A new type of brd4 protein targeted degrader, its preparation method and application

Abstract

The application discloses a novel BRD4 protein targeted degradation agent, a preparation method and application thereof, and belongs to the field of medicinal chemistry. Active test results show that the novel PROTAC molecule for targeting and degrading BRD4 of the application directly degrades BRD4 protein by using a ubiquitin-proteasome pathway, has a remarkable degradation efficiency, can directly kill tumor cells with high expression of BRD4 protein, and has a better effect than a positive control drug (+)‑JQ‑1. Meanwhile, the PROTAC molecules can avoid the regulation of feedback increase of target protein content caused by a conventional small molecule inhibitor (+)‑JQ‑1 of BRD4.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a novel BRD4 protein-targeting degrader, its preparation method, and its application. Background Technology

[0002] Histone lysine acetylation is an important post-transcriptional modification of genes. Bromodomain and extra-terminal (BET) proteins can recognize and bind to acetylated lysine residues in histones and non-histone proteins, regulating chromatin dynamics, cellular processes, and disease progression. BRD4, one of the four subtypes of the BET family, has been the most extensively studied. BRD4 activates transcriptional initiation response genes, regulates transcription factors such as C-MYC and BCL2, and participates in processes such as cell cycle, cell proliferation, and apoptosis. Upregulation of BRD4 expression leads to abnormal expression of its downstream genes, playing a crucial role in the occurrence and development of tumors such as leukemia, breast cancer, melanoma, liver cancer, and lung cancer. Currently, research on BRD4 inhibitors is booming. (+)-JQ-1 was the earliest reported BRD4 inhibitor, and several other drugs have entered clinical trials, such as OTX-015, GSK525762, I-BET151, and ABBV-075.

[0003] BRD4 inhibitors can reduce the expression of its downstream proteins, thereby controlling the disease. However, inhibition of BRD4 protein can cause a feedback increase in BRD4 protein expression, significantly affecting the efficacy of the inhibitors. Furthermore, during research on BRD4 inhibitors, it was discovered that some tumors (such as triple-negative breast cancer) can develop resistance to BRD4-targeted therapies. Therefore, developing drugs with novel mechanisms targeting BRD4 has practical clinical significance.

[0004] Proteolytic Targeting Chimera (PROTAC) technology links the ligand of an E3 ubiquitin ligase to a target protein molecule via a linker chain, forming a ternary complex of the target protein, the PROTAC molecule, and the E3 ubiquitin ligase. The target protein is ubiquitinated by the E3 ubiquitin ligase and further degraded by the proteasome, thus losing its function. Compared to traditional small molecule inhibitors, PROTAC technology has advantages such as applicability to targets that are traditionally difficult to develop into drugs, high protein degradation efficiency, and less likelihood of inducing negative feedback increases in the target protein. Therefore, addressing the clinical bottlenecks encountered by BRD4 small molecule inhibitors, developing PROTAC molecules targeting the BRD4 protein can directly overcome the limitation of compensatory increases in the target protein caused by BRD4 small molecule inhibitors and avoid the potential drug resistance problem. Summary of the Invention

[0005] The purpose of this invention is to provide a class of compounds that target the degradation of BRD4 protein, their preparation and application, specifically pyrrolopyrazinone PROTAC molecules based on Cereblon E3 ubiquitin ligands and their preparation methods, as well as the application of such compounds as BRD4 protein degraders in the treatment or prevention of diseases such as tumors, inflammation, and metabolism.

[0006] The purpose of this invention is to provide some new pyrrolopyrazinone small molecules or their stereoisomers, tautomers and their pharmaceutically acceptable salts, hydrates, prodrugs and drug combinations with the compound as the active ingredient.

[0007] The purpose of this invention is to provide a novel class of antitumor compounds, the target tumors of which may be, but are not limited to, multiple myeloma, gastric cancer, lung cancer, breast cancer, esophageal cancer, colon cancer, medulloblastoma, acute myeloid leukemia, chronic leukemia, melanoma, prostate cancer, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, skin cancer, ovarian cancer, colon cancer, glioma, thyroid cancer, or pancreatic cancer.

[0008] The present invention also aims to provide a method for preparing novel bifunctional small molecules of pyrrolopyrazinones.

