPOTAC.

[0247] 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 and enhance its targeting. Based on this result, it is expected that compounds with better activity will be obtained by modifying the original drug, trying more types of connection chains, and increasing the types of E3 ubiquitin ligases.

[0248] 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. A PROTAC compound with a carbon chain as a connecting chain developed based on PROTAC technology, characterized in that: Its general structural formula is Formula 1: In the formula, R is -(CH2) n -, n is 3 to 8.

2. The PROTAC compound according to claim 1, characterized in that: The structure of the compound is specifically one of the following:

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

4. The preparation method according to claim 3, characterized in that: The synthesis steps of compound B3C are as follows: Synthesis of intermediate 1d: 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, 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 = 1:1, volume ratio, to obtain compound 1d. Synthesis of intermediate 1e: In a round-bottom flask, 1d (500 mg, 1 eq) was dissolved in N,N-dimethylformamide, and K2CO3 (342.2 mg, 1.2 eq) and pomalidomide (322 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. The eluent system was dichloromethane: methanol = 150:1, volume ratio, to obtain compound 1e. Synthesis of intermediate 1f: 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 temperature was slowly raised to room temperature for 3 h. The reaction was detected by TLC technology, and after the reaction was completed, column chromatography was used for purification, with the eluent system of petroleum ether: ethyl acetate = 3:1, volume ratio, to obtain compound 1f; Synthesis of the final product B3C: In a round-bottom flask, 1f (50 mg, 0.9 eq) was dissolved in N,N-dimethylformamide, 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, and 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, and the eluent system was dichloromethane: methanol = 150:1, volume ratio, to obtain compound B3C; Alternatively, the synthesis steps of B4C are as follows: Synthesis of intermediate 2d In a dry round-bottom flask, compound 1,4-butanediol (8 g, 1 eq) was dissolved in dichloromethane, and p-toluenesulfonyl chloride (15.23 g, 0.9 eq) and 8 mL of triethylamine were added at 0°C. The temperature was slowly raised to room temperature and the reaction was carried out for 3 h. The reaction was detected by TLC technology. After the reaction was completed, the mixture was purified by column chromatography with the eluent system of petroleum ether: ethyl acetate = 1:1, volume ratio, to obtain compound 2d. Synthesis of intermediate 2e: 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 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, with the eluent system of dichloromethane:methanol = 150:1, volume ratio, to obtain compound 2e. Synthesis of intermediate 2f: 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 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, with the eluent system of petroleum ether: ethyl acetate = 3:1, volume ratio, to obtain compound 2f; Synthesis of the final product B4C: 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, 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 B4C; Alternatively, the synthesis steps of B5C are as follows: Synthesis of intermediate 3d: 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 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, the compound was purified by column chromatography, and the eluent system was petroleum ether: ethyl acetate = 1:1, volume ratio, to obtain compound 3d; Synthesis of intermediate 3e: 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 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. 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, and the eluent system was dichloromethane: methanol = 150: 1, volume ratio, to obtain compound 3e; Synthesis of intermediate 3f 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, 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 3f. Synthesis of the final product B5C 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, 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 B5C; Alternatively, the synthesis steps of B6C are as follows: Synthesis of intermediate 4d: 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 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 = 1:1, volume ratio, to obtain compound 4d. Synthesis of intermediate 4e: 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 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. 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, 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, 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 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 4f. Synthesis of the final product B6C: 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, and 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, 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, 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 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, the compound was purified by column chromatography (eluent system: petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain compound 5d; Synthesis of intermediate 5e: 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 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 5e; Synthesis of intermediate 5f 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 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 5f. Synthesis of the final product B7C: 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, 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 B7C; Alternatively, the B8C synthesis steps are as follows: Synthesis of intermediate 6d: 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 temperature was slowly raised to room temperature and 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 6d; Synthesis of intermediate 6e: 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 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 6e; Synthesis of intermediate 6f: 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 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 6f. Synthesis of the final product B8C: 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, 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 B8C.

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

6. Use of the PROTAC compound according to claim 1 or 2 in the preparation of an anti-non-alcoholic fatty hepatitis drug.

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