Degradation agents based on inhibitor fusion covalent fragments and uses

Abstract

The application discloses a degradation agent based on inhibitor fusion covalent fragments and application, and belongs to the technical field of medicines. Specifically, the degradation agent is obtained by fusing covalent fragments of an inhibitor (+) JQ-1 of BRD4 protein. The degradation agent can target BRD4 protein and degrade BRD4 protein. The degradation activity of the degradation agent is screened through two experiments of high-content screening technology and Western blotting, and the best compound M4 in degradation activity is obtained. The compound M4 can induce significant degradation of BRD4 protein within 6 hours, and presents dose dependence; the mechanism of action is to degrade BRD4 protein through an ubiquitin-proteasome pathway mediated by E3 ubiquitin ligase DCAF11. Cell experiments prove that the compound M4 has a broad-spectrum anticancer ability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, and in particular to a degradation agent based on inhibitor fusion covalent fragments and applications. BACKGROUND

[0002] Targeted Protein Degradation (TPD) is an emerging therapy that selectively degrades the Protein of Interest (POI) through the intrinsic protein degradation system in cells, which has the advantages of targeting traditional undruggable targets and overcoming small molecule inhibitor resistance. At present, degradation agents based on the ubiquitin-proteasome system mainly include two types: Proteolysis Targeting Chimeras (PROTACs) and molecular glue degraders. Among them, molecular glue degraders exert their effects by enhancing or creating Protein-Protein Interaction (PPI), compared with PROTACs, the molecular glue with smaller molecular weight is more in line with the characteristics of drugs.

[0003] Molecular glue degraders have physicochemical properties like traditional small molecule drugs, most of which fall within the scope of the Rule of five (RO5, also known as Lipinski's rule), and are better than PROTACs in terms of bioavailability and pharmacokinetics. From the perspective of mechanism of action, molecular glue degraders exhibit unique advantages. Unlike traditional inhibitors, molecular glue degraders do not need to have high affinity to both POI and E3 ubiquitin ligase. They only need to show affinity to either POI or E3 ubiquitin ligase to function. Specifically, molecular glue degraders bind to the surface of POI or E3 ubiquitin ligase and form a new protein surface with the help of the interaction of surrounding amino acid residues. This surface effectively promotes the interaction between POI and E3 ubiquitin ligase, thereby driving the ubiquitination and degradation of the target protein. This mechanism makes the potential targeting range of molecular glue degraders wider than that of proteolytic targeting chimeras. From the progress of clinical application, molecular glue degraders have made significant breakthroughs, and some drugs have been successfully approved for marketing. Among them, thalidomide, lenalidomide and pomalidomide, as typical molecular glue degraders, have been widely used in clinical treatment. The good clinical performance of these drugs has effectively proved the effectiveness and feasibility of molecular glue degraders in clinical application. Molecular glue degraders can be defined in a broad sense as monovalent small molecules that bind cooperatively at protein-protein interaction interfaces (PPIs). They exhibit affinity to at least one protein and enhance the stability of protein-protein interactions after binding. However, due to the lack of in-depth understanding of PPI interfaces, the mechanism of molecular glue degraders presents a complex diversity, making it difficult to achieve rational design through traditional methods. On the other hand, PPI interfaces usually have the characteristics of large area, flatness and lack of specific binding pockets, which further increases the complexity of rational design of molecular glue degraders. Although molecular glue degraders have shown significant effects in clinical practice, they involve fewer targets, lack methods for rational design, and have a small overall number.

[0004] Bromodomain-containing protein 4 (BRD4 protein) is an important epigenetic regulatory factor, which plays an important role in the occurrence and development of various cancers such as acute myeloid leukemia, breast cancer and non-small cell lung cancer.

[0005] Therefore, the development of molecular glue degraders targeting BRD4 protein not only helps to expand the types of molecular glue degraders, but also provides a new strategy for treating cancers related to abnormal function of BRD4 protein, which is of great significance. SUMMARY

[0006] Based on the deficiencies of the prior art, the purpose of the present application is to provide a degradation agent based on inhibitor fusion covalent fragment and application, aiming at taking BRD4 protein as a target, performing covalent fragment modification of target cysteine at the solvent exposure zone benzene ring position of the inhibitor (+)-JQ-1, completing the change of the inhibitor to the degradation agent, so as to realize the targeted degradation of BRD4 protein for treating related cancers. The structural formula of the inhibitor (+)-JQ-1 is ; wherein Me is methyl.

[0007] The technical scheme of the present application is as follows:

[0008] In the first aspect of the present application, a degradation agent based on inhibitor fusion covalent fragment is provided, which comprises a compound represented by formula (I) and its structural isomers:

[0009] Formula (I);

[0010] Wherein, R is a covalent fragment capable of forming a covalent bond with the sulfhydryl group of cysteine in E3 ubiquitin ligase (i.e. covalent fragment R); Me is methyl.

[0011] As Figure 1 shown, Figure 1 POI in the above formula is a target protein (i.e. an undruggable target), a pentagram represents a molecular glue degradation agent, E3 represents E3 ubiquitin ligase, E2 represents E2 ubiquitin binding enzyme, and Ub represents ubiquitin. Figure 1 A mechanism for degrading undruggable targets by a molecular glue degradation agent is shown, which is as follows: the molecular glue degradation agent induces new protein-protein interactions (PPIs) on the target protein (POI) and E3 ubiquitin ligase, significantly enhances the binding affinity between the two, and thus stabilizes the formation of a ternary complex. The ternary complex can be recognized by E2 ubiquitin binding enzyme, which promotes the polyubiquitination (Ub) of the target protein. The ubiquitinated target protein is further recognized and degraded by 26S proteasome, thereby achieving effective clearance of the target protein.

[0012] Based on the principle of enhancing PPI by the molecular glue degradation agent shown in Figure 1 , the present application adopts covalent modification to introduce covalent fragment R (covalent fragment R can form a covalent bond with a specific residue of E3 ubiquitin ligase) into the structure of the inhibitor (+)-JQ-1 of BRD4 protein, thereby obtaining a series of BRD4 protein targeted degradation agents based on inhibitor fusion covalent fragment. As Figure 2As shown, the BRD4 protein targeted degradation agent based on inhibitor fusion covalent fragment can recruit E3 ubiquitin ligase and induce BRD4 protein ubiquitination, and the ubiquitinated BRD4 protein is finally degraded after being recognized by proteasome. Therefore, the BRD4 protein targeted degradation agent based on inhibitor fusion covalent fragment can target BRD4 protein and degrade BRD4 protein. Among them, the covalent fragment R forms a covalent interaction with specific residues of E3 ubiquitin ligase in a reversible or irreversible manner, and the type, position and spatial orientation of the covalent fragment R can be adjusted according to specific needs, so as to achieve maximum degradation and minimum toxicity.

[0013] In the selection of modified E3 ubiquitin ligase specific residues, it is found that cysteine has the highest nucleophilicity among all natural amino acids due to its thiol residue under physiological conditions. Therefore, the covalent fragment R selected by the present application will be coupled with different types, different reactivities and different binding directions of covalent fragment R and inhibitor (+)-JQ-1 at the cysteine connection site. According to the chemical reaction type of the covalent bond formed between the covalent fragment R and the thiol group of cysteine, the following types of compounds are designed and synthesized, which are compounds M3-M10, in which the covalent fragment R is combined with cysteine by Michael addition reaction; compounds M11-M14, M16 and M20, in which the covalent fragment R is combined with cysteine by nucleophilic reaction; compound M15, in which the covalent fragment R is combined with cysteine by oxidation reaction; and compounds M17-M19, in which the covalent fragment R is formed by extending the direction of the reaction handle. For compounds M1-M20, degradation activity screening is performed by high content screening technology (HCS) and Western blot (WB) technology, and through the screening of the two methods, it is found that compounds M1-M20 exhibit better degradation activity on BRD4 protein, and among them, compound M4 exhibits the best degradation activity on BRD4 protein. The structural formula of compounds M1-M20 is shown below.

