PROTAC compound for targeted degradation of AR / AR-V7 as well as preparation method and application of PROTAC compound

Abstract

The invention relates to a PROTAC compound for targeted degradation of AR / AR-V7, the general formula of the PROTAC compound is # imgabs0 #, and R is-CN,-NO2 or-CI; the Linker is a linking group and comprises an alkylene group or an alkoxy group. The PROTAC compound is innovatively designed, the limitation of a traditional small-molecule inhibitor is overcome by inducing degradation of target protein, and a brand new path is provided for prostate cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to a PROTAC compound for targeted degradation of AR / AR-V7 and a preparation method and application thereof. Background Art

[0002] Prostatic cancer (PCa) affects millions of men worldwide and is the second most common cancer in men worldwide. With the aging of the population and changes in dietary structure in my country, the incidence and mortality of prostate cancer have increased year by year, and the situation is grim. Due to many factors such as point mutations in the androgen receptor ligand binding domain and expression of splice variants, existing prostate cancer treatments have become ineffective one after another, so new strategies are urgently needed for the treatment of prostate cancer. In recent years, targeted protein degradation technology has made great progress. Among them, androgen receptor degraders block the androgen receptor signaling pathway by promoting the degradation of androgen receptors, which is expected to solve the problem of drug resistance in clinical prostate cancer patients.

[0003] In the process of vigorous development in the field of biomedicine, technological progress often promotes the innovation of concepts. In turn, the upgrading of concepts also accelerates the change of technology. As a result, a new technology - Proteolysis Targeting Chimera (PROTAC) came into being. As the name suggests, PROTAC is a type of chimeric small molecule with multiple functional groups. It shows the effect of selective degradation of target proteins in biological activity. Due to its unique mode of action, it can make up for the shortcomings of traditional drug treatment, thereby accelerating its development.

[0004] However, the current research in this area is insufficient and further studies on such compounds are needed. Summary of the invention

[0005] Under this background, the present invention proposes a PROTAC compound for targeted degradation of AR / AR-V7 and a preparation method and application thereof. The PROTAC compound has a significant target protein degradation effect on drug-resistant cells 22RV1 and a significant anti-tumor activity effect on tumor-resistant cells.

[0006] In order to overcome at least one of the above disadvantages of the prior art, on the one hand, the present invention provides a PROTAC compound for targeting degradation of AR / AR-V7, the general formula of which is as follows

[0007] Wherein, said R is -CN, -NO2 or -CI; The linker is a connecting group, including an alkylene group or an alkoxy group.

[0008] Further, the alkylene group is any one of -(CH2)n-, alkenylene, alkynylene or cycloalkylene; The alkoxy group is -(CH2) n CO-, -(CH2CH2O) n -or- (CH2CH2O) n Any one of CH2CO-; wherein n represents a natural number from 1 to 6.

[0009] Furthermore, the PROTAC compound is selected from any one of the following structures:

[0010] The second aspect of the present invention provides a method for preparing a PROTAC compound that targets and degrades AR / AR-V7. Wherein, the preparation method of the compound A4a, A4b, A4c or A4d is as follows: 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, nucleophilic substitution occurs under alkaline conditions of potassium carbonate and connects with the secondary amine of 5,5-dimethylhydantoin to obtain intermediate A1; 2) Under the alkaline condition of NaH, different lengths of aliphatic carbon chains with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergo affinity substitution with intermediate A1 to obtain intermediate A2a, A2b, A2c or A2d; 3) The intermediate A2a, A2b, A2c or A2d is subjected to trifluoroacetic acid to remove the Boc to obtain the intermediate A3a, A3b, A3c or A3d; 4) The intermediate A3a, A3b, A3c or A3d undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A4a, A4b, A4c or A4d.

[0011] The preparation method of the compound A7a, A7b or A7c is as follows: 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, the intermediate A1 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, polyethylene glycol chains of different lengths with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end are subjected to affinity substitution with intermediate A1 to obtain intermediate A5a, A5b or A5c; 3) After intermediate A5a, A5b or A5c is subjected to trifluoroacetic acid to remove Boc, intermediate A7a, A7b or A7c is obtained; 4) The intermediate A7a, A7b or A7c undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A7a, A7b or A7c.

[0012] The preparation method of the compound A11a, A11b, A11c or A11d is: 1) Using 2-nitro-5-fluorobenzotrifluoride as the starting material, the intermediate A8 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, different lengths of aliphatic carbon chains with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergo affinity substitution with intermediate A8 to obtain intermediate A9a, A9b, A9c or A9d; 3) intermediate A9a, A9b, A9c or A9d after removal of Boc by trifluoroacetic acid to give intermediate A10a, A10b, A10c or A10d; 4) The intermediate A10a, A10b, A10c or A10d undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A11a, A11b, A11c or A11d.

[0013] The preparation method of the compound A14a, A14b or A14c is as follows: 1) Using 2-nitro-5-fluorobenzotrifluoride as the starting material, the intermediate A8 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, a polyethylene glycol chain of different lengths with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end is subjected to affinity substitution with the intermediate A8 to obtain the intermediate A12a, A12b or A12c; 3) After the intermediate A12a, A12b or A12c is stripped of Boc by trifluoroacetic acid, the intermediate A13a, A13b or A13c is obtained; 4) The intermediate A13a, A13b or A13c undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A14a, A14b or A14c.

[0014] The preparation method of the compound A17a or A17b is as follows: 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, the intermediate A1 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, a polycyclic chain with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergoes affinity substitution with intermediate A1 to obtain intermediate A15a or A15b; 3) After intermediate A15a or A15b is stripped of Boc by trifluoroacetic acid, intermediate A16a or A16b is obtained; 4) The intermediate A16a or A16b undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A17a or A17b.

[0015] The third aspect of the present invention provides a use of the above-mentioned PROTAC compound for targeted degradation of AR / AR-V7 in the preparation of anti-tumor drugs.

[0016] Furthermore, the application is the use of PROTAC compounds that target the degradation of AR / AR-V7 in anti-prostate cancer, lung cancer or liver cancer drugs.

[0017] The beneficial effects of the present invention are: The present invention innovatively designs a PROTAC compound that targets and degrades AR / AR-V7. The compound has a strong proliferation inhibitory effect on 22RV1 cells by inducing the degradation of the target protein, and is concentration-dependent. It has no proliferation inhibitory effect on 3T3 cells, indicating that the compound provided by the present invention has the effect of targeted degradation of AR, overcomes the limitations of traditional small molecule inhibitors, and provides a new path for the treatment of prostate cancer. Experiments show that the PROTAC compound can efficiently and selectively degrade AR and AR-V7, inhibit the proliferation of prostate cancer cells, and exhibit good anti-tumor activity in in vitro experiments, laying the foundation for the development of new drugs for the treatment of prostate cancer and other androgen receptor-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0019] Figure 1 The degradation activity results of compounds A4a-d, A7a-c, A11a-d, A14a-c and A17a-b on AR / AR-V7 protein in 22rv1 cells. DETAILED DESCRIPTION

[0020] It should be mentioned before discussing the exemplary embodiments in more detail that some exemplary embodiments are described as processes or methods depicted as flow charts. Although the flow charts describe the operations as sequential processes, many of the operations therein can be implemented in parallel, concurrently or simultaneously. In addition, the order of the operations can be rearranged. The process can be terminated when its operation is completed, but can also have additional steps not included in the accompanying drawings. The process can correspond to a method, function, procedure, subroutine, subprogram, etc.

[0021] It should be understood that, although the terms "first", "second", etc. may be used herein to describe various units, these units should not be limited by these terms. These terms are used only to distinguish one unit from another unit. For example, without departing from the scope of the exemplary embodiments, the first unit may be referred to as the second unit, and similarly the second unit may be referred to as the first unit. The term "and / or" used herein includes any and all combinations of one or more of the listed associated items.

[0022] A PROTAC compound targeting degradation of AR / AR-V7, the general formula is as follows

[0023] Wherein, said R is -CN, -NO2 or -CI; The linker is a connecting group, including an alkylene group or an alkoxy group.

[0024] Further, the alkylene group is any one of -(CH2)n-, alkenylene, alkynylene or cycloalkylene; The alkoxy group is -(CH2) n CO-, -(CH2CH2O) n -or- (CH2CH2O) n Any one of CH2CO-; wherein n represents a natural number from 1 to 6.