[0009] Another object of the present invention is to provide a pharmaceutical formulation containing a novel bifunctional small molecule of pyrrolopyrazinone.

[0010] A novel class of PROTAC molecules that target and degrade BRD4, wherein the PROTAC molecules that target and degrade BRD4 are selected from compounds represented by general formula (I) or their stereoisomers, tautomers, and pharmaceutically acceptable salts, hydrates, or prodrugs:

[0011]

[0012] in:

[0013] R 1 Or R 2 Selected from C 1-12 Alkylene, -CH2(CH2OCH2) n CH2-, n = integers from 1 to 10,

[0014] R 3 Selected from -H, C l-5 alkyl,

[0015] As a preferred embodiment of the present invention

[0016] R 1 Selected from C 1-5 Alkylene;

[0017] R 2 Selected from C 2-l2 Alkylene, -CH2(CH2OCH2) n CH2-, n = integers from 1 to 5;

[0018] R 3 Selected from C l-5 alkyl.

[0019] As a further preferred embodiment of the present invention,

[0020] R 1 Selected from C 2-4 Alkylene;

[0021] R 2 Selected from C 4-10 Alkylene, -CH2(CH2OCH2) n CH2-, n = 2-4 integers;

[0022] R 3 Selected from C l-3 alkyl.

[0023] In some preferred embodiments of the present invention, the PROTAC molecule targeting the degradation of BRD4 is selected from compounds represented by the following formula or their stereoisomers, tautomers, and pharmaceutically acceptable salts, hydrates, or prodrugs:

[0024]

[0025] R 1 Selected from -(CH2)2- and -(CH2)3-;

[0026] R 2 Selected from C 4-l0 Alkylene, -CH2(CH2OCH2) n CH2-, n = integers from 1 to 4;

[0027] R 3 Selected from methyl.

[0028] In some other preferred embodiments of the present invention, the PROTAC molecule targeting the degradation of BRD4 is selected from compounds represented by the following formula or their stereoisomers, tautomers, and pharmaceutically acceptable salts, hydrates, or prodrugs:

[0029]

[0030] R 1 Selected from -(CH2)2-;

[0031] R 2 Selected from C 4-l0Alkylene, -CH2(CH2OCH2) n CH2-, n = integers from 1 to 4;

[0032] R 3 Selected from methyl.

[0033] In a preferred embodiment of the present invention, the PROTAC molecule targeting the degradation of BRD4 is selected from the following compounds or their stereoisomers, tautomers, and pharmaceutically acceptable salts, hydrates, or prodrugs:

[0034]

[0035]

[0036] Furthermore, its stereoisomers, tautomers, and pharmaceutically acceptable salts are selected from acetates, adipic acid salts, aspartate salts, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates / sulfates, borates, camphor sulfonates, citrates, cyclohexanesulfonates, ethanedisulfonates, ethanesulfonates, formates, fumarates, glucohepanoates, glucuronates, hexafluorophosphates, hydrochlorides / oxides, hydrobromates / bromines, hydroiodates / iodides, hydroxyethyl sulfonates, lactates, malates, maleic anhydride, and malates. Salts, methanesulfonates, methyl sulfates, naphthates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, dihydronaphthalates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, glycosides, stearates, succinates, tannins, tartrates, toluenesulfonates, trifluoroacetates and sine, aluminum salts, arginine salts, benzathine penicillin salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, sodium salts, potassium salts, ammonium salts, tromethamine salts and zinc salts;

[0037] Furthermore, its stereoisomers, tautomers, and pharmaceutically acceptable salts, hydrates, and prodrugs can be used alone as tumor treatment drugs or in combination with existing tumor treatment drugs to exert synergistic effects for tumor treatment and prevention.

[0038] Furthermore, the dosage forms of its stereoisomers, tautomers, and pharmaceutical compositions can be tablets, capsules, pills, suppositories, soft capsules, oral liquids, suspensions, injections, and other commonly used pharmaceutical dosage forms, and contain a therapeutically effective amount of the compound and pharmaceutically acceptable excipients;

[0039] Furthermore, the method for preparing the compound, the method following the reaction route:

[0040]

[0041] Where R 1 R 2 As defined in any of the technical solutions,

[0042] The method includes the following steps:

[0043] Using ethyl 3-methyl-1H-pyrrole-2-carboxylate, 2-bromo-1-fluoro-4-nitrobenzene, 3-fluorophthalimide, and 4-fluorophthalimide as raw materials, the final product compound 1-11 was obtained through a 13-step synthetic reaction.