[0014] Among them, the structural formula of compound M1 is , and the structural formula of compound M2 is ; wherein Me is methyl.

[0015] Alternatively, as shown in Figure 3 , the covalent fragment R can be divided into covalent fragment R 1 , covalent fragment R 2 , covalent fragment R 3 , and covalent fragment R 4 according to the reaction type of the covalent bond formed between the covalent fragment R and cysteine; covalent fragment R 1 is combined with cysteine by Michael addition reaction; covalent fragment R 2Through nucleophilic reaction with cysteine; covalent fragment R 3 Through oxidative reaction with cysteine; covalent fragment R 4 Through meta-substituted Michael addition reaction with cysteine. Specifically as follows:

[0016] Covalent fragment R 1 BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R 1 The BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R ;

[0017] Wherein, Me is methyl.

[0018] Covalent fragment R 2 Through nucleophilic reaction with cysteine; BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R 2 The BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R

[0019] ;

[0020] Wherein, Me is methyl.

[0021] The structural formula of compound M20 is .

[0022] Covalent fragment R 3 Through oxidative reaction with cysteine; BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R 3 The BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R ; wherein, Me is methyl. Compound M15 is designed based on the reversible exchange reaction mechanism of thiol-disulfide bond, which mechanism involves the nucleophilic attack of the sulfur atom of thiol group to the disulfide bond, resulting in the breakage of the original S-S bond and the formation of a new disulfide bond structure.

[0023] Covalent fragment R 4 Through meta-substituted Michael addition reaction with cysteine; BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R 4 The BRD4 protein targeted degrader based on inhibitor fusion covalent fragment R

[0024] ;

[0025] Wherein, Me is methyl.

[0026] The second aspect of the application provides a use of the degrader based on inhibitor fusion covalent fragment in the preparation of a drug for treating cancer.

[0027] Further, the cancer includes at least one of breast cancer, glioma.

[0028] In a third aspect of the present application, a medicament is provided, which includes the inhibitor fusion covalent fragment-based degrader and a pharmaceutically acceptable carrier.

[0029] Further, the pharmaceutically acceptable carrier includes at least one of an excipient, a glidant, a diluent, a preservative, a colorant, a flavoring agent, a wetting agent, a suspending agent, a stabilizer, an isotonic agent, a solvent, an emulsifying agent.

[0030] Beneficial effects: The present application is based on the principle of covalently modifying enhanced protein-protein interaction (PPI), and designs a BRD4 protein targeted degrader based on inhibitor fusion covalent fragment with cysteine as the covalent reaction target. The covalent fragment of the BRD4 protein targeted degrader is covalently combined with cysteine through Michael addition reaction, nucleophilic reaction, oxidation reaction or meta-substitution reaction, and a series of candidate compounds with structural diversity are formed by coupling a variety of covalent fragments with inhibitor (+)-JQ-1. Among the candidate compounds, M4 shows the best BRD4 protein degradation activity. Thus, the present application not only expands the design space of molecular glue degraders, improves the degradation efficiency of targeted BRD4 protein, but also is expected to provide a new direction for drug research for the treatment of related cancers. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Schematic diagram of the action mechanism of the molecular glue degrader based on ubiquitin-proteasome pathway to degrade target protein.

[0032] Figure 2 Schematic diagram of the action mechanism of the BRD4 protein targeted degrader based on inhibitor fusion covalent fragment to degrade BRD4 protein.

[0033] Figure 3 Synthetic route map of the BRD4 protein targeted degrader based on inhibitor fusion covalent fragment of the present application.

[0034] Figure 4 Construction of the high-content screening platform and activity test results of each compound, wherein (a) is a degradation effect heat map of the compound on high content; (b) is a degradation effect column chart of the compound on high content; (c) is a BRD4 protein degradation effect chart of the top four compounds in high content degradation effect on endogenous cells; (d) is a semi-quantitative chart of the BRD4 protein degradation effect of the top four compounds in high content degradation effect on endogenous cells.

[0035] Figure 5Figure of the target specificity, dose dependence and time dependence of compound M4 to BET family proteins (also referred to as BET proteins herein); wherein (a) is the structure of compound M4; (b) is the degradation effect of compound M4 to BET family proteins at different concentrations; (c) is the semi-quantitative degradation of BET family proteins by compound M4; (d) is the semi-quantitative fitting curve of the degradation of BET family proteins by compound M4; (e) is the degradation effect of compound M4 to BRD4 protein at different time periods; (f) is the semi-quantitative degradation effect of compound M4 to BRD4 protein at different time periods.

[0036] Figure 6 Figure of the degradation mechanism research results of compound M4, wherein (a) is the cell state after co-treatment of compound M4 and inhibitors in the autophagy-lysosome and ubiquitin-proteasome processes in the high-content experiment; (b) is the semi-quantitative figure of the high-content experiment results after co-treatment of degradation inhibitors; (c) and (d) are the degradation effect and quantitative figure after co-treatment of compound M4 and inhibitors in the autophagy-lysosome process in the Western blot experiment; (e) and (f) are the degradation effect and quantitative figure after co-treatment of compound M4 and inhibitors in the ubiquitin-proteasome process in the Western blot experiment.

[0037] Figure 7 Figure of the dose dependence of compound M4 to human breast cancer cell line (MDA-MB-231) and human glioma cell line (U251). DETAILED DESCRIPTION

[0038] The present application provides a degradation agent based on inhibitor fusion covalent fragments and applications, in order to make the purpose, technical scheme and effect of the present application more clear and definite, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by those skilled in the art to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing the specific embodiments and are not intended to limit the present application.

[0039] The present application is further described below through specific examples.

[0040] The following examples and related tests do not specify the specific technology or conditions, according to the technology or conditions described in the literature in the art, or according to the product manual. All reagents or instruments not specified by the manufacturer are conventional products that can be purchased through commercial channels.

[0041] Example 1 Synthesis of inhibitor (+)-JQ-1

[0042] The synthetic route is as follows:

[0043]

[0044] In the formula, Me in compound 5 and compound 7 is methyl, and Me appearing in the following formula is methyl.

[0045] (1) Synthesis of compound 3

[0046] In a round-bottom flask, Fmoc-L-aspartic acid 4-methyl ester (compound 2, 7.4 g, 20 mmol), N,N'-dicyclohexyl carbodiimide (DCC, 8.3 g, 40 mmol), 1-hydroxybenzotriazole (HoBt, 5.4 g, 40 mmol), dichloromethane (DCM, 100 mL), and N,N'-diisopropylethylamine (DIPEA, 10 mL) were sequentially added and stirred for 10 minutes of activation. 2-amino-3-(4-chlorobenzoyl)-4,5-dimethylthiophene (compound 1, 5.3 g, 20 mmol) was added and stirred overnight. The reaction was detected by thin layer chromatography (TLC, petroleum ether: ethyl acetate = 4:1). The reaction solution was filtered under vacuum by circulating water pump, extracted with dichloromethane (3 x 50 mL), washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Then, compound 3 (6.66 g, yield 54%) was obtained in the form of yellow oil after column chromatography separation and purification.

[0047] (2) Synthesis of compound 4

[0048] To compound 3 (6.17 g, 10 mmol), 16 mL of dichloromethane (DCM) and 4 mL of piperidine deprotection group were added. After half an hour of reaction, TLC (with petroleum ether: ethyl acetate = 2:1) was used to detect the reaction. After concentration under reduced pressure, compound 4 (3.6 g, 9 mmol, yield 90%) was obtained in the form of yellow oil after column chromatography separation and purification.

[0049] (3) Synthesis of compound 5

[0050] To compound 4 (3.6 g, 9 mmol), ethanol (EtOH, 9 mL) and glacial acetic acid (AcOH, 1.5 mL) were added. After being heated to 80°C and refluxed and stirred overnight, TLC (petroleum ether: ethyl acetate = 2:1) was used to detect the reaction. After concentration under reduced pressure, compound 5 (3.1 g, 8.1 mmol, yield 90%) was obtained in the form of yellow oil.