[0025] Furthermore, the PROTAC compound is selected from any one of the following structures: .

[0026] A method for preparing a PROTAC compound for targeted degradation of AR / AR-V7, Wherein, the preparation route of the compound A4a, A4b, A4c or A4d is

[0027] Scheme1-1 Among them, a: K2CO3, DMF, 45℃, 24h; b: NaH, DMF, 55℃, 12h; c: TFA, DCM,rt, 2h; d: DIPEA, NMP, 90℃, 18h. The preparation method is 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, nucleophilic substitution occurs under alkaline conditions of potassium carbonate and connects with the secondary amine of 5,5-dimethylhydantoin to obtain intermediate A1; 2) Under the alkaline condition of NaH, different lengths of aliphatic carbon chains with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergo affinity substitution with intermediate A1 to obtain intermediate A2a, A2b, A2c or A2d; 3) intermediate A2a, A2b, A2c or A2d after removal of Boc by trifluoroacetic acid to give intermediate A3a, A3b, A3c or A3d; 4) The intermediate A3a, A3b, A3c or A3d undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A4a, A4b, A4c or A4d.

[0028] The synthetic route of the compound A7a-c is:

[0029] Scheme 1-2 Among them, a: K2CO3, DMF, 45℃, 24h; b: NaH, DMF, 55℃, 12h; c: TFA, DCM,rt, 2h; d: DIPEA, NMP, 90℃, 18h. The preparation method is 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, the intermediate A1 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, polyethylene glycol chains of different lengths with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end are subjected to affinity substitution with intermediate A1 to obtain intermediate A5a, A5b or A5c; 3) After intermediate A5a, A5b or A5c is stripped of Boc by trifluoroacetic acid, intermediate A6a, A6b or A6c is obtained; 4) The intermediate A6a, A6b or A6c undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A7a, A7b or A7c.

[0030] The synthetic route of the compound A11a, A11b, A11c or A11d is:

[0031] Scheme 1-3 Among them, a: K2CO3, DMF, 45℃, 24h; b: NaH, DMF, 55℃, 12h; c: TFA, DCM,rt, 2h; d: DIPEA, NMP, 90℃, 18h. The preparation method is 1) Using 2-nitro-5-fluorobenzotrifluoride as the starting material, the intermediate A8 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, different lengths of aliphatic carbon chains with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergo affinity substitution with intermediate A8 to obtain intermediate A9a, A9b, A9c or A9d; 3) The intermediate A9a, A9b, A9c or A9d is treated with trifluoroacetic acid to remove the Boc to obtain the intermediate A10a, A10b, A10c or A10d; 4) The intermediate A10a, A10b, A10c or A10d undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A11a, A11b, A11c or A11d.

[0032] The synthetic route of the compound A14a-c is:

[0033] Scheme 1-4 Among them, a: K2CO3, DMF, 45℃, 24h; b: NaH, DMF, 55℃, 12h; c: TFA, DCM,rt, 2h; d: DIPEA, NMP, 90℃, 18h. The preparation method is 1) Using 2-nitro-5-fluorobenzotrifluoride as the starting material, the intermediate A8 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, a polyethylene glycol chain of different lengths with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end is subjected to affinity substitution with the intermediate A8 to obtain the intermediate A12a, A12b or A12c; 3) The intermediate A12a, A12b or A12c is subjected to trifluoroacetic acid to remove the Boc to obtain the intermediate A13a, A13b or A13c; 4) The intermediate A13a, A13b or A13c undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A14a, A14b or A14c.

[0034] The synthetic route of the compound A17a-b is

[0035] Scheme 1-5 Among them, a: K2CO3, DMF, 45℃, 24h; b: NaH, DMF, 55℃, 12h; c: TFA, DCM,rt, 2h; d: DIPEA, NMP, 90℃, 18h. The preparation method is 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, the intermediate A1 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, a polycyclic chain with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergoes affinity substitution with intermediate A1 to obtain intermediate A15a or A15b; 3) After the intermediate A15a or A15b is subjected to trifluoroacetic acid to remove the Boc, the intermediate A16a or A16b is obtained; 4) The intermediate A16a or A16b undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A17a or A17b.

[0036] Example 1 Preparation of 4-(3-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A4a) Step 1: Preparation of 4-(4,4-dimethyl-2,5-dioxoimidazolin-1-yl)-2-trifluoromethylbenzonitrile (A1): 5,5-dimethylhydantoin (12.80 g, 100.0 mmol) was dissolved in DMF (30.0 ml), K2CO3 (4.2 g, 30 mmol) was added, and the mixture was stirred at room temperature for 30 min. 2-cyano-5-fluorotrifluorotoluene (3.78 g, 20.0 mmol) was slowly added, and the temperature was raised to 45°C after the addition was completed. After TLC detection, the reaction solution was poured into 300 ml of water, extracted three times with ethyl acetate (100 ml*3), the ethyl acetate phases were combined, washed once with saturated brine (200 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and separated by silica gel column chromatography to obtain compound A1 (4.98 g, white solid, 56% yield).

[0037] 1 H NMR (400 MHz, DMSO) δ 8.86 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.19(d, J = 2.1 Hz, 1H), 8.03 (dd, J = 8.4, 2.0 Hz, 1H), 1.43 (s, 6H). 13 C NMR (101MHz, DMSO) δ 175.78, 153.01, 137.02, 136.06, 131.23, 130.04, 124.14, 123.66,120.94, 115.29, 106.59, 58.01, 24.62. Step 2: Preparation of tert-butyl (2-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)ethyl)carbamate (A2a): Under nitrogen protection, compound A1 (100 mg, 0.34 mmol) was dissolved in DMF (2 ml), cooled to 0°C, and then NaH (21 mg, 0.51 mmol, 60% content) was slowly added and stirred at room temperature for 1-2 h, followed by slow addition of N-BOC-bromoethylamine (114 mg, 0.51 mmol), and the temperature was raised to 55°C after the addition was complete. After TLC detection, the reaction solution was poured into 20 ml of water, extracted three times with ethyl acetate (10 ml*3), the ethyl acetate phases were combined, washed once with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and separated by silica gel column chromatography to obtain compound A2a (121 mg, white solid, 81% yield).

[0038] 1 H NMR (400 MHz, DMSO) δ 8.33 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 2.1 Hz,1H), 8.04 (dd, J = 8.3, 2.2 Hz, 1H), 6.97 (t, J = 6.0 Hz, 1H), 3.32 (d, J = 6.2 Hz,2H), 3.22 (q, J = 6.5 Hz, 2H), 1.44 (s, 6H), 1.37 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 174.65, 155.67, 152.61, 136.81, 136.17, 131.27, 130.95, 129.88,123.89, 123.64, 120.92, 118.44, 115.24, 106.70, 92.72, 77.85, 61.62, 38.37,28.23, 22.41. Step 3: Preparation of 4-(3-(2-aminoethyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A3a): Compound A2a (100 mg, 0.227 mmol) was dissolved in DCM (3 ml), trifluoroacetic acid (1 ml) was slowly added dropwise, and the mixture was stirred at room temperature for 2 h. After TLC detection, the reaction was completed and the mixture was concentrated under reduced pressure to obtain compound A3a (71 mg, 0.209 mmol, 91% yield), which was used directly in the next step without further purification.

[0039] Step 4: Preparation of 4-(3-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A4a): Compound A3a (71 mg, 0.209 mmol) and DIPEA (29.7 mg, 0.223 mmol) were added to NMP (3 ml), stirred at room temperature for 30 min, and then 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (63.5 mg, 0.223 mmol) was slowly added. After the addition was completed, the temperature was raised to 90°C. After the reaction was completed by TLC detection, the reaction solution was poured into 30 ml of water, extracted three times with ethyl acetate (10 ml*3), the ethyl acetate phases were combined, washed once with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and separated by silica gel column chromatography to obtain compound A4a (102 mg, yellow solid, 82% yield).