[0044] (1) Using ethyl 3-methyl-1H-pyrrole-2-carboxylic acid a and bromoacetaldehyde diethanol condensate as raw materials, a substitution reaction was carried out under the action of NaH to obtain b;

[0045] (2) Compound b undergoes ester hydrolysis under alkaline conditions to give c;

[0046] (3) Compound c reacts with ammonium chloride under alkaline conditions via an amide condensation agent to yield d;

[0047] (4) Compound d undergoes self-cyclization under acidic conditions to give e;

[0048] (5) Compound e undergoes a bromination reaction to give f;

[0049] (6) Using 2-bromo-1-fluoro-4-nitrobenzene g as a reactant, it reacts with phenol to obtain h;

[0050] (7) Compound h is reduced with iron powder to obtain i;

[0051] (8) Compound i reacts with pinacol diboronate via palladium catalyst to yield j;

[0052] (9) Compound j undergoes a Suzuki coupling reaction with compound f to give k;

[0053] (10) Compound k was condensed with succinic anhydride to form an amide, yielding l;

[0054] (11) n-1 is obtained by a substitution reaction between 3-fluorophthalimide m-1 and 3-amino-2,6-piperidinide hydrochloride.

[0055] (12) Compound n-1 reacts with N-Boc-diamine to give compounds o-1 to o-8;

[0056] (13) Compounds o-1 to o-8 were condensed with compound l in a condensing agent catalysis to give the final product compound 1-8;

[0057] The preparation of compounds 9-11 differs from that of compounds 1-8 in that (11) 3-fluorophthalimide m-1 is replaced with 4-fluorophthalimide m-2.

[0058] The application of the PROTAC molecule that targets and degrades BRD4 described in this invention in the preparation of drugs for treating and / or preventing tumors; the tumor is preferably any one of multiple myeloma, gastric cancer, lung cancer, breast cancer, esophageal cancer, medulloblastoma, acute myeloid leukemia, chronic leukemia, melanoma, prostate cancer, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, skin cancer, ovarian cancer, colon cancer, glioma, thyroid cancer, and pancreatic cancer.

[0059] As a preferred embodiment of the present invention, the PROTAC molecule that targets and degrades BRD4 is used as the sole active ingredient, or together with other antitumor compounds, as an active ingredient in the preparation of medicaments for the treatment and / or prevention of tumors.

[0060] A pharmaceutical formulation composition comprising a compound of general formula (I) or a pharmaceutically acceptable salt thereof as an active agent and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier refers to one or more inert, non-toxic solid or liquid fillers, diluents, adjuvants, etc., which do not react adversely with the active compound or the patient.

[0061] Furthermore, the dosage form can be a commonly used pharmaceutical dosage form such as tablets, capsules, pills, suppositories, soft capsules, oral liquids, suspensions, and injections. Oral medications and capsules contain traditional excipients such as fillers, diluents, lubricants, dispersants, and binders. They can be prepared according to methods well known in the art.

[0062] The amount of the compound of the present invention administered will depend on the individual being treated, the rate of administration, the disposal of the compound, and the judgment of the prescribing physician. Generally, the proven advantageous amount for achieving the desired results is a total amount of the compound of formula (I) administered over 24 hours per kilogram of body weight, preferably a total amount of about 0.1-40 mg / kg. If necessary, it may be administered in the form of several single doses.

[0063] Beneficial effects:

[0064] The novel PROTAC molecules of this invention target and degrade BRD4 directly via the ubiquitin-proteasome pathway, exhibiting significant degradation efficiency and directly killing tumor cells with high BRD4 protein expression, demonstrating superior efficacy compared to the positive control drug (+)-JQ-1. Furthermore, these PROTAC molecules avoid the feedback increase in target protein levels caused by the application of the conventional small molecule inhibitor of BRD4, (+)-JQ-1. Attached Figure Description

[0065] Figure 1 Compounds (+)-JQ-1 and 7 effectively reduced the expression levels of BRD4 protein and its downstream protein c-Myc in MV4-11 cells; * p<0.05, ** p<0.01, *** p<0.001, compared with the control group. Detailed Implementation

[0066] To make the objectives and technical solutions of this invention clearer, the invention is further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, specific experimental methods not mentioned in the following embodiments were performed according to conventional experimental methods.