[0051] (4) Synthesis of compound 7

[0052] Compound 5 (3.1 g, 8.1 mmol) was degassed under nitrogen protection, 10 mL of super dry tetrahydrofuran (THF) and potassium tert-butoxide (t-BuOK, 8 mL) were added at -78 ℃ and stirred at room temperature for 30 minutes, and then diphenyl chlorophosphonate (PO(OPh)2Cl, 2 mL, 9.7 mmol) was added at -78 ℃ and slowly warmed to -10 ℃ for 1 hour, and then compound 6 (acetyl hydrazine, AcNHNH2, 1.2 g, 16.2 mmol) was added at -10 ℃ and stirred for 1 hour, and then 10 mL of tert-butanol was added and heated to 90 ℃ and refluxed overnight, and then TLC (petroleum ether: ethyl acetate = 1:1) was used to detect the reaction, and then ethyl acetate was extracted, dried, concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain compound 7 (inhibitor (+)-JQ-1, 2 g, 4.8 mmol, yield 59%) in the form of yellow powder.

[0053] The nuclear magnetic resonance hydrogen spectrum data thereof are 1 H NMR (500 MHz, CDCl3) δ 7.40 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 4.61 (m, 1H), 3.76 (s, 3H), 3.64 - 3.60 (m, 2H), 2.67 (s, 3H), 2.40 (s, 3H), 1.68 (s, 3H). The nuclear magnetic resonance carbon spectrum data thereof are 13 C NMR (126 MHz, CDCl3) δ 171.8, 164.1, 155.0, 151.6, 150.2, 137.0, 131.6, 129.9, 129.3, 128.7, 124.1, 120.3, 53.5, 51.9, 36.4, 14.4, 13.1, 11.5. The high resolution mass spectrum data thereof are HRMS. ESI (m / z): [M+H] + calcd for C 20 H 20 N4O2ClS + :417.0942, found 417.0961 (calcd for represents the theoretical calculation result, and found represents the actual analysis result. The meanings of calcd for and found are the same below).

[0054] Example 2 Synthesis of BRD4 protein targeted degrader based on inhibitor fusion covalent fragment (compounds M1, M2 and M17)

[0055] The synthesis route is as follows:

[0056]

[0057] Wherein Boc in compound 9 is tert-butyloxycarbonyl, and Boc appearing in the following structural formula is tert-butyloxycarbonyl.

[0058] (1) Synthesis of compound 9

[0059] In a round-bottom flask, compound 7 (166 mg, 0.4 mmol), benzenboronic acid pinacol ester (compound 8, 141 mg, 0.44 mmol), palladium acetate (Pd(OAc)2, 9 mg, 0.04 mmol), potassium fluoride (KF, 70 mg, 1.2 mmol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos, 17 mg, 0.04 mmol), 1.5 mL of super dry tetrahydrofuran (THF) and 30 μL of water (H2O) were added successively, and the reaction was stirred at 80 ℃ overnight under the protection of nitrogen. TLC (dichloromethane:methanol = 20:1) was used to detect the reaction, and then ethyl acetate was used for extraction, drying, concentration under reduced pressure, and then column chromatography on silica gel was used for separation and purification to obtain compound 9 (205 mg, 0.36 mmol, yield 90%) in the form of black brown powder. Its nuclear magnetic resonance hydrogen spectrum data are 1 H NMR (600 MHz, CDCl3) δ 7.55 - 7.48 (m, 6H), 7.44 (d, J = 8.3 Hz, 2H), 6.72 (s, 1H), 4.64 (m, 1H), 3.78 (d, J = 1.1 Hz, 3H), 3.70 - 3.62 (m, 2H), 2.68 (s, 3H), 2.41 (s, 3H), 1.73 (s, 3H), 1.53 - 1.50 (m, 9H). Its nuclear magnetic resonance carbon spectrum data are 13 C NMR (151 MHz, CDCl3) δ 172.1, 164.7, 155.4, 152.6, 149.9, 142.7, 138.3, 136.5, 134.6, 131.9, 131.2, 130.8, 130.5, 128.9, 127.6, 126.6, 118.7, 65.8, 53.7, 51.9, 36.7, 28.3, 15.2, 14.4, 13.1. Its high resolution mass spectrum data are HRMS. ESI (m / z): [M+H] + calcd for C 31 H 34 N5O4S + : 572.2326, found 572.2301. Purity: 95.42 % (purity represents purity, and the meaning of purity is the same below).

[0060] (2) Synthesis of compound M1

[0061] To the compound 9 (205 mg, 0.36 mmol) in 1 mL of dichloromethane (DCM) and 1 mL of trifluoroacetic acid (TFA) to deprotect the group, after half an hour of reaction, TLC (dichloromethane:methanol = 10:1) to detect the product, extract to adjust pH, and concentrate under reduced pressure, then use silica gel column chromatography to separate and purify to obtain the compound M1 (150 mg, 0.32 mmol, yield 90%) in dark yellow powder. Its nuclear magnetic resonance hydrogen spectrum data are 1 H NMR (600 MHz, CDC13) δ 7.53 - 7.49 (m, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 4.63 (m, 1H), 3.85 (s, 1H), 3.77 (s, 3H), 3.69 - 3.62 (m, 2H), 2.67 (s, 3H), 2.41 (s, 3H), 1.73 (s, 3H). Its nuclear magnetic resonance carbon spectrum data are 13 C NMR (151 MHz, CDC13) δ 172.1, 164.7, 155.5, 149.8, 146.5, 143.2, 135.8, 131.8, 131.2, 130.9, 130.3, 130.0, 128.8, 127.9, 126.0, 115.3, 53.7, 51.8, 36.7, 14.4, 13.0, 11.8. Its high resolution mass spectrum data are HRMS. ESI (m / z): [M+H] + calcd for C 26 H 26 N5O2S + : 472.1802, found 472.1807.

[0062] (3) Synthesis of compound M2

[0063] According to the synthesis method of compound M1, the compound 8 is replaced by 4-(tert-butoxycarbonyl)phenyl boronic acid pinacol ester, and the rest of the conditions remain unchanged, that is, 4-(tert-butoxycarbonyl)phenyl boronic acid pinacol ester reacts with compound 7, and finally compound M2 is obtained. Its nuclear magnetic resonance hydrogen spectrum data are 1 H NMR (400 MHz, CDC13) δ 8.18 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.56 (d, J= 8.2 Hz, 2H), 6.29 (s, 1H), 4.68 (m, 1H), 3.79 (s, 3H), 3.71 - 3.64 (m, 2H), 2.75 (s, 3H), 2.43 (s, 3H), 1.75 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) δ 172.0, 170.4, 164.7, 155.2, 150.1, 144.9, 142.1, 137.7, 131.7, 131.2, 131.0, 130.9, 130.7, 129.1, 129.1, 127.3, 127.1, 53.7, 52.0, 36.6, 14.4, 13.1, 11.7. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 27 H 25 N4O4S + : 501.1591, found 501.1592, Purity: 96.87%.

[0064] (4) Synthesis of compound M17

[0065] According to the synthesis method of compound M1, compound 8 is replaced by 3- (BOC-amino) phenylboronic acid pinacol ester, and the rest is unchanged, that is, 3- (BOC-amino) phenylboronic acid pinacol ester reacts with compound 7, and finally compound M17 is obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (400 MHz, CDC13) δ 7.57 - 7.48 (m, 4H), 7.25 - 7.18 (m, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.91 - 6.86 (m, 1H), 6.69 (d, J = 8.0 Hz, 1H), 4.64 (m, 1H), 3.78 (d, J = 1.6 Hz, 3H), 3.68 - 3.65 (m, 2H), 2.79 (s, 2H), 2.68 (s, 3H), 2.42 (s, 3H), 1.73 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13C NMR (151 MHz, CDC13) δ 172.1, 164.7, 155.4, 146.8, 143.5, 141.3, 136.9, 131.2, 130.5, 129.7, 128.8, 127.0, 117.5, 114.6, 113.7, 103.4, 53.7, 51.9, 36.7, 14.4, 13.1, 11.8. Its high resolution mass spectrometry data is HRMS. ESI (m / z): [M+H] + calcd for C 26 H 26 N5O2S + : 472.1802, found 472.1792, Purity: 95.98%.

[0066] Example 3 Synthesis of BRD4 protein targeted degraders based on covalent fragment (compounds M3, M8, M9, M10, M12, M15)

[0067] The synthesis route is as follows:

[0068]

[0069] (1) Synthesis of compound M3

[0070] In a round-bottom flask, compound 10 (36 mg, 0.2 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 91 mg, 0.24 mmol), N,N-dimethylformamide (DMF) and N,N-diisopropyl ethylamine (DIPEA, 0.1 mL, 0.6 mmol) were added in turn, and compound M1 (94 mg, 0.2 mmol) was added after 10 minutes of activation. Stir at room temperature, detect the reaction by TLC (dichloromethane:methanol = 20:1), extract with ethyl acetate, concentrate under reduced pressure, and then separate and purify by silica gel column chromatography to obtain compound M3 (107 mg, 0.17 mmol, yield 83%) in the form of a deep white powder. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 8.22 (s, 1H), 7.73 (d, J = 7.7 Hz, 2H), 7.68 (s, 1H), 7.58 - 7.48 (m, 6H), 7.46 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.56 (d, J= 15.4 Hz, 1H), 4.65 (m, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.68 - 3.63 (m, 2H), 2.67 (s, 3H), 2.42 (s, 3H), 1.73 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) δ 172.1, 164.8, 161.0, 155.5, 150.0, 146.5, 142.7, 139.2, 138.5, 136.5, 131.7, 131.2, 130.9, 130.8, 129.6, 128.9, 127.9, 127.5, 126.6, 126.1, 120.2, 115.3, 114.2, 55.3, 53.7, 51.9, 36.7, 14.4, 13.1, 11.7. Its high resolution mass spectrum data is HRMS. ESI ([M+H]+): 632.2326, found 632.2329. m / z ) : [M+H]+ + calcd for C 36 H 34 N5O4S + : 632.2326, found 632.2329.

[0071] (2) Synthesis of compound M8

[0072] According to the synthesis method of compound M3, compound 10 is replaced by α-cyanocinnamic acid, and the rest of the conditions remain unchanged, that is, α-cyanocinnamic acid reacts with compound M1 to obtain compound M8. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (600 MHz, CDC13) δ 8.46 (s, 1H), 8.10 (s, 1H), 8.02 - 7.97 (m, 2H), 7.72 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.60 - 7.51 (m, 7H), 4.67 (m, 1H), 3.79 (s, 3H), 3.70 - 3.64 (m, 2H), 2.72 (s, 3H), 2.44 (s, 3H), 1.75 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13C NMR (151 MHz, CDC13) δ 172.1, 164.7, 158.1, 155.4, 154.1, 142.4, 137.1, 136.9, 136.7, 133.2, 132.1, 131.7, 131.3, 130.9, 130.8, 129.9, 129.4, 129.1, 128.6, 127.8, 126.9, 120.8, 117.0, 104.0, 53.8, 51.9, 36.7, 29.7, 14.5, 13.2, 11.9. Its high resolution mass spectrometry data is HRMS. ESI (m / z): [M+H] + calcd for C 36 H 31 N6O3S + : 627.2173, found 627.2172, Purity: 95.15%.

[0073] (3) Synthesis of compound M9

[0074] According to the synthesis method of compound M3, compound 10 is replaced by 3-maleimide propionic acid, and the rest of the conditions remain unchanged, that is, compound M9 is obtained by reacting 3-maleimide propionic acid with compound M1. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (500 MHz, CDC13 δ 8.06 (s, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.54 - 7.49 (m, 6H), 6.68 (d, J = 1.2 Hz, 2H), 4.65 (m, 1H), 3.92 (t, J = 7.1 Hz, 2H), 3.77 (d, J = 1.2 Hz, 3H), 3.67 - 3.63 (m, 2H), 2.77 - 2.72 (m, 2H), 2.69 (d, J = 1.1 Hz, 3H), 2.42 (s, 3H), 1.73 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (126 MHz, CDC13) δ 172.0, 170.5, 168.4, 164.8, 155.4, 150.0, 142.7, 137.9, 136.5, 135.6, 134.1, 131.7, 131.2, 130.9, 130.7, 128.9, 127.4, 126.6, 120.3, 53.6, 51.9, 36.6, 35.6, 34.1, 14.4, 13.1, 11.7. Its high resolution mass spectral data is HRMS. ESI (m / z): [M+H] + calcd for C 33 H 31 N6O5S + : 623.2071, found 623.2075. Purity: 99.75%.

[0075] (4) Synthesis of compound M10

[0076] According to the synthesis method of compound M3, compound 10 is replaced by 2-fluoroacrylic acid, and the rest of the conditions remain unchanged, that is, compound M10 is obtained by reacting 2-fluoroacrylic acid with compound M1. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 8.17 (s, 1H), 7.71 - 7.67 (m, 1H), 7.59 - 7.55 (m, 2H), 7.55 - 7.48 (m, 3H), 7.47 (d, J = 8.1 Hz, 1H), 7.41 - 7.39 (m, 1H), 6.73 (d, 2H), 4.64 (m, 1H), 3.78 (d, J = 3.2 Hz, 3H), 3.67 - 3.63 (m, 2H), 2.67 (s, 3H), 2.41 (s, 3H), 1.73 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13 CNMR (151 MHz, CDC13) δ 172.1, 165.5, 164.5, 155.4, 150.0, 143.5, 141.9, 138.7, 137.6, 133.8, 132.9, 131.9, 131.0, 130.8, 130.7, 129.1, 127.9, 127.3, 127.2, 119.8, 117.7, 83.4, 53.8, 51.9, 36.7, 14.5, 13.1, 11.7. Its high resolution mass spectral data is HRMS. ESI (m / z): [M+H] + calcd for C 29 H 27 N5O3SF+ : 544.1813, found 544.1818. Purity: 95.57%.

[0077] (5) Synthesis of compound M12

[0078] Following the synthetic method for compound M3, compound 10 was replaced with p-aldehyde benzoic acid, with all other conditions remaining unchanged. This resulted in the reaction of p-aldehyde benzoic acid with compound M1 to obtain compound M12. Its 1H NMR spectral data are as follows: 1 H NMR (500MHz, CDCl3) δ 10.06 (d, J = 17.7 Hz, 1H), 8.67 (s, 1H), 8.07 (d, J = 8.1 Hz, 2H), 7.94 (d, J =7.9 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.59 – 7.54 (m, 4H), 7.51 (d, J = 8.2 Hz, 2H), 4.65 (m, 1H), 3.75 (s, 3H), 3.68 – 3.61 (m, 2H), 2.67 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). Its carbon NMR data are as follows: 13 C10 NMR (151 MHz, CDCl3) δ 191.5, 172.0, 164.7, 155.4, 150.0, 142.6, 139.9, 138.2, 137.7, 136.6, 136.3, 131.6, 131.2, 130.9, 130.5, 129.8, 129.4, 129.0, 128.0, 127.6, 126.7, 120.8, 53.6, 51.9, 36.6, 14.4, 13.1, 11.7. Its high-resolution mass spectrometry data are HRMS.ESI (m / z): [M+H] + calcd for C 34 H 30 N5O4S + : 604.2013, found 604.2011. Purity: 98.80%.

[0079] (6) Synthesis of compound M15

[0080] Following the synthetic method for compound M3, compound 10 was replaced with α-lipoic acid, with all other conditions remaining unchanged. This resulted in the reaction of α-lipoic acid with compound M1 to obtain compound M15. Its 1H NMR spectral data are as follows: 1H NMR (500 MHz, CDC13) δ 7.76 (s, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.52 (q, J = 8.4 Hz, 6H), 4.65 (m, 1H), 3.77 (s, 3H), 3.69 - 3.62 (m, 2H), 3.58 - 3.53 (m, 1H), 3.18 - 3.07 (m, 2H), 2.69 (s, 3H), 2.44 (s, 1H), 2.42 (s, 3H), 2.41 - 2.36 (m, 2H), 1.92 - 1.88 (m, 1H), 1.83 - 1.75 (m, 2H), 1.74 (s, 3H), 1.72 - 1.67 (m, 2H), 1.55 - 1.47 (m, 2H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (126 MHz, CDC13) δ 172.0, 171.5, 164.7, 155.4, 149.9, 142.7, 138.1, 136.5, 135.4, 131.7, 131.2, 130.8, 130.7, 128.9, 127.4, 126.6, 120.1, 56.3, 53.6, 51.9, 40.1, 38.4, 37.2, 36.7, 34.5, 28.8, 25.2, 14.4, 13.1, 11.7. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 34 H 38 N5O3S3 + : 660.2131, found 660.2138. Purity: 99.88%.

[0081] Example 4 Synthesis of BRD4 protein targeted degraders based on inhibitor fusion covalent fragments (compounds M4, M11, M14, M18, M20)

[0082] The synthesis route is as follows:

[0083]

[0084] (1) Synthesis of compound M4

[0085] In a round-bottom flask, after adding compound M1 (94 mg, 0.2 mmol), nitrogen protection, then adding 5 mL of dichloromethane (DCM), triethylamine (Et3N, 56 μL, 0.4 mmol), stirring at 0 ℃ for 20 min, adding acryloyl chloride (compound 11, 25 μL, 0.3 mmol), stirring at room temperature, TLC (dichloromethane:methanol = 20:1) detection reaction, ethyl acetate extraction, vacuum concentration, and then using silica gel column chromatography to separate and purify, compound M4 (95 mg, 0.18 mmol, yield 92%) in the form of a deep white powder was obtained. Its nuclear magnetic resonance hydrogen spectrum data are 1 H NMR (500 MHz, CDCl3) δ 8.23 (s, 1H), 7.69 (d, J = 8.2Hz, 2H), 7.55 - 7.49 (m, 6H), 6.45 (dd, J = 16.9, 1.6 Hz, 1H), 6.35 (dd, J = 16.9, 10.0 Hz, 1H), 5.74 (dd, J = 10.0, 1.6 Hz, 1H), 4.65 (m, 1H), 3.77 (s, 3H), 3.70 - 3.61 (m, 2H), 2.67 (s, 3H), 2.42 (s, 3H), 1.73 (s, 3H). Its nuclear magnetic resonance carbon spectrum data are 13 C NMR (101 MHz, CDCl3) δ 172.1, 164.7, 163.9, 155.5, 150.0, 143.3, 138.8, 138.1, 136.6, 135.8, 131.8, 131.2, 130.9, 130.7, 128.9, 127.7, 127.5, 126.7, 120.3, 54.1, 51.9, 37.7, 14.4, 13.1, 11.3. Its high-resolution mass spectrum data are HRMS. ESI ( m / z ) : [M+H] + calcd for C 29 H 28 N5O3S + : 526.1907, found 526.1901 Purity: 98.19%.

[0086] (2) Synthesis of compound M11

[0087] According to the synthesis method of compound M4, compound 11 is replaced by chloroacetyl chloride, and the remaining conditions are unchanged, i.e. compound M11 is obtained by the reaction of chloroacetyl chloride and compound M1. Its nuclear magnetic resonance hydrogen spectrum data are 1H NMR (400 MHz, CDC13) δ 8.49 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.60 - 7.49 (m, 6H), 4.65 (m, 1H), 4.20 (s, 2H), 3.77 (s, 3H), 3.71 - 3.60 (m, 2H), 2.68 (s, 3H), 2.42 (s, 3H), 1.73 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) δ 172.1, 164.6, 164.1, 155.4, 149.9, 142.4, 136.9, 136.7, 136.7, 131.9, 131.1, 130.8, 130.6, 129.0, 127.6, 126.7, 120.4, 53.7, 51.9, 42.9, 36.7, 14.4, 13.1, 11.8. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 28 H 27 N5O3SCl + : 548.1518, found 548.1523. Purity: 99.86%.

[0088] (3) Synthesis of compound M14

[0089] According to the synthesis method of compound M4, compound 11 is replaced by 2,4,5-trifluorobenzoyl chloride, and the remaining conditions are unchanged, that is, compound M14 is obtained by reaction of 2,4,5-trifluorobenzoyl chloride with compound M1. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (400 MHz, CDC13) δ 8.49 (s, 1H), 7.64 (d, J = 15.0 Hz, 1H), 8.05 - 7.98 (m, 1H), 7.74 - 7.70 (m, 2H), 7.62 - 7.59 (m, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.09 - 7.03 (m, 1H), 4.65 (m, 1H), 3.78 (s, 3H), 3.67 - 3.64 (m, 2H), 2.67 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13C NMR (126 MHz, CDC13) δ 172.19, 164.63, 159.29, 155.48, 149.92, 142.40, 137.13, 137.04, 136.84, 132.07, 131.20, 130.80, 130.55, 129.05, 127.77, 126.80, 120.97, 120.29, 120.12, 106.59, 106.42, 106.34, 106.17, 53.81, 51.92, 36.79, 14.49, 13.13, 11.86. Its high resolution mass spectrometry data is HRMS. ESI (m / z): [M+H] + calcd for C 33 H 27 N5O3F3S + : 630.1781, found 630.1772. Purity: 98.64%.

[0090] (4) Synthesis of compound M18

[0091] According to the synthesis method of compound M4, compound M1 is replaced by compound M17, and the remaining conditions are unchanged, that is, compound 11 reacts with compound M17 to obtain compound M18. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 8.48 (s, 1H), 7.91 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.57 - 7.54 (m, 2H), 7.50 (dd, J = 8.0, 3.7 Hz, 2H), 7.37 (t, J = 7.8 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 6.46 - 6.43 (m, 1H), 6.38 - 6.33 (m, 1H), 5.73 (dd, J = 10.1, 1.5 Hz, 1H), 4.66 (m, 1H), 3.77 (s, 3H), 3.71 - 3.61 (m, 2H), 2.67 (s, 3H), 2.42 (s, 3H), 1.72 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) δ 172.1, 164.8, 163.9, 155.4, 150.0, 142.9, 140.8, 138.7, 137.0, 131.8, 131.2, 130.9, 130.7, 129.4, 128.9, 127.7, 122.9, 119.4, 118.7, 53.8, 51.9, 36.7, 14.4, 13.1, 11.7. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 29 H 28 N5O3S + : 526.1907, found 526.1905. Purity: 97.90%.

[0092] (5) Synthesis of compound M20

[0093] According to the synthesis method of compound M4, compound 11 is replaced by propionyl chloride, and the rest of the conditions remain unchanged, that is, compound M20 is obtained by the reaction of propionyl chloride and compound M1. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 8.26 (s, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.50 (q, J = 8.1 Hz, 6H), 4.64 (m, 1H), 3.75 (s, 3H), 3.69 - 3.61 (m, 2H), 2.66 (s, 3H), 2.40 (s, 3H), 2.32 - 2.16 (m, 2H), 1.72 (s, 3H), 1.24 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13 C NMR (151 MHz, CDC13) δ 172.1, 164.7, 155.4, 149.9, 142.7, 139.2, 138.1, 136.6, 135.4, 131.8, 131.2, 130.8, 130.6, 128.9, 127.5, 126.6, 120.0, 53.7, 51.9, 36.7, 30.6, 14.4, 13.1, 11.8, 9.6. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 29 H 30 N5O3S + : 528.2064, found 528.2070. Purity: 99.82%.

[0094] Example 5. Synthesis of BRD4 protein targeted degrader based on inhibitor fusion covalent fragment (Compound M5)

[0095] The synthetic route is as follows:

[0096]

[0097] (1) Synthesis of Compound 13

[0098] Compound 13 (147 mg, 0.22 mmol, yield 89%) was obtained as yellow oil after compound M2 (125 mg, 0.25 mmol), HBTU (113 mg, 0.3 mmol), DMF (5 mL) and DIPEA (97 μL, 0.75 mmol) were activated for 10 minutes, compound 12 (47 mg, 0.25 mmol) was added, stirred at room temperature, TLC (dichloromethane:methanol = 20:1) was used to detect the reaction, ethyl acetate was used for extraction, concentrated and then purified by silica gel column chromatography.

[0099] (2) Synthesis of Compound 14

[0100] Compound 14 (119 mg, 0.21 mmol, yield 98%) was obtained as yellow oil after compound 13 (147 mg, 0.22 mmol) was deprotected with dichloromethane (DCM, 1 mL) and trifluoroacetic acid (TFA, 1 mL), the reaction was detected by TLC (dichloromethane:methanol = 10:1) after half an hour, extracted to adjust pH, concentrated under reduced pressure and then purified by silica gel column chromatography.

[0101] (3) Synthesis of Compound M5

[0102] According to the synthesis method of compound M4, compound M1 was replaced by compound 14, and the rest of the conditions were unchanged, i.e. compound 11 reacted with compound 14 to obtain compound M5 in white powder. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 8.2 Hz, 2H), 7.57 (t, J = 7.3 Hz, 4H), 7.49 (d, J = 8.1 Hz, 2H), 6.59 (d, J = 8.3 Hz, 1H), 6.33 (dd, J = 16.8, 1.9 Hz, 1H), 5.75 (d, J= 10.7 Hz, 1H), 4.65 (m, 1H), 3.78 (s, 3H), 3.70 - 3.61 (m, 8H), 3.55 - 3.43 (m, 2H), 2.68 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) δ 172.1, 170.3, 165.6, 157.8, 155.3, 149.9, 142.0, 132.1, 131.1, 130.6, 129.2, 129.1, 128.8, 127.8, 127.3, 127.1, 126.9, 118.8, 114.0, 103.4, 55.5, 53.8, 51.9, 36.7, 29.6, 14.4, 13.1, 11.8. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 34 H 35 N6O4S + : 623.2435, found 623.2437. Purity: 98.04 %.

[0103] Example 6 Synthesis of BRD4 protein targeted degrader based on inhibitor fusion covalent fragment (compounds M6, M7, M19)

[0104] The synthesis route is as follows:

[0105]

[0106] (1) Synthesis of compound M7

[0107] In a round-bottom flask, compound M1 (94 mg, 0.2 mmol), triethylene diamine (DABCO, 0.04 mmol), compound 15 (52 mg, 0.24 mmol) and dichloromethane (DCM, 5 mL) were stirred at room temperature, TLC (dichloromethane:methanol = 20:1) was used to detect the reaction, ethyl acetate was used for extraction, concentration, separation and purification, and white powdery compound M7 (97 mg, 0.17 mmol, yield 87%) was obtained. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (500 MHz, CDC13) δ 7.51 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 6.98 (s, 0H), 6.68 (d, J= 8.4 Hz, 2H), 6.29 (s, 1H), 5.81 (s, 1H), 4.64 (m, 1H), 4.24 (q, J = 7.2 Hz, 2H), 4.08 (s, 2H), 3.79 (d, J = 6.2 Hz, 4H), 3.67 - 3.63 (m, 2H), 2.69 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13 C NMR (151 MHz, CDC13) δ 172.2, 171.7, 166.7, 164.7, 155.5, 149.8, 147.3, 143.2, 136.9, 135.7, 131.8, 131.3, 131.0, 130.3, 129.2, 128.8, 127.9, 125.9, 113.2, 60.7, 53.7, 51.9, 51.8, 36.9, 14.2, 13.1, 11.8. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 31 H 32 N5O4S + : 570.2170 found 570.2173. Purity: 99.85%

[0108] (2) Synthesis of compound M6

[0109] According to the synthesis method of compound M7, compound M1 is replaced by compound 14, and the remaining conditions are unchanged, that is, compound 14 reacts with compound 15 to obtain white powdery compound M6. Its hydrogen nuclear magnetic resonance spectrum data is 1 H NMR (400 MHz, CDC13) δ 7.57 (ddt, J = 21.6, 8.8, 4.4 Hz, 6H), 7.46 (dd, J = 8.2, 3.2 Hz, 2H), 6.28 (s, 1H), 5.78 (s, 1H), 4.64 (m, 1H), 3.89 - 3.72 (m, 8H), 3.68 - 3.61 (m, 2H), 3.51 - 3.40 (m, 2H), 3.29 - 3.19 (m, 2H), 2.67 (s, 3H), 2.63 - 2.43 (m, 4H), 2.41 (s, 3H), 1.73 (s, 3H). Its carbon nuclear magnetic resonance spectrum data is 13C NMR (151 MHz, CDC13) δ 172.1, 169.9, 167.1, 164.5, 155.4, 149.9, 142.2, 141.5, 139.3, 137.5, 136.4, 135.1, 132.1, 131.1, 130.6, 130.5, 129.0, 127.7, 127.2, 127.1, 58.3, 53.8, 53.3, 52.6, 51.9, 51.9, 36.7, 14.4, 13.1, 11.8. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 36 H 39 N6O5S + : 667.2697, found 667.2697. Purity: 98.25 %.

[0110] (3) Synthesis of compound M19

[0111] According to the synthesis method of compound M7, compound M1 is replaced by compound M17, and the remaining conditions are unchanged, that is, compound M17 reacts with compound 15 to obtain white powdery compound M19. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (500 MHz, CDC13) δ 7.51 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 6.98 (s, OH), 6.68 (d, J = 8.4 Hz, 2H), 6.29 (s, 1H), 5.81 (s, 1H), 4.64 (m, 1H), 4.24 (q, J = 7.2 Hz, 2H), 4.08 (s, 2H), 3.79 (d, J = 6.2 Hz, 4H), 3.67 - 3.63 (m, 2H), 2.69 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) δ 172.2, 171.7, 166.7, 164.7, 155.5, 149.8, 147.3, 143.2, 136.9, 135.7, 131.8, 131.3, 131.0, 130.3, 129.2, 128.8, 127.9, 125.9, 113.2, 60.7, 53.7, 51.9, 51.8, 36.9, 14.2, 13.1, 11.8. Its high resolution mass spectrum data is HRMS. ESI (m / z): [M+H] + calcd for C 31 H 32 N5O4S + : 570.2170 found 570.2173.Purity: 99.85%。

[0112] Example 7 Synthesis of BRD4 protein targeted degrader based on inhibitor fusion covalent fragment (compound M13)

[0113]

[0114] In a round-bottom flask, compound M1 (24 mg, 0.05 mmol), compound 16 (13 mg, 0.055 mmol) were added in turn, and the flask was protected by nitrogen gas exchange. Dichloromethane (DCM, 5 mL) was added, and the mixture was stirred at room temperature. The reaction was detected by TLC (dichloromethane:methanol = 20:1), extracted with ethyl acetate, concentrated, and separated and purified to obtain compound M13 (24 mg, 0.048 mmol, yield 95%) in the form of white powder. Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (600 MHz, CDC13) δ 7.61 (d, J = 8.5Hz, 2H), 7.58 (s, 4H), 7.34 (d, J = 8.4 Hz, 2H), 4.70 (m, 1H), 3.83 (s, 3H), 3.72 - 3.69 (m, 2H), 2.73 (s, 3H), 2.47 (s, 3H), 1.78 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) δ 172.2, 164.4, 155.4, 149.9, 141.7, 139.1, 137.6, 135.9, 132.1, 131.1, 130.8, 130.6, 130.6, 129.1, 128.2, 126.9, 126.2, 53.8, 51.9, 36.7, 14.5, 13.1, 11.9. Its high resolution mass spectrometry data is HRMS. ESI (m / z): [M+H] + calcd for C 27 H 24 N5O2S2 + : 514.1366, found 514.1353. Purity: 95.14%.

[0115] Example 8 Synthesis of BRD4 protein targeted degrader based on inhibitor fusion covalent fragment (compound M16)

[0116]

[0117] In a round-bottom flask, compound M1 (47 mg, 0.1 mmol), compound 17 (13 mg, 0.1 mmol), HATU (46 mg, 0.12 mmol), 5 mL DMF were added in turn, stirred at room temperature, TLC (dichloromethane:methanol = 20:1) detection reaction, ethyl acetate extraction, concentration, separation and purification to obtain white powder compound M16 (48 mg, 0.09 mmol, yield 90%). Its nuclear magnetic resonance hydrogen spectrum data is 1 H NMR (400 MHz, CDC13) δ 7.92 (s, 1H), 7.63 - 7.45 (m, 7H), 7.42 (s, 1H), 6.74 (d, J = 8.3 Hz, 1H), 4.64 (m, 1H), 3.78 (s, 3H), 3.69 - 3.63 (m, 2H), 3.59 (dd, J = 4.7, 2.5 Hz, 0.5H), 3.48 (t, J = 7.0 Hz, 0.5H), 3.11 (t, J = 5.0 Hz, 0.5H), 2.94 (dd, J = 5.6, 2.8 Hz, 0.5H), 2.68 (s, 3H), 2.42 (s, 3H), 1.74 (s, 3H). Its nuclear magnetic resonance carbon spectrum data is 13C NMR (151 MHz, CDC13) δ 172.2, 172.2, 166.5, 155.5, 155.4, 149.9, 143.2, 142.4, 136.5, 129.0, 128.9, 128.0, 127.7, 126.7, 126.1, 120.1, 115.3, 53.7, 51.9, 49.9, 47.7, 36.7, 14.4, 13.1, 11.8. Its high resolution mass spectrometry data is HRMS. ESI (m / z): [M+H] + calcd for C 29 H 28 N5O4S + : 542.1857, found 542.1844. Purity: 96.38%.

[0118] Test Example:

[0119] (1) High content screening test

[0120] Digest and count the well-transfected HEK-293T cells at a density of 1 x 10 4 cells / well into a 96-well plate and incubate overnight. Observe the cell adhesion density under an inverted microscope to reach 70%-80%. Dilute the test compound (i.e., the compound prepared in Example 1-Example 8) with DMEM high-sugar medium to prepare compound solutions at two concentration gradients of 1 µM and 100 nM. Discard the old culture medium, add the compound solution in the preset number order, 3 replicate wells for each concentration, and add an equal amount of PBS buffer at the edge of the hole to eliminate the edge effect. Incubate in the incubator for 12 h, then take out and gently shake to ensure uniform distribution of the cells. Place the 96-well plate in the high content analyzer and perform automatic imaging and data acquisition on each well according to the preset program.

[0121] (2) Western blotting experiment

[0122] Rinse the cell plate with PBS buffer, add Repa lysis buffer (containing protease inhibitors), scrape the cells and transfer them to a centrifuge tube for crushing (use ultrasonic crusher, 20% power, ultrasonic 8 times, 1-2 s each time, interval 5 s). Centrifuge at 12000 rpm for 20 min at 4 °C. Transfer the supernatant to a new centrifuge tube. Take the above supernatant (fixed volume), add 5x loading buffer, mix well and heat at 95 °C for 8 min, use or store at -80 °C. Perform protein quantification. Prepare protein electrophoresis gel at different concentrations as needed.

[0123] Install the electrophoresis device, add the electrophoresis liquid to the top, pull out the comb to leak the loading hole, add the pre-dyed protein (Maker) and the protein sample in turn, set the electrophoresis conditions (70 V, 30 min), run the gel, and when the mixture of the pre-dyed protein and the protein sample is flush, separate them under the voltage condition (120 V, 60 min), and stop when the double-color loading buffer (Loading Buffer) reaches the bottom. Soak the polyvinylidene fluoride membrane (PVDF membrane) of appropriate size in methanol for 1 min, rinse it with the membrane transfer solution, cover the PVDF membrane to the gel block after electrophoresis, and cut off the excess gel block; transfer the gel block covered with the PVDF membrane to the electrotransfer clamp to discharge the bubbles, clamp it tightly in the electrotransfer tank, and transfer the membrane under the condition of 230 mA constant current and ice bath. Set the electrotransfer time according to the protein molecular weight.

[0124] After electrotransfer, place the PVDF membrane in 5% skimmed milk at room temperature for 1 h. After blocking, remove the PVDF membrane, rinse it with 1x TBST buffer for 3 times, 20 min each time, cut the PVDF membrane at the position of the target protein and mark it, and incubate it uniformly in the primary antibody at 4 °C overnight. After taking it out, rinse it with 1x TBST buffer for 3 times, 20 min each time, incubate the PVDF membrane in the secondary antibody at room temperature for 1 h. After taking out the PVDF membrane, rinse it with 1x TBST buffer for 3 times, 20 min each time, mix equal volumes of A and B in the developing reagent kit to prepare the developing solution in the light-proof box, soak and incubate it for 3 min, take out the developing solution, and analyze it.

[0125] (3) Cell viability assay

[0126] After cell digestion and counting, inoculate 3000-5000 cells per well in a 96-well plate, and incubate it in an incubator overnight. Prepare the compound with complete culture medium by gradient dilution, replace the culture medium in the 96-well plate, 3 replicates for each concentration, add PBS to the edge wells of the plate, and incubate it in an incubator for 48 h. Add 10 μL of cell counting kit-8 (CCK-8) reagent to the 96-well plate, shake well, and incubate it in a cell incubator for 1-2 h. After taking it out, detect the absorbance OD value at 450 nm with a microplate reader, and calculate the cell survival rate.

[0127] The results are as follows:

[0128] (1) Construction of high-content screening platform and activity test of each compound

[0129] To detect whether the inhibitor can be converted into a degrading agent under the modification of covalent fragments, high-content screening experiments were first carried out in HEK-293T cells stably expressing BD1 domain, BD2 domain and BD1-BD2 domain, respectively. The HEK-293T cells were inoculated in a 96-well plate at 1x10 4The cells were seeded at a density of 1 μM / well in 96-well plates and incubated overnight. Once the adhesion density reached 70%-80%, each candidate compound was treated with two concentration gradients of 1 μM and 100 nM, with three replicates for each condition. Compound HL435 (structural formula: [insert structural formula here]) was used. The molecular formula is C 47 H 48 BrCF3N7O7S) was used as a positive control. After incubation for 12 hours, images were acquired using a high-content imaging system. The degradation efficiency of BRD4 protein after compound treatment was obtained by measuring the fluorescence ratio of mScarlet to enhanced green fluorescent protein (EGFP) in each well, and a heatmap was plotted. Figure 4 (a) and bar chart ( Figure 4 In (b), the high-content screening used overexpressed fragments, which typically exhibited lower degradation effects than endogenous expression models. Results showed that some compounds showed significant protein degradation activity at both concentrations, with compounds M4, M5, M7, and M11 showing superior effects. These screening results were then further validated in MDA-MB-231 cells. Western blot analysis was performed on MDA-MB-231 cells after 12 hours of treatment to assess the effects of different compound concentrations (0.1 μM and 1 μM) on endogenous BRD4 protein levels. The results indicated that treatment with compounds M4, M5, M7, and M11 significantly reduced BRD4 protein expression. Figure 4 In (c), the degree of degradation is dose-dependent at different concentrations. Figure 4 The results of the middle (d) screening are basically consistent with those of the high-content screening.

[0130] (2) Target specificity, dose dependence and time dependence of compound M4 on BET family proteins

[0131] Digest and count well-transfected HEK-293T cells, then add compound M4 (structure shown in [link to compound]) at concentrations of 0, 1, 5, 10, 50, and 100 nM. Figure 5HEK-293T cells were treated with compound M4 at different concentrations (a) and different time (b) respectively for 12 hours. After the treatment of HEK-293T cells, the HEK-293T cells were lysed and total proteins were extracted for Western blot and protein degradation semi-quantitative analysis. The following primary antibodies were used in Western blot: BRD2 antibody (purchased from Abeam company, named Anti-BRD2 antibody [EPR7642]-CHIP Grade), BRD3 antibody (purchased from Abeam company, named Anti-BRD3 antibody [EPR23743-226]), BRD4 antibody (purchased from CST company, named BRD4 (E2A7X) Rabbit mAb), BRDT antibody (purchased from absin company, named Rabbit anti-BRDT Polyclonal Antibody (N-term)), a-tubulin (purchased from CiteAb company, named Anti-a-Tubulin (T6047)). The corresponding HRP-labeled secondary antibodies were used, i.e. anti-rabbit IgG for BRD2 antibody, BRD3 antibody, BRD4 antibody, BRDT antibody, and anti-mouse IgG for a-tubulin; the secondary antibodies were diluted 5000 times with the antibody dilution buffer (1X TBST).

[0132] The results are shown in Figure 5 Fig. 3 (b) and Figure 5 Fig. 3 (c), which show the degradation effect of compound M4 on BET family proteins at different concentrations; compound M4 has a concentration-dependent degradation effect on BRD4 protein and BRD3 protein. Further fitting of the dose-response curve Figure 5 Fig. 3 (d), in which the DC 50 of BRD3 protein and BRD4 protein are 3.443 nM and 5.781 nM respectively, which indicates that compound M4 has good selective degradation activity. In order to evaluate the time dependence, 100 nM of compound M4 was treated for different times (1, 3, 6, 9, 12, 24 hours), and the same Western blot process as above was used to detect the changes of BRD4 protein Figure 5 Fig. 3 (e), and protein degradation semi-quantitative analysis Figure 5 Fig. 3 (f). The results show that compound M4 can induce significant degradation of BRD4 protein within 6 hours, and presents a dose-dependent effect (DC 50 = 5.781 nM).

[0133] (3) Mechanism of degradation of compound M4

[0134] HEK-293T-BD1 reporter cell line stably expressing BD1 domain of BRD4 was constructed and seeded in 96-well plates and incubated overnight. Then 100 nM of compound M4 and different degradation pathway inhibitors were added to each well respectively. Figure 6 in (a), Figure 6 in (b), and the high content test results showed that the addition of autophagy pathway related inhibitors chloroquine (CQ), bafilomycin Al (Baf Al) could not inhibit the degradation of BRD4 protein by compound M4, while the addition of ubiquitin-proteasome pathway inhibitors (Z-Leu-Leu-Leu-al): N-[(benzyloxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide (MG132), bortezomib (PS341), 4-[4-[(5-nitro-2-furanyl)methylene]-3,5-dioxo-1-pyrazolidinyl]benzoic acid ethyl ester (PYR41), [(1S,2S,4R)-4-[4-[[(1S)-2,3-dihydro-1H-inden-1-yl]amino]-7H-pyrrolo[2,3-D]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl sulfamate (MLN4924) would inhibit the degradation of BRD4 protein by compound M4, indicating that the degradation of BRD4 protein by compound M4 was achieved through the ubiquitin-proteasome pathway. MDA-MB-231 cells were seeded in 96-well plates and incubated overnight, then treated with the following combinations for 6 hours: compound M4 (100 nM), CQ (20 μM), Baf Al (125 nM), CQ+M4, Baf Al+M4; by using autophagy-lysosome pathway inhibitors chloroquine (CQ) and bafilomycin Al (Baf Al) to pretreat the cells, it was found that the degradation of BRD4 protein by compound M4 was not affected, which ruled out the possibility that compound M4 degraded BRD4 protein through the autophagy-lysosome pathway Figure 6 in (c), Figure 6 in (d). To further verify the above conclusion, as shown in Figure 6 in (e), Figure 6 in (f), the following treatment groups were additionally set up, and the above WB procedure was repeated: compound M4 (100 nM), MG132 (5 μM), PS341 (50 nM), MLN4924 (2 μM), PYR41 (30 μM), and compound M4 combined with the above inhibitors (M4+MG132, M4+PS341, M4+MLN4924, M4+PYR41), and the results showed that the above four inhibitors could significantly inhibit the degradation of BRD4 protein mediated by compound M4, indicating that compound M4 achieved target protein degradation through the ubiquitin-proteasome system. In summary, compound M4 degraded BRD4 protein through the ubiquitin-proteasome pathway mediated by E3 ubiquitin ligase DCAF11.

[0135] (4) Verification of the inhibitory effect of compound M4 on the proliferation of various cancer cells and cytotoxicity

[0136] Logarithmic growth phase cells were counted and then inoculated into 96-well plates at a density of 3000-5000 cells per well, and incubated at 37°C overnight. When the cell monolayer confluence was greater than 70%, the old culture medium was removed, and different concentrations of the test compound prepared by logarithmic dilution method (complete culture medium was prepared, and 3 replicate wells were prepared for each concentration) were added. The edge wells were filled with PBS to prevent evaporation, and incubated at 37°C for 48 h. Then 10% CCK-8 reagent was added to each well, and shaken to mix well, and then incubated at 37°C for 1-2 h in the dark. The OD of each well was read at 450 nm by an enzyme-labeled instrument, and the cell survival rate was calculated according to the formula in the CCK-8 instruction manual.

[0137] The results are shown in Table 1. Figure 7 As shown in Table 1, compound M4 inhibited the human breast cancer cell line (MDA-MB-231) and the human glioma cell line (U251) in a dose-dependent manner within 48 h, and the cell viability continuously decreased with the increase of the dose. The IC 50 was 0.54 μM (MDA-MB-231) and 1.39 μM (U251), respectively. Among them, the inhibitory activity of compound M4 on MDA-MB-231 was particularly outstanding (IC 50 < 1 μM). The results show that compound M4 not only has a significant anti-tumor effect on breast cancer and glioma, which are two malignant tumors with different pathological origins, but also exhibits potential broad-spectrum anticancer properties.

[0138] In summary, compound M4 provided by the present application can induce significant degradation of BRD4 protein within 6 hours, and shows a dose-dependent effect (DC 50 = 5.781 nM). Further mechanism studies show that compound M4 degrades BRD4 protein through the ubiquitin-proteasome pathway mediated by E3 ubiquitin ligase DCAF11. In addition, cell experiments confirm that compound M4 has broad-spectrum anticancer ability.

[0139] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A degrader based on inhibitor fusion covalent fragment, characterized in that, The degradation agent based on inhibitor fusion covalent fragments includes a compound shown in formula I and its structural isomers: Formula I; wherein R is a covalent moiety capable of forming a covalent bond with the thiol of a cysteine in an E3 ubiquitin ligase; Me is a methyl group; R is selected from one of the following structures: 、 、 、 、 、 、 、 、 , , , , , , , ; wherein represents the point of attachment.

2. Use of a degrader based on an inhibitor fusion covalent moiety according to claim 1 for the preparation of a medicament for the treatment of cancer. The cancer is selected from at least one of breast cancer, glioma.

3. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The medicament comprises a degrader based on an inhibitor fusion covalent moiety according to claim 1 and a pharmaceutically acceptable carrier.

4. The medicament according to claim 3, characterized in that, The pharmaceutically acceptable carrier comprises at least one of an excipient, a glidant, a diluent, a preservative, a colorant, a flavoring agent, a wetting agent, a suspending agent, a stabilizer, an isotonic agent, a solvent, an emulsifying agent.

Images