[0040] 1 H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 2.0 Hz, 1H), 7.99 (dd, J = 8.5, 2.0Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.56 (dd, J = 8.6, 7.2 Hz, 1H), 7.16 (d, J = 7.1Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 4.91 (dd, J = 12.0, 5.4 Hz, 1H), 3.67 (d, J =6.6 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 2.78 – 2.71 (m, 2H), 2.18 – 2.00 (m,2H), 1.54 – 1.52 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 174.32, 171.18, 168.44,167.51, 153.63, 151.30, 136.71, 135.56, 132.72, 128.21, 123.24, 121.11,116.53, 112.61, 110.89, 108.66, 101.25, 91.98, 70.81, 62.16, 54.12, 49.08,41.24, 39.49, 31.53, 26.02, 23.53, 22.83.HRMS (ESI) m / z Calculated forC 28 H 23 F3N6O6 + [M+H] + 596.1631, found 596.1533. Example 2 The preparation of 4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)propyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A4b) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0041] Step 2: The preparation of tert-butyl (3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)propyl)carbamate (A2b) was carried out in the same manner as in Step 2 of Example 1, except that N-Boc-3-aminopropyl bromide was used instead of N-BOC-bromoethylamine.

[0042] 1 H NMR (400 MHz, DMSO) δ 8.31 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 2.1 Hz,1H), 8.03 (dd, J = 8.4, 2.0 Hz, 1H), 6.88 (d, J = 6.0 Hz, 1H), 3.31 (d, J = 7.7 Hz,2H), 2.99 (q, J = 6.5 Hz, 2H), 1.74 (p, J = 7.2 Hz, 2H), 1.46 (s, 6H), 1.38 (s, 9H). 13C NMR (101 MHz, DMSO) δ 174.69, 155.65, 152.58, 136.88, 136.07, 131.20,130.88, 130.03, 124.13, 124.08, 123.65, 120.93, 115.26, 106.66, 77.57, 61.69,37.65, 37.38, 29.39, 28.27, 22.49. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0043] 1 H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.99(dd, J = 8.5, 2.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.56 (dd, J = 8.6, 7.2 Hz,1H), 7.16 (d, J = 7.1 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 4.91 (dd, J = 12.0, 5.4 Hz, 1H), 3.67 (d, J = 6.6 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 2.91 –2.78 (m, 2H), 2.78 – 2.70 (m, 2H), 2.15 – 2.03 (m, 2H), 1.54 – 1.51 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 174.32, 171.19, 169.54, 168.49, 167.51, 153.63,150.73, 146.25, 145.38, 136.70, 135.56, 132.72, 128.21, 125.61, 116.53,115.12, 112.61, 110.88, 108.66, 100.77, 98.42, 62.16, 49.07, 41.24, 39.49,38.09, 31.53, 23.53, 22.84. HRMS (ESI) m / z Calculated for C 29 H 25 F3N6O6 + [M+H] + 610.1788, found 610.1897. Example 3 The preparation method of 4-(3-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)butyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A4c) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0044] Step 2: The preparation of tert-butyl (4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)butyl)carbamate (A2c) was the same as in Step 2 of Example 1, except that tert-butyl N-(4-bromobutyl)carbamic acid was used instead of N-BOC-bromoethylamine.

[0045] 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 2.0 Hz, 1H), 7.99 (dd, J = 8.4, 2.0Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 3.40 – 3.34 (m, 2H), 3.18 (t, J = 6.8 Hz, 2H),1.78 – 1.68 (m, 2H), 1.57 (dd, J = 8.4, 6.4 Hz, 2H), 1.52 (s, 6H), 1.43 (s, 9H). The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0046] 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 2.0 Hz, 1H), 7.99 (dd, J = 8.5, 2.0Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 7.1 Hz,1H), 6.89 (d, J = 8.5 Hz, 1H), 6.25 (t, J = 5.8 Hz, 1H), 4.90 (dd, J = 12.1, 5.3Hz, 1H), 3.41 (t, J = 7.3 Hz, 2H), 3.35 (q, J = 6.5 Hz, 2H), 2.92 – 2.80 (m, 2H), 2.80 – 2.67 (m, 2H), 2.16 – 2.08 (m, 1H), 1.83 (q, J = 8.3 Hz, 2H), 1.76 (q, J =7.2 Hz, 2H), 1.54 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 180.99, 174.63, 171.14,169.65, 168.49, 167.63, 153.08, 146.84, 136.39, 135.43, 132.61, HRMS (ESI) m / z Calculated for C 30 H 27 F3N6O6 + [M+H] + 624.1944, found 624.1836. Example 4 The preparation method of 4-(3-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)hexyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A4d) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0047] Step 2: The preparation of tert-butyl (6-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)hexyl)carbamate (A2d) was the same as in Step 2 of Example 1, except that tert-butyl (6-bromohexyl)carbamate was used instead of N-BOC-bromoethylamine.

[0048] 1 H NMR (400 MHz, DMSO) δ 8.31 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 1.9 Hz,1H), 8.03 (dd, J = 8.4, 2.0 Hz, 1H), 6.79 (t, J = 5.7 Hz, 1H), 3.29 (t, J = 7.7 Hz,2H), 2.89 (q, J = 6.5 Hz, 2H), 1.64 – 1.56 (m, 2H), 1.45 (s, 6H), 1.36 (s, 9H), 1.28 (tt, J = 10.4, 4.9 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 202.86, 174.77,166.69, 155.63, 152.52, 136.92, 136.08, 131.22, 130.90, 130.03, 124.13,124.08, 123.67, 120.95, 115.29, 106.64, 77.35, 61.67, 29.43, 28.95, 28.30,26.06, 25.99, 22.58. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0049] 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 2.1 Hz, 1H), 8.00 (dd,J = 8.4, 2.1Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.48 (dd, J = 8.5, 7.1 Hz, 1H), 7.07 (d, J = 7.1Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 6.23 (t, J = 5.7 Hz, 1H), 4.90 (dd, J = 11.9,5.4 Hz, 1H), 3.38 – 3.31 (m, 2H), 3.27 (q, J = 6.5 Hz, 2H), 2.91 – 2.66 (m,3H), 2.11 (dd, J = 11.8, 5.0 Hz, 1H), 1.71 (dp, J = 14.6, 7.4 Hz, 5H), 1.50 –1.40 (m, 4H). 13 C NMR (101 MHz, CDCl3) δ 174.77, 171.25, 169.65, 168.58,167.69, 152.91, 147.02, 136.59, 136.27, 135.38, 133.81, 132.59, 128.01,123.44, 123.06, 120.72, 116.71, 115.16, 111.59, 110.02, 108.27, 61.98, 48.96,42.59, 40.40, 31.50, 29.54, 29.19, 26.88, 26.68, 23.61, 22.90.HRMS (ESI) m / zCalculated for C 32 H 31 F3N6O6 + [M+H] + 652.2257, found 652.2158. Example 5 The preparation method of 4-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)ethyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A7a) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0050] Step 2: The preparation of tert-butyl (2-(2-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)ethoxy)ethyl)carbamate (A5a) was carried out in the same manner as in Step 2 of Example 1, except that tert-butyl bromo-monopolyethylene glycol-amino was used in place of N-BOC-bromoethylamine.

[0051] 1 H NMR (400 MHz, DMSO) δ 8.32 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 2.1 Hz,1H), 8.04 (dd, J = 8.4, 2.0 Hz, 1H), 6.80 (t, J = 5.7 Hz, 1H), 3.58 (t, J = 6.0 Hz,2H), 3.48 (t, J = 6.2 Hz, 2H), 3.42 (d, J = 5.8 Hz, 2H), 3.08 (q, J = 5.9 Hz, 2H),1.45 (s, 6H), 1.36 (d, J = 9.4 Hz, 9H). 13 C NMR (101 MHz, DMSO) δ 194.48, 188.84,174.81, 155.66, 152.78, 149.05, 136.83, 136.14, 131.28, 130.09, 124.17,120.95, 115.29, 106.79, 77.65, 69.17, 67.66, 61.66, 36.32, 28.26, 22.60. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0052] 1 H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 2.1 Hz, 1H), 7.96 (dd, J = 8.4, 2.0Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.50 (dd, J = 8.6, 7.1 Hz, 1H), 7.10 (d,J = 7.1Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.49 (t, J = 5.5 Hz, 1H), 4.77 (dd, J = 12.4,5.4 Hz, 1H), 3.79 (dq, J = 14.6, 4.9 Hz, 2H), 3.74 (t, J = 5.2 Hz, 2H), 3.63 –3.54 (m, 2H), 3.47 (q, J = 5.3 Hz, 2H), 3.38 (t, J = 7.1 Hz, 1H), 2.81 (d, J = 23.7Hz, 2H), 2.70 – 2.58 (m, 2H), 2.37 (t, J = 8.1 Hz, 1H), 1.51 (d, J = 3.3 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 191.55, 180.97, 174.78, 170.92, 169.47, 168.22,153.17, 146.63, 136.27, 135.31, 132.51, 128.21, 119.06, 116.67, 115.03,111.96, 108.59, 69.13, 68.21, 62.11, 49.48, 48.83, 42.17, 40.39, 31.34,30.73, 23.43, 22.67, 17.68, 12.28. HRMS (ESI) m / z Calculated for C 30 H 27 F3N6O7 + [M+H] + 640.1893, found 640.1794. Example 6 The preparation method of 4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethoxy)ethyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A7b) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0053] Step 2: The preparation of tert-butyl (2-(2-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)ethoxy)ethyl)carbamate (A5b) was carried out in the same manner as in Step 2 of Example 1, except that tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate was used instead of N-BOC-bromoethylamine.