[0067] The structure of the compound was determined by nuclear magnetic resonance spectroscopy (NMR). 1 H NMR, 13 The reaction was confirmed by C NMR and LC-MS; the reaction was monitored by thin-layer chromatography (TLC) or LC-MS, and the developing solvent systems used were: dichloromethane and methanol system, n-hexane and ethyl acetate system, and petroleum ether and ethyl acetate system.

[0068] The compound is purified using column chromatography, which typically uses 200-300 mesh silica gel as the stationary phase. The eluent systems include dichloromethane and methanol, and n-hexane and ethyl acetate. The volume ratio of the solvent is adjusted according to the different polarities of the compound.

[0069] In the following examples, unless otherwise specified, the reaction temperature is room temperature (20°C to 30°C).

[0070] The present invention will be further described below with reference to embodiments. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the present invention. Various modifications made by those skilled in the art based on the teachings of the present invention should be within the scope of protection claimed in the claims of this application.

[0071] Example 1

[0072] Preparation of compound b

[0073] Compound a (1.0 mmol, 153.18 mg), bromoacetaldehyde diethanol (1.5 mmol, 226 μL), and Cs₂CO₃ (1.3 mmol, 423.56 mg) were dissolved in 5 mL of DMF and reacted at 110 °C for 24 h under nitrogen protection. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 234.2 mg of a yellow oily liquid compound b, in 87.0% yield. 1 H NMR (300MHz, CDCl3) δ (ppm) 6.77 (d, J = 2.5Hz, 1H), 5.94 (d, J = 2.5Hz, 1H), 4.64 (t, J = 5.3Hz, 1H), 4.29 ( m,4H),3.67(m,2H),3.38(m,2H),2.32(s,3H),1.36(t,J=7.1Hz,3H),1.14(t,J=7.0Hz,6H).LC-MS(ESI + )calcd.for C 14 H 23 NO4Na[M+Na] + :292.15,found292.19.

[0074] Example 2

[0075] Preparation of compound c

[0076] Compound b (0.5 mmol, 134.67 mg) and lithium hydroxide monohydrate (2.5 mmol, 104.9 mg) were dissolved in a mixed solvent of 3 mL ethanol and 3 mL water, and reacted at 75 °C for 18 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature, extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 100.3 mg of white solid compound c, with a yield of 83.2%. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 12.21 (s, 1H), 6.89 (d, J = 2.5Hz, 1H), 5.93 (d, J = 2.3Hz, 1H), 4.59 (t, J = 5.3 Hz,1H),4.26(d,J=5.3Hz,2H),3.57(m,2H),3.29(m,3H),2.23(s,3H),1.03(t,J=7.0Hz,6H).LC-MS(ESI - )calcd.for C 12 H 18 NO4[MH]- :240.13, found 240.24.

[0077] Example 3

[0078] Preparation of compound d

[0079] Compound c (0.4 mmol, 96.5 mg), ammonium chloride (64.19 mg, 1.2 mmol), and HATU (0.6 mmol, 228.14 mg) were dissolved in 3 mL of DMF solvent, and then DIPEA (2.0 mmol, 348 μL) was added. The reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the product was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 82.1 mg of white solid compound d, with a yield of 85.5%. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 7.05 (s, 2H), 6.74 (d, J = 2.5Hz, 1H), 5.84 (d, J = 2 .4Hz,1H),4.59(t,J=5.3Hz,1H),4.17(d,J=5.3Hz,2H),3.57(m,2H),3.32(m, 2H),2.19(s,3H),1.05(t,J=7.0Hz,6H).LC-MS(ESI + )calcd.for C 12 H 20 N₂O₃Na[M+Na] + :263.14,found263.09.