[0054] 1 H NMR (400 MHz, DMSO) δ 8.32 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 2.1 Hz,1H), 8.04 (dd, J = 8.4, 2.0 Hz, 1H), 6.76 (t, J = 5.7 Hz, 1H), 3.60 (t, J = 6.1 Hz,2H), 3.55 (dd, J = 6.2, 3.4 Hz, 2H), 3.50 (dd, J = 6.5, 3.9 Hz, 4H), 3.36 (d, J =5.9 Hz, 2H), 3.04 (q, J = 6.1 Hz, 2H), 1.46 (s, 6H), 1.36 (s, 9H). 13 C NMR (101MHz, DMSO) δ 202.60, 174.59, 170.85, 155.41, 152.57, 136.61, 135.93, 131.05,130.73, 129.86, 123.94, 123.89, 123.45, 120.73, 115.06, 106.56, 77.43, 69.50,69.30, 69.01, 67.82, 61.44, 28.04, 22.37 The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0055] 1 H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 8.13 (s, 1H), 7.98 (d, J = 8.4Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.49 (t,J = 7.8 Hz, 1H), 7.11 (d, J = 7.1 Hz,1H), 6.90 (d, J = 8.5 Hz, 1H), 4.90 (dd, J = 11.7, 5.4 Hz, 1H), 4.12 (q, J = 7.2Hz, 1H), 3.73 (dt, J = 10.3, 5.3 Hz, 4H), 3.66 (s, 3H), 3.56 (t, J = 5.7 Hz, 2H),3.45 (t, J = 5.3 Hz, 2H), 2.98 – 2.72 (m, 3H), 2.18 – 2.07 (m, 1H), 2.04 (d, J =1.5 Hz, 1H), 1.52 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 178.67, 176.36, 170.86,169.15, 167.33, 152.96, 151.80, 146.49, 136.26, 135.95, 135.13, 132.60,127.82, 122.90, 116.60, 111.71, 102.21, 70.55, 70.20, 69.25, 68.59, 65.57,61.82, 48.67, 42.26, 40.24, 31.17, 23.18, 22.63, 14.01, 7.22, 1.73.HRMS (ESI)m / z Calculated for C 32 H 31 F3N6O8 + [M+H] + 684.2155, found 684.2157. Example 7 The preparation method of 4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethoxyethoxyethyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A7c) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0056] Step 2: The preparation of tert-butyl (2-(2-(5,5-dimethyl-3-(4-cyano-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethoxy)ethyl)ethyl) carbamate (A5c) was carried out in the same manner as in Step 2 of Example 1, except that tert-butyloxycarbonyltetraethylene glycol bromide was used instead of N-BOC-bromoethylamine.

[0057] 1 H NMR (400 MHz, DMSO) δ 8.31 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 2.0 Hz,1H), 8.03 (dd, J = 8.4, 2.1 Hz, 1H), 6.76 (t, J = 5.8 Hz, 1H), 3.61 (t, J = 6.1 Hz,2H), 3.55 (dd, J = 5.4, 2.8 Hz, 2H), 3.52 (d, J = 5.0 Hz, 2H), 3.50 – 3.48 (m,4H), 3.35 (t, J = 6.1 Hz, 4H), 3.04 (q, J = 6.0 Hz, 2H), 1.46 (s, 6H), 1.36 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 177.04, 174.82, 155.65, 152.78, 136.83, 136.15,131.29, 130.96, 130.07, 124.16, 123.67, 120.95, 115.29, 106.77, 77.65, 69.79,69.76, 69.56, 69.21, 68.01, 61.67, 28.26, 22.58. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0058] 1 H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 8.12 (d, J = 2.1 Hz, 1H), 7.98(dd, J = 8.5, 2.1 Hz, 1H), 7.90 (d, J= 8.5 Hz, 1H), 7.53 – 7.43 (m, 1H), 7.09(dd, J = 9.8, 7.1 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.54 – 6.43 (m, 1H), 4.90(dd, J = 11.9, 5.4 Hz, 1H), 3.75 – 3.66 (m, 4H), 3.65 (d, J = 3.0 Hz, 8H), 3.54(t, J = 5.7 Hz, 2H), 3.45 (q, J = 5.3 Hz, 2H), 2.90 – 2.65 (m, 2H), 2.11 (dd, J =11.8, 5.1 Hz, 1H), 1.52 (s, 5H), 1.27 (d, J = 15.1 Hz, 1H), 0.85 (ddp, J = 8.8,6.5, 2.3 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ 174.90, 171.24, 169.30, 168.52,167.61, 153.18, 146.76, 136.38, 136.13, 135.33, 132.51, 128.10, 116.77,115.10, 111.77, 110.27, 108.25, 77.28, 74.38, 70.86, 70.62, 70.51, 69.40,68.49, 62.07, 48.85, 42.33, 40.41, 31.44, 24.30, 23.44, 22.81, 20.17, 12.80.HRMS (ESI) m / z Calculated for C 34 H 35 F3N6O9 + [M+H] + 728.2418, found 728.2315. Example 8 Preparation of 4-(2-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (A11a) Step 1: Preparation of 5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)imidazolidine-2,4-dione (A8) 5,5-dimethylhydantoin (12.80 g, 100.0 mmol) was dissolved in DMF (30.0 ml), K2CO3 (4.2 g, 30 mmol) was added, and the mixture was stirred at room temperature for 30 min. 2-nitro-5-fluorotrifluorotoluene (4.18 g, 20.0 mmol) was slowly added, and the temperature was raised to 45°C after the addition was completed. After the reaction was completed by TLC detection, the reaction solution was poured into 300 ml of water, extracted three times with ethyl acetate (100 ml*3), the ethyl acetate phases were combined, washed once with saturated brine (200 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and separated by silica gel column chromatography to obtain compound A1 (4.70 g, light yellow solid, 74% yield).

[0059] 1 H NMR (400 MHz, DMSO) δ 8.85 (s, 1H), 8.32 (d, J = 8.7 Hz, 1H), 8.21(d, J = 2.2 Hz, 1H), 8.07 (dd, J = 8.8, 2.2 Hz, 1H), 1.43 (s, 6H). 13 C NMR (101MHz, DMSO) δ 175.81, 153.07, 145.23, 136.57, 131.16, 126.47, 125.31, 125.26,122.04, 121.70, 58.07, 24.62. Step 2: Preparation of tert-butyl (2-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethyl)carbamate (A9a): Under nitrogen protection, compound A8 (100 mg, 0.32 mmol) was dissolved in DMF (2 ml), cooled to 0°C, and then NaH (19 mg, 0.47 mmol, 60% content) was slowly added and stirred at room temperature for 1-2 h, followed by slow addition of N-BOC-bromoethylamine (107 mg, 0.48 mmol), and the temperature was raised to 55°C after the addition was completed. After TLC detection, the reaction solution was poured into 20 ml of water, extracted three times with ethyl acetate (10 ml*3), the ethyl acetate phases were combined, washed once with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and separated by silica gel column chromatography to obtain compound A9a (128 mg, light yellow liquid, 87% yield).

[0060] 1 H NMR (400 MHz, DMSO) δ 8.34 (d, J = 8.7 Hz, 1H), 8.20 (d, J = 2.1 Hz,1H), 8.07 (dd, J = 8.8, 2.2 Hz, 1H), 3.59 – 3.53 (m, 2H), 2.61 (dd, J = 8.2, 6.8Hz, 2H), 1.46 (s, 6H), 1.42 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 174.70, 170.24,152.65, 145.32, 136.33, 131.16, 126.59, 125.27, 123.22, 122.10, 121.76,120.51, 80.35, 61.76, 35.31, 34.50, 27.78, 27.64, 22.37. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0061] 1 H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 8.01 (s, 2H), 7.55 (t, J = 7.9Hz, 1H), 7.14 (d, J = 7.2 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 4.90 (dd, J = 12.0,5.3 Hz, 1H), 3.68 (t, J = 6.6 Hz, 2H), 3.60 (t, J = 6.5 Hz, 2H), 3.43 (t, J = 6.8Hz, 1H), 2.76 (dd, J = 13.5, 4.0 Hz, 2H), 2.49 (t, J = 7.7 Hz, 1H), 2.09 (dd, J =9.6, 5.6 Hz, 1H), 1.53 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 174.37, 171.29,169.54, 168.55, 167.51, 153.66, 146.27, 136.66, 135.98, 132.73, 129.00,126.19, HRMS (ESI) m / z Calculated forC 27 H 23 F3N6O8 + [M+H] + 616.2418, found 616.1423. Example 9 The preparation method of 4-((3-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)propyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (A11b) comprises the following steps: Step 1: Same as step 1 in Example 8.

[0062] Step 2: The preparation of tert-butyl (3-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)propyl)carbamate (A9b) was carried out in the same manner as in Step 2 of Example 8, except that N-Boc-3-aminopropyl bromide was used instead of N-BOC-bromoethylamine.

[0063] 1 H NMR (400 MHz, DMSO) δ 8.33 (d, J = 8.7 Hz, 1H), 8.21 (d, J = 2.1 Hz,1H), 8.08 (dd, J = 8.7, 2.2 Hz, 1H), 6.88 (t, J = 5.7 Hz, 1H), 3.32 (d, J = 7.6 Hz,2H), 3.00 (q, J = 6.5 Hz, 2H), 1.75 (p, J = 7.0 Hz, 2H), 1.46 (s, 6H), 1.38 (s, 9H). 13C NMR (101 MHz, DMSO) δ 174.71, 155.65, 152.63, 145.23, 136.42, 131.13,126.45, 125.27, 125.22, 123.20, 122.01, 121.68, 120.48, 77.56, 61.73, 37.65,37.39, 29.40, 28.25, 22.48. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0064] 1 H NMR (400 MHz, DMSO) δ 8.34 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 2.1 Hz,1H), 8.09 (dd, J = 8.8, 2.1 Hz, 1H), 7.59 (dd, J = 8.6, 7.0 Hz, 1H), 7.16 (d, J =8.6 Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.75 (t, J = 6.1 Hz, 1H), 5.05 (dd, J =12.8, 5.4 Hz, 1H), 3.44 (q, J = 7.8 Hz, 5H), 2.88 (ddd, J = 16.9, 13.8, 5.3 Hz,1H), 2.62 – 2.55 (m, 1H), 2.01 (dp, J = 12.2, 3.8 Hz, 1H), 1.91 (p, J = 7.3 Hz,3H), 1.48 (s, 6H). 13C NMR (101 MHz, DMSO) δ 174.71, 172.83, 170.10, 168.81,167.28, 152.94, 146.22, 145.22, 139.67, 136.25, 132.24, 131.21, HRMS (ESI) m / z Calculated for C 28 H 25 F3N6O8 + [M+H] + 630.1686, found 630.1584. Example 10 The preparation method of 4-(4-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)butyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (A11c) comprises the following steps: Step 1: Same as step 1 in Example 8.

[0065] Step 2: The preparation of tert-butyl (4-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)butyl)carbamate (A9c) was the same as in Step 2 of Example 8, except that tert-butyl N-(4-bromobutyl)carbamic acid was used instead of N-BOC-bromoethylamine.

[0066] 1 H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 2.1 Hz, 1H), 8.03 – 7.99 (m, 2H), 3.37 (t, J = 7.8 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H), 1.77 (d, J = 5.6 Hz, 4H), 1.54(s, 6H). The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0067] 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.05 –7.97 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 7.1 Hz, 1H), 6.89 (d, J = 8.6Hz, 1H), 4.94 – 4.86 (m, 1H), 3.42 (t, J = 7.3 Hz, 2H), 3.35 (t, J = 6.7 Hz, 2H),2.89 – 2.80 (m, 1H), 2.79 – 2.70 (m, 2H), 2.10 (td, J = 7.3, 3.9 Hz, 1H), 1.84(p, J = 7.5 Hz, 2H), 1.75 (t, J = 8.0 Hz, 2H), 1.54 (s, 6H), 1.44 (s, 1H). 13 C NMR(101 MHz, CDCl3) δ 174.68, 171.35, 169.64, 168.64, 167.64, 153.12, 146.84,145.99, 136.36, 136.23, 132.61, 128.82, 126.10, 124.28, 116.71, 111.86,110.24, 62.08, 53.57, 48.98, 42.21, 40.02, 31.49, 29.81, 28.51, 27.15, 26.81,23.62, 22.87. HRMS (ESI) m / z Calculated for C 29 H 27 F3N6O8 + [M+H] + 644.1842, found644.1744. Embodiment 11 The preparation method of 4-(6-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)hexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (A11d) comprises the following steps: Step 1: Same as step 1 in Example 8.

[0068] Step 2: The preparation of tert-butyl (6-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)hexyl)carbamate (A9d) was the same as in Step 2 of Example 8, except that tert-butyl (6-bromohexyl)carbamate was used instead of N-BOC-bromoethylamine.

[0069] 1 H NMR (400 MHz, DMSO) δ 8.32 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 2.1 Hz,1H), 8.08 (dd, J = 8.8, 2.2 Hz, 1H), 6.78 (t, J = 5.7 Hz, 1H), 3.30 (t, J = 7.7 Hz,2H), 2.90 (q, J = 6.5 Hz, 2H), 1.61 (p, J = 7.4 Hz, 2H), 1.46 (s, 6H), 1.39 (d, J =2.1 Hz, 2H), 1.36 (s, 9H), 1.32 – 1.26 (m, 4H). 13 C NMR (101 MHz, DMSO) δ174.75, 161.44, 155.61, 152.55, 145.22, 136.46, 131.07, 126.43, 125.21,125.15, 123.20, 122.02, 121.68, 120.48, 77.29, 61.67, 29.40, 28.94, 28.26,26.35, 26.04, 25.96, 22.55. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0070] 1 H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.48 (ddd, J = 9.2, 7.2, 2.1 Hz,1H), 7.07 (t, J = 6.7 Hz, 1H), 6.87 (dd, J= 8.6, 2.0 Hz, 1H), 6.30 (d, J = 7.0 Hz,1H), 6.23 (t, J = 5.7 Hz, 1H), 4.91 (dd, J = 12.0, 5.4 Hz, 1H), 3.64 (dd, J = 11.7,5.4 Hz, 3H), 3.26 (q, J = 6.6 Hz, 2H), 2.89 – 2.70 (m, 2H), 2.53 (q, J = 7.4 Hz,1H), 2.37 (t, J = 7.0 Hz, 1H), 2.16 – 2.08 (m, 1H), 1.68 (t, J = 7.0 Hz, 2H),1.58 (q, J = 6.6 Hz, 2H), 1.54 – 1.37 (m, 6H). 13 C NMR (101 MHz, CDCl3) δ 195.93,193.60, 182.62, 171.17, 169.56, 168.49, 167.68, 160.78, 156.72, 147.00,136.18, 132.48, 116.70, 111.44, 109.81, 83.95, 77.27, 62.78, 56.84, 52.87,48.86, 42.57, 32.55, 31.44, 30.86, 29.64, 29.16, 26.75, 25.50, 24.34, 22.82.HRMS (ESI) m / z Calculated for C 31 H 31 F3N6O8 + [M+H] + 672.2155, found 672.2052. Example 12 The preparation method of 4-(2-(5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (A14a) comprises the following steps: Step 1: Same as step 1 in Example 8.

[0071] Step 2: Under nitrogen protection, compound A8 (100 mg, 0.32 mmol) was dissolved in DMF (2 ml), cooled to 0°C, and then NaH (19 mg, 0.47 mmol, 60% content) was slowly added and stirred at room temperature for 1-2 h, followed by slow addition of bromo-monopolyethylene glycol-amino tert-butyl ester (128 mg, 0.48 mmol), and the temperature was raised to 55°C after the addition was completed. After TLC detection, the reaction solution was poured into 20 ml of water, extracted three times with ethyl acetate (10 ml*3), the ethyl acetate phases were combined, washed once with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified and separated by silica gel column chromatography to obtain compound A12a (135 mg, light yellow liquid, 84% yield).

[0072] 1 H NMR (400 MHz, DMSO) δ 8.33 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 2.2 Hz,1H), 8.09 (dd, J = 8.7, 2.2 Hz, 1H), 6.78 (t, J = 5.7 Hz, 1H), 3.59 (t, J = 6.0 Hz,2H), 3.49 (t, J = 6.0 Hz, 2H), 3.42 (t, J = 5.9 Hz, 2H), 3.09 (q, J = 5.9 Hz, 2H), 1.47 (s, 6H), 1.35 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 174.79, 155.61, 152.80,145.29, 136.34, 131.14, 126.47, 125.25, 125.20, 123.19, 122.05, 121.71,120.47, 77.59, 69.14, 67.66, 61.66, 36.29, 28.21, 24.60, 22.56. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0073] 1 H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.48 (dd, J = 8.5, 7.1 Hz, 1H),7.08 (d,J = 7.1 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.23 (t, J = 5.7 Hz, 1H), 4.91(dd, J = 12.0, 5.4 Hz, 1H), 3.68 – 3.61 (m, 2H), 3.26 (q, J = 6.5 Hz, 2H), 2.91 –2.67 (m, 3H), 2.16 – 2.08 (m, 1H), 1.68 (t, J = 7.1 Hz, 4H), 1.58 (p, J = 6.6 Hz,2H), 1.51 (d, J = 3.9 Hz, 1H), 1.49 – 1.37 (m, J = 5.6 Hz, 4H). 13 C NMR (101 MHz, CDCl3) δ 171.22, 169.55, 168.52, 167.69, 147.00, 136.18, 132.47, 116.70,113.05, 111.44, 109.81, 108.93, 62.78, 51.90, 48.86, 42.57, 32.55, 31.44,29.16, 26.74, 25.50, 22.82, 12.87. HRMS (ESI) m / z Calculated for C 29 H 27 F3N6O9 + [M+H] + 660.1792, found 660.1681. Embodiment 13 The preparation method of 4-(2-(2-(5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethoxy)ethyl)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (A14b) comprises the following steps: Step 1: Same as step 1 in Example 8.

[0074] Step 2: The preparation of tert-butyl (2-(2-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethoxy)ethyl)carbamate (A9b) is the same as that in Step 2 of Example 12, except that tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate is used instead of tert-butyl bromo-monopolyethylene glycol-amino.

[0075] 1 H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.91(d, J = 8.4 Hz, 1H), 4.95 (s, 1H), 3.65 (t, J = 5.6 Hz, 2H), 3.57 – 3.52 (m, 4H), 3.52 – 3.48 (m, 2H), 3.44 (t, J = 5.4 Hz, 2H), 3.21 (t, J = 5.5 Hz, 2H), 1.47 (s, 6H), 1.35 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 174.80, 155.60, 152.81, 151.88,145.30, 144.15, 140.80, 136.35, 131.10, 127.78, 126.48, 125.23, 125.18,123.19, 121.89, 77.61, 69.70, 69.49, 69.21, 68.04, 61.67, 28.21, 22.55. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0076] 1 H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 8.04 – 7.94 (m, 2H), 7.51 –7.44 (m, 1H), 7.08 (dd, J = 7.0, 1.9 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 4.89 (dd, J = 11.7, 5.4 Hz, 1H), 3.73 (dq, J= 11.1, 5.3 Hz, 4H), 3.66 (s, 3H), 3.61 (t, J =4.2 Hz, 1H), 3.55 (t, J = 6.1 Hz, 2H), 3.44 (t, J = 5.5 Hz, 2H), 2.89 – 2.64 (m,2H), 2.21 – 1.98 (m, 2H), 1.51 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 175.04,171.41, 169.44, 168.70, 167.66, 153.26, 146.84, 136.29, 136.22, 132.61,128.89, 126.05, 124.39, 124.33, 116.83, 111.87, 110.43, 70.80, 70.48, 69.55,68.85, 68.77, 62.16, 48.95, 42.49, 40.52, 31.46, 23.57, 23.48, 23.46, 22.90.HRMS (ESI) m / z Calculated for C 31 H 31 F3N6O 10 + [M+H] + 704.2054, found 704.1954. Embodiment 14 The preparation method of 4-(2-(2-(5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethoxyethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (A14c) comprises the following steps: Step 1: Same as step 1 in Example 8.

[0077] Step 2: The preparation of tert-butyl (2-(2-(5,5-dimethyl-3-(4-nitro-3-(trifluoromethyl)phenyl)-2,4-dioxoimidazolidin-1-yl)ethoxy)ethyl)ethyl) carbamate (A12c) is the same as in Step 2 of Example 12, except that tert-butyloxycarbonyltetraethylene glycol bromide is used instead of bromo-monoethylene glycol-tert-butyl amino ester.

[0078] 1 H NMR (400 MHz, CDCl3) δ 8.17 (d, J= 2.1 Hz, 1H), 8.03 – 8.00 (m, 2H), 4.99 (s, 1H), 3.73 (t, J = 5.6 Hz, 3H), 3.65 – 3.64 (m, 4H), 3.62 – 3.60 (m,4H), 3.56 (t, J = 5.6 Hz, 3H), 3.52 (t, J = 5.1 Hz, 2H), 1.54 (s, 6H), 1.43 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 174.80, 155.59, 152.80, 151.88, 145.28, 144.15,140.80, 136.36, 131.09, 127.79, 126.48, 125.23, 125.17, 123.19, 121.89,120.47, 116.81, 77.59, 69.77, 69.53, 69.18, 68.01, 61.67, 28.21, 22.54. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0079] 1 H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.14 (d, J = 2.1 Hz, 1H), 8.05 –7.95 (m, 2H), 7.46 (q, J = 6.6 Hz, 1H), 7.08 (d, J = 7.2 Hz, 1H), 6.89 (d, J = 8.5Hz, 1H), 6.45 (q, J = 6.1 Hz, 1H), 4.97 – 4.84 (m, 1H), 3.70 (d, J = 5.8 Hz, 4H),3.64 (d, J = 2.5 Hz, 8H), 3.54 (t, J = 5.7 Hz, 2H), 3.44 (q, J = 5.5 Hz, 2H), 2.88– 2.79 (m, 2H), 2.79 – 2.68 (m, 2H), 1.52 (s, 6H). 13C NMR (101 MHz, CDCl3) δ174.93, 171.41, 169.30, 168.65, 167.62, 153.18, 152.11, 146.75, 145.86,136.15, 136.10, 132.49, 128.87, 125.99, 124.34, 124.28, 116.77, 111.71,110.24, 70.81, 70.61, 70.50, 70.49, 69.41, 68.49, 62.10, 49.48, 48.84, 42.32,40.42, 31.42, 23.41, 22.79. HRMS (ESI) m / z Calculated for C 35 H 35 F3N6O 11 + [M+H] + 748.2316, found 748.2216. Embodiment 15 The preparation method of 4-(3-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)azetidin-3-yl)methyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A17a) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0080] Step 2: Preparation of tert-butyl 3-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)azetidine-1-carboxylate (A15a) was carried out in the same manner as in Step 2 of Example 1, except that 1-BOC-3-(bromomethyl)azetidine was used instead of N-BOC-bromoethylamine.

[0081] 1 H NMR (400 MHz, DMSO) δ 8.31 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 1.9 Hz,1H), 8.03 (dd, J = 8.4, 2.0 Hz, 1H), 3.88 (s, 2H), 3.66 (s, 2H), 3.59 (d, J = 7.7Hz, 2H), 2.89 (dp, J= 11.1, 3.9 Hz, 1H), 1.45 (s, 6H), 1.37 (s, 9H). 13 C NMR(101 MHz, DMSO) δ 174.62, 155.66, 153.23, 136.83, 136.10, 131.24, 130.92,130.15, 124.20, 124.15, 123.67, 120.94, 115.27, 106.77, 78.52, 78.29, 62.50,61.64, 42.74, 28.25, 28.11, 22.53. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0082] 1 H NMR (600 MHz, DMSO) δ 11.06 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 8.20(d, J = 2.0 Hz, 1H), 8.05 (dd, J = 8.3, 2.0 Hz, 1H), 7.58 (dd, J = 8.5, 7.0 Hz,1H), 7.12 (d, J = 7.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 5.05 (dd, J = 12.9, 5.5Hz, 1H), 4.31 (s, 2H), 4.02 (dd, J = 8.9, 3.7 Hz, 2H), 3.67 (d, J = 7.5 Hz, 2H),3.08 (p, J = 6.5 Hz, 1H), 2.86 (ddd, J = 17.0, 13.7, 5.2 Hz, 1H), 2.57 (dt, J =17.0, 3.1 Hz, 1H), 2.08 – 2.04 (m, 1H), 1.98 (d, J = 3.8 Hz, 1H), 1.49 (s, 6H). 13C NMR (151 MHz, DMSO) δ 174.62, 172.81, 170.03, 167.23, 166.48, 153.19,148.06, 136.82, 136.08, 134.97, 133.29, 130.13, 124.15, 123.18, 121.37,119.96, 115.24, 111.75, 110.16, 106.75, 61.66, 48.63, 42.96, 31.29, 30.96,29.28, 29.02, 22.54, 22.10, 13.97. HRMS (ESI) m / z Calculated for C 30 H 25 F3N6O6 + [M+H] + 622.1788, found 622.1862. Example 16 The preparation method of 4-(3-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)piperidin-4-yl)methyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (A17b) comprises the following steps: Step 1: Same as step 1 in Example 1.

[0083] Step 2: Preparation of tert-butyl 4-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)methyl)piperidine-1-carboxylate (A15b) was carried out in the same manner as in Step 2 of Example 5, except that 1-BOC-4-bromomethylpiperidine was used instead of N-BOC-bromoethylamine.

[0084] 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 2.0 Hz, 1H), 7.99 (dd, J = 8.5, 2.1Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 4.14 (s, 2H), 3.20 (s, 2H), 2.68 (s, 2H), 2.02 (ddh, J = 11.4, 7.7, 3.8 Hz, 1H), 1.70 (d, J = 17.0 Hz, 4H), 1.52 (s, 6H), 1.44 (s, 9H).13 C NMR (101 MHz, CDCl3) δ 174.60, 154.79, 153.54, 136.47,135.39, 133.90, 133.57, 128.00, 123.41, 123.06, 120.69, 115.12, 108.40,79.74, 67.73, 62.15, 54.63, 46.29, 43.32, 38.92, 35.91, 30.25, 28.54, 23.69. The operations of step 3 and step 4 are the same as those of step 3 and step 4 in embodiment 1.

[0085] 1 H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 8.15 (d, J = 2.1 Hz, 1H), 8.01(dd, J = 8.5, 2.1 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.58 (dd, J = 8.4, 7.2 Hz,1H), 7.39 (d, J = 7.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 5.01 – 4.91 (m, 1H), 3.77 (t, J = 12.6 Hz, 2H), 3.48 (s, 1H), 3.28 (d, J = 7.5 Hz, 2H), 2.94 – 2.83(m, 3H), 2.82 – 2.70 (m, 2H), 2.17 – 2.06 (m, 2H), 1.90 (d, J = 12.0 Hz, 2H),1.60 (d, J = 2.9 Hz, 1H), 1.55 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 174.35,170.81, 168.06, 167.15, 166.49, 153.26, 150.41, 136.16, 135.45, 135.11,133.89, 133.28, 127.73, 123.52, 122.84, 117.33, 115.52, 114.81, 108.14,88.90, 77.05, 61.91, 51.32, 51.02, 48.91, 46.11, 34.78, 31.21, 30.09, 23.41,22.48. HRMS (ESI) m / z Calculated for C 32 H 29 F3N6O6 + [M+H] + 650.2101, found650.2003. Experimental Example 1 Tumor cell inhibitory activity 1. Experimental drugs Different concentrations of AR-degrading PROTACs (A4a-d, A7a-c, A11a-d, A14a-c, A17a-b).

[0086] 2. Cell lines Human prostate cancer cells (22RV1) and mouse embryonic cells (3T3) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were routinely cultured in 1640 and DMEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator, with the medium replaced every 24 hours. When the cells reached saturation, they were digested and passaged with 0.25% trypsin, and passaged once every 2-3 days. Cells in the logarithmic growth phase were selected for the experiment.

[0087] 3. Preparation of DMEM / 1640 culture medium 5 mL of fetal bovine serum, 0.5 mL of penicillin-streptomycin solution (200,000 U / mL), add DMEM / 1640 culture medium and finally make up to 50 mL.

[0088] 4. Cell Viability Detection The MTT [3-(4,5)-dimethyl-2-thiazol-(2,5)-phenyl tetrazolium blue bromide] method was used to determine the effects of cucurbitacin B ester derivatives on the proliferation activity of human prostate cancer cell 22RV1 and mouse embryonic cell 3T3.

[0089] Adding drugs: dilute the test drugs into six different concentrations from high to low and add them to each well, with 4 parallel groups for each concentration. The experiment is divided into drug test group (adding different concentrations of test drugs), control group (adding only culture medium and cells, without adding test drugs) and blank group (adding only culture medium, without adding cells and test drugs). Place the 96-well plate after adding drugs in a 37°C, 5% CO2 incubator for 48 hours.

[0090] Determination of viable cells: After 48 hours of culture, add 20ul of MTT (5mg / mL in PBS buffer) to each well of a 96-well plate. After 4 hours at 37°C, remove the supernatant. Add 200ul of DMSO to each well and shake for 15-30 minutes. Finally, use an automatic microplate reader to detect the optical density (OD value) of each well at a wavelength of 490nm.

[0091] Growth inhibition rate = (1-survival rate) × 100% = [1-(OD test-OD blank) / (OD control-OD blank)] × 100% (where OD test represents the average optical density of the test drug group, OD control represents the average optical density of the control group, and OD blank represents the average optical density of the control group) The inhibitory activity of AR-degrading PROTACs on 22RV1 cells and 3T3 cells is shown in Table 1.

[0092]

[0093]

[0094] a IC 50 Values ​​are expressed as the mean ± standard deviation (SD) of three independent experiments.

[0095] Table 1 shows the half inhibition rate IC of each concentration of PROTACs of the present invention on tumor cells 50 Value (uM).

[0096] The results showed that all 16 PROTACs had a strong inhibitory effect on the proliferation of 22RV1 cells, and were concentration-dependent. And the inhibitory effect of most PROTACs was greater than that of Enzalutamide. Among them, A4b-d, A7b, A11a-c, and a14a all had anti-cancer activity, and the effects of A4d, A7b, A11c, and A14a were more significant, and the effect against 22RV1 prostate cancer cells was more obvious. The 16 PROTACs had no inhibitory effect on the proliferation of 3T3 cells, indicating that our compounds have the effect of targeted degradation of AR.

[0097] The 22RV1 cytotoxicity experiment showed that among rigid chains, PROTACs with a linker length of 4-6 carbon chains had the best inhibitory effect on proliferation, while PROTACs with cyclic linkers showed average effects among our PROTACs. At the same time, among flexible linkers, PROTACs with 1-2 ethylene glycol chains showed the best inhibitory effect on proliferation.

[0098] Experimental Example 2 Androgen receptor protein degradation activity of PROTACs 1. Experimental drugs Different concentrations of AR-degrading PROTACs (A4a-d, A7a-c, A11a-d, A14a-c, A17a-b).

[0099] 2. Cell lines Human prostate cancer cells (22RV1) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were routinely cultured in a 37°C, 5% CO2 incubator using 1640 medium containing 10% fetal bovine serum, with the medium replaced every 24 hours. When the cells reached saturation, they were digested and passaged using 0.25% trypsin, and passaged once every 2-3 days. Cells in the logarithmic growth phase were selected for the experiment.

[0100] 3. Preparation of 1640 culture medium: 5 mL of fetal bovine serum, 0.5 mL of penicillin-streptomycin solution (200,000 U / mL), add 1640 culture medium and finally make up to 50 mL.

[0101] 4. Androgen receptor protein research Western blotting (WB) was used to detect the degradation effect of different concentrations of PROTACs on the androgen receptor protein of human prostate cancer cell 22RV1. 22RV1 cells were seeded in 6-well plates at a density of 2×105 cells per well and cultured overnight. Then, different drugs were diluted with culture medium and incubated with cells for 24 hours. After the incubation, the culture medium was removed and the cells were washed three times with 1mL 4℃ PBS (pH 7.4, 10mM). Then, 150μL of RIPA strong acid lysis buffer containing 1% protease inhibitor was used per well to lyse 22RV1 cells in an ice water bath. After 5 minutes, the cell fluid was collected in a 1.5mL centrifuge tube and lysed for 30 minutes. Subsequently, the cell fluid was centrifuged at 12000rpm for 5 minutes, and the supernatant was collected as the total protein solution. The protein concentration was determined using a BCA kit, and all protein samples were adjusted to the same concentration with PBS. Finally, 25% (v / v) protein loading buffer was added to the protein solution and stored at -80℃ for later use.

[0102] After the extracted protein samples were denatured in a boiling water bath for 5-10 minutes, equal amounts of proteins were separated by 8% SDS-PAGE in a VE-180 vertical electrophoresis tank, and then the separated proteins were transferred from the separation gel to a polyvinylidene fluoride (PVDF) membrane in a transfer electrophoresis tank. The PVDF membrane was blocked with a Tris-buffered saline solution containing 5% skim milk powder for 2 hours. Then, 5 mL of AR antibody and β-actin antibody (for detecting the internal reference protein β-actin) were incubated with the PVDF membrane at 4°C overnight. After the incubation, the primary antibody was recovered, and the PVDF membrane was washed in a SLK-O3000-S digital circular shaker for 15 minutes with TBS buffer containing 0.1% Tween-20, i.e., TBST buffer, and repeated three times. Then, the PVDF membrane was incubated with HRP-labeled goat anti-mouse IgG at room temperature for 1.5 hours. After the incubation, the secondary antibody was recovered and TBST buffer was used three times. Finally, Immobilon Western chemiluminescent HRP substrate and chemiluminescent imaging analysis system 5200Multl were used to detect and image protein bands. Cells incubated with medium without drugs were used as the control group, β-actin was used as the internal reference protein, and four groups were set up in parallel for each group of samples.

[0103] Western blotting (WB) Figure 1 The results showed that all 16 PROTACs had strong degradation effects on AR and AR-V7, and were concentration-dependent. Among them, A4d, A7a, A7c, A11a-d and A14c all had degradation effects on AR and AR-V7 proteins, and A4d, A11a, A11b and A11d had significant effects.

[0104] In 22RV1 cells, it was shown that among rigid chains, PROTACs with linkers of 4-6 carbon chains had the best degradation effect on AR and AR-V7, while PROTACs with cyclic linkers showed average degradation effect on AR and AR-V7 among our PROTACs. At the same time, among flexible linkers, PROTACs with 3 ethylene glycol chains had the best degradation effect on AR and AR-V7.

Claims

1. A PROTAC compound targeting AR / AR-V7 degradation, characterized in that: The general formula is as follows Wherein, said R is -CN, -NO2 or -CI; The Linker is a connecting group.

2. The PROTAC compound targeting AR / AR-V7 degradation according to claim 1, characterized in that: The linker is -(CH2)n-, alkenylene, alkynylene, cycloalkylene, -(CH2) n CO-, -(CH2CH2O) n - and - (CH2CH2O) n Any one or a combination of CH2CO-; wherein n represents a natural number from 1 to 6.

3. The PROTAC compound targeting AR / AR-V7 degradation according to any one of claim 2, characterized in that: The PROTAC compound is selected from any one of the following structures: 。 4. A method for preparing a PROTAC compound for targeting degradation of AR / AR-V7 as claimed in claim 3, characterized in that: The preparation method of the compound A4a, A4b, A4c or A4d is as follows: 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, nucleophilic substitution occurs under alkaline conditions of potassium carbonate and connects with the secondary amine of 5,5-dimethylhydantoin to obtain intermediate A1; 2) Under the alkaline condition of NaH, different lengths of aliphatic carbon chains with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergo affinity substitution with intermediate A1 to obtain intermediate A2a, A2b, A2c or A2d; 3) The intermediate A2a, A2b, A2c or A2d is subjected to trifluoroacetic acid to remove the Boc to obtain the intermediate A3a, A3b, A3c or A3d; 4) The intermediate A3a, A3b, A3c or A3d undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A4a, A4b, A4c or A4d.

5. A method for preparing a PROTAC compound for targeting degradation of AR / AR-V7 as claimed in claim 3, characterized in that: The preparation method of the compound A7a, A7b or A7c is as follows: 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, the intermediate A1 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, polyethylene glycol chains of different lengths with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end are subjected to affinity substitution with intermediate A1 to obtain intermediate A5a, A5b or A5c; 3) After intermediate A5a, A5b or A5c is subjected to trifluoroacetic acid to remove Boc, intermediate A6a, A6b or A6c is obtained; 4) The intermediate A6a, A6b or A6c undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A7a, A7b or A7c.

6. A method for preparing a PROTAC compound for targeted degradation of AR / AR-V7 as claimed in claim 3, characterized in that: The preparation method of the compound A11a, A11b, A11c or A11d is: 1) Using 2-nitro-5-fluorobenzotrifluoride as the starting material, the intermediate A8 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, different lengths of aliphatic carbon chains with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergo affinity substitution with intermediate A8 to obtain intermediate A9a, A9b, A9c or A9d; 3) The intermediate A9a, A9b, A9c or A9d is treated with trifluoroacetic acid to remove the Boc to obtain the intermediate A10a, A10b, A10c or A10d; 4) The intermediate A10a, A10b, A10c or A10d undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A11a, A11b, A11c or A11d.

7. A method for preparing a PROTAC compound for targeting degradation of AR / AR-V7 as claimed in claim 3, characterized in that: The preparation method of the compound A14a, A14b or A14c is as follows: 1) Using 2-nitro-5-fluorobenzotrifluoride as the starting material, the intermediate A8 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, a polyethylene glycol chain of different lengths with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end is subjected to affinity substitution with the intermediate A8 to obtain the intermediate A12a, A12b or A12c; 3) The intermediate A12a, A12b or A12c is subjected to trifluoroacetic acid to remove the Boc to obtain the intermediate A13a, A13b or A13c; 4) The intermediate A13a, A13b or A13c undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A14a, A14b or A14c.

8. A method for preparing a PROTAC compound for targeted degradation of AR / AR-V7 as claimed in claim 3, characterized in that: The preparation method of the compound A17a or A17b is as follows: 1) Using 2-cyano-5-fluorotrifluorotoluene as the starting material, the intermediate A1 is obtained by nucleophilic substitution with the secondary amine of 5,5-dimethylhydantoin under alkaline conditions of potassium carbonate; 2) Under the alkaline condition of NaH, a polycyclic chain with a bromine atom at one end and an amino group protected by tert-butyl methyl ester (Boc) at the other end undergoes affinity substitution with intermediate A1 to obtain intermediate A15a or A15b; 3) After the intermediate A15a or A15b is subjected to trifluoroacetic acid to remove the Boc, the intermediate A16a or A16b is obtained; 4) The intermediate A16a or A16b undergoes affinity substitution reaction with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione under alkaline conditions of DIPEA to finally obtain the final product A17a or A17b.

9. Use of a PROTAC compound targeting AR / AR-V7 degradation as claimed in any one of claims 1 to 3 in the preparation of an anti-tumor drug.

10. Use of the PROTAC compound targeting AR / AR-V7 degradation according to claim 9 in the preparation of anti-tumor drugs, characterized in that: The tumor drug is an anti-prostate cancer, lung cancer or liver cancer drug.

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