[0080] Example 4

[0081] Preparation of compound e

[0082] Compound d (0.5 mmol, 120 mg) was dissolved in 4 mL of glacial acetic acid and reacted at 100 °C for 1 h. After the reaction was completed, the mixture was cooled to room temperature, extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 62 mg of white solid compound e, with a yield of 83.7%. 1 HNMR (300MHz, CDCl3) δ (ppm) 10.42 (s, 1H), 7.00 (d, J = 2.6 Hz, 1H), 6.87 (d, J = 5. 8Hz,1H),6.40(t,J=5.2Hz,1H),6.36(d,J=2.9Hz,1H),2.60(s,3H).LC-MS(ESI +)calcd.forC8H8N2O[M+H] + :149.07, found 149.45.

[0083] Example 5

[0084] Preparation of compound f

[0085] Compound e (0.34 mmol, 50 mg) was dissolved in a mixed solvent of 2 mL dichloromethane and 1 mL trifluoroacetic acid. The mixture was first stirred in an ice bath at 0 °C, and then N-bromosuccinimide (0.31 mmol, 55 mg) was added, and the reaction was carried out in an ice bath at 0 °C for 30 min. After the reaction was completed, the solvent was evaporated, and a saturated sodium bicarbonate solution was added. The mixture was stirred at room temperature for 20 min, and a white solid precipitated. The solid was filtered, the filter cake was washed with water, and dried to give 71 mg of a white solid compound f, with a yield of 92.4%. 1 H NMR (300MHz, CDCl3) δ (ppm) 10.30 (s, 1H), 7.00 (d, J = 6.0Hz, 1H), 6.53 (t, J = 6.0Hz, 1H), 6.43 (s, 1H), 2.60 (s, 3H). LC-MS (ESI + )calcd.for C8H8BrN2O[M+H] + 228.07, found 228.93.

[0086] Example 6

[0087] Preparation of compound h

[0088] Compound g (1.5 mmol, 330 mg), phenol (1.8 mmol, 158 μL), and potassium carbonate (4.5 mmol, 621.94 mg) were dissolved in 4 mL of DMF and reacted at 90 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid h 420.12 mg, with a yield of 95.6%. 1 H NMR (300MHz, CDCl3) δ (ppm) 8.55 (d, J = 2.7Hz, 1H), 8.09 (dd, J = 9.1, 2.7Hz, 1H), 7.52–7 .39(m,2H),7.34–7.25(m,1H),7.10(d,J=7.4Hz,2H),6.83(d,J=9.1Hz,1H).LC-MS(ESI - )calcd.for C 12 H8BrNO3[MH] -291.98, found 291.96.

[0089] Example 7

[0090] Preparation of compound i

[0091] Compound h (1.5 mmol, 441 mg), iron powder (10.5 mmol, 588 mg), and glacial acetic acid (10.5 mmol, 606 μL) were dissolved in 12 mL of ethanol and the mixture was heated under reflux at 85 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 175.8 mg of a yellow oily liquid compound i, with a yield of 44.6%. 1 H NMR (300MHz, CDCl3) δ (ppm) 7.42–7.22 (m, 2H), 7.05 (t, J = 7.4Hz, 1H), 6.99 (d, J = 2.7Hz, 1H), 6.91 (d, J = 8.9Hz, 3H), 6.64 (dd, J = 8.6, 2.7Hz, 1H). LC-MS (ESI + )calcd.for C 12 H 11 BrNO[M+H] + :264.00, found 264.07.

[0092] Example 8

[0093] Preparation of compound j

[0094] Compound i (0.5 mmol, 131.5 mg), pinacol diboronate (1.0 mmol, 253.9 mg), Pd(dppf)Cl2 (0.025 mmol, 18.30 mg), and potassium acetate (1.0 mmol, 98.14 mg) were dissolved in 5 mL of anhydrous 1,4-dioxane solvent. The mixture was heated to reflux at 100 °C for 12 hours under nitrogen protection. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 74.9 mg of a brown oily liquid j, with a yield of 48.1%. LC-MS (ESI) + )calcd.forC 18 H 22 BNO3[M+H] + 312.18, found 312.56.

[0095] Example 9

[0096] Preparation of compound k

[0097] Compound f (0.22 mmol, 48.9 mg), compound j (0.24 mmol, 74 mg), Pd(PPh3)4 (0.01 mmol, 12.50 mg), and K2CO3 (0.43 mmol, 59.8 mg) were dissolved in 3 mL of a mixed solvent of 1,4-dioxane and 1 mL of H2O. The mixture was heated to reflux at 100 °C for 12 h under nitrogen protection. After the reaction was completed by TLC monitoring, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give compound k 54.9 mg as a white solid, with a yield of 75.4%. 1 H NMR(300MHz,DMSO-d6)δ(ppm)10.37–10.07(d,1H),7.19(t,J=7.5Hz,2H),6.88(dt,J=15.7,6.8Hz ,3H),6.78–6.51(m,4H),6.44(t,J=5.1Hz,1H),6.25(s,1H),5.21(s,2H),2.38(s,3H).LC-MS(ESI + )calcd.forC 20 H 18 N3O2[M+H] + 332.14, found 332.87.

[0098] Example 10

[0099] Preparation of compound l

[0100] Compound K (0.08 mmol, 26.48 mg) and succinic anhydride (0.096 mmol, 9.60 mg) were dissolved in 3 mL of toluene. The mixture was refluxed at 115 °C for 6 h under nitrogen protection. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature, and a solid precipitated. The precipitate was filtered, washed three times with n-hexane, and dried to give 130.5 mg of a white solid, with a yield of 88.4%. LC-MS (ESI) yielded 30.5 mg. + )calcd.for C 24 H 22 N3O5[M+H] + :432.16, found 432.12.

[0101] Example 11

[0102] Preparation of compound n-1

[0103] Compound m-1 (0.5 mmol, 83.06 mg) and 3-amino-2,6-piperidinedione (0.5 mmol, 82.30 mg) were dissolved in 6 mL of acetic acid, and sodium acetate (0.6 mmol, 49.22 mg) was added. The mixture was heated to reflux at 120 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 102.69 mg of white solid n-1, with a yield of 74.4%. LC-MS (ESI) + )calcd.for C 13 H 10 FN₂O₄[M+H] + :277.06, found 277.14.

[0104] Example 12

[0105] Preparation of compound n-2

[0106] Compound m-2 (0.5 mmol, 83.06 mg) and 3-amino-2,6-piperidinedione (0.5 mmol, 82.30 mg) were dissolved in 6 mL of acetic acid, and sodium acetate (0.6 mmol, 49.22 mg) was added. The mixture was heated to reflux at 120 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 90.68 mg of white solid n-2, with a yield of 65.7%. LC-MS (ESI) + )calcd.for C 13 H 10 FN₂O₄[M+H] + :277.06, found 277.14.

[0107] Example 13

[0108] Preparation of compound o-1

[0109] Compound n-1 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,4-butanediamine (0.585 mmol, 110.13 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the reaction mixture was cooled to room temperature. The mixture was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid n-1 (105.46 mg), with a yield of 52.76%. LC-MS (ESI) - )calcd.for C 22 H 27 N4O6[MH] - :443.20, found 443.29.

[0110] Example 14

[0111] Preparation of compound o-2

[0112] Compound n-1 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,5-pentanediamine (0.585 mmol, 118.27 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the reaction mixture was cooled to room temperature. The mixture was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-2108.38 mg, with a yield of 52.6%. LC-MS (ESI) - )calcd.for C 23 H 29 N4O6[MH] - :457.22,found 457.33.

[0113] Example 15

[0114] Preparation of compound o-3

[0115] Compound n-1 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,6-hexanediamine (0.585 mmol, 126.47 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-3152.58 mg, with a yield of 71.8%. LC-MS (ESI) - )calcd.for C 24 H 31 N4O6[MH] - :471.23,found 471.33.

[0116] Example 16

[0117] Preparation of compound o-4

[0118] Compound n-1 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,8-octanediamine (0.585 mmol, 142.87 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-4153.75 mg, with a yield of 68.4%. LC-MS (ESI) - )calcd.for C 26 H 35 N4O6[MH] - :499.26, found 499.31.

[0119] Example 17

[0120] Preparation of compound o-5

[0121] Compound n-1 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,10-decanediamine (0.585 mmol, 159.26 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid of 0.5149.53 mg, with a yield of 63.0%. LC-MS (ESI) - )calcd.for C 28 H 39 N4O6[MH] - :527.29,found527.43.

[0122] Example 18

[0123] Preparation of compound o-6

[0124] Compound n-1 (0.45 mmol, 124.30 mg), N-tert-butoxycarbonyl-2-(2-aminoethoxy)ethylamine (0.585 mmol, 119.43 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-6 110.79 mg, with a yield of 53.5%. LC-MS (ESI) - )calcd.for C 22 H 27 N4O7[MH] - :459.20,found459.11.

[0125] Example 19

[0126] Preparation of compound o-7

[0127] Compound n-1 (0.45 mmol, 124.30 mg), N-tert-butoxycarbonyl-2,2′-(ethylenedioxy)diethylamine (0.585 mmol, 145.18 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-7 112.54 mg, with a yield of 49.6%. LC-MS (ESI) - )calcd.for C 24 H 31 N4O8[MH] - :503.22,found503.26.

[0128] Example 20

[0129] Preparation of compound o-8

[0130] Compound n-1 (0.45 mmol, 124.30 mg), 13-amino-5,8,11-trioxa-2-azatridecanoic acid-1,1-tert-butyl ester (0.585 mmol, 170.94 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-8 137.42 mg, with a yield of 55.8%. LC-MS (ESI) - )calcd.for C 26 H 35 N4O9[MH] - :547.25, found 547.31.

[0131] Example 21

[0132] Preparation of compound o-9

[0133] Compound n-2 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,5-pentanediamine (0.585 mmol, 118.27 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid of 0.953.47 mg, with a yield of 25.9%. LC-MS (ESI) - )calcd.for C 23 H 29 N4O6[MH] - :457.22,found 457.29.

[0134] Example 22

[0135] Preparation of compound o-10

[0136] Compound n-2 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,8-octanediamine (0.585 mmol, 142.87 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-10129.44 mg, with a yield of 57.5%. LC-MS (ESI) - )calcd.for C 26 H 35 N4O6[MH] - :499.26, found 499.38.

[0137] Example 23

[0138] Preparation of compound o-11

[0139] Compound n-2 (0.45 mmol, 124.30 mg), N-(tert-butoxycarbonyl)-1,10-decanediamine (0.585 mmol, 159.26 mg), and DIPEA (1.35 mmol, 235 μL) were dissolved in 6 mL of N-methylpyrrolidone and reacted under reflux at 100 °C for 12 h. After the reaction was complete as monitored by TLC, the mixture was cooled to room temperature. The product was extracted with water and ethyl acetate, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow oily liquid o-11145.97 mg, with a yield of 61.4%. LC-MS (ESI) - )calcd.for C 28 H 39 N4O6[MH] - :527.29,found527.39.

[0140] Example 24

[0141] N 1 -(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-amino)butyl)-N 4 Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 1)

[0142]

[0143] Compound O-1 (0.06 mmol, 26.65 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 22.83 mg of the target compound 1 as a yellow solid, with a yield of 50.2%. Mp: 178–180 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 10.24 (d, J = 5.3Hz, 1H), 10.13 (s, 1H), 7.92 (t, J = 5.5Hz, 1H), 7.78 ( d,J=2.5Hz,1H),7.59(m,2H),7.27(t,J=8.0Hz,2H),7.04(m,4H),6.84(t,J=5.7Hz,3H),6.55(t,J=5.8Hz,1H), 6.45(t,J=5.7Hz,1H),6.34(s,1H),5.06(dd,J=12.8,5.3Hz,1H),3.29(dd,J=12.6,6.3Hz,2H),3.09(dd,J=12. 0,6.2Hz,2H),2.98–2.80(m,1H),2.65–2.52(m,4H),2.45(m,2H),2.41(s,3H),2.08–2.00(m,1H),1.52(m,4H). 13 C NMR(75MHz,DMSO-d6)δ(ppm)173.30,171.47,171.02,170.59,169.38,167.76,157.6 4,157.36,149.28,146.82,136.71,136.14,132.63,130.27,125.65,123.43,123.03 ,122.38,121.25,120.84,120.78,117.88,117.66,114.89,114.43,110.83,109.43, 106.27,48.99,41.98,38.59,32.14,31.44,30.73,27.02,26.62,22.62,12.72.HPLC purity:>95.0%.LC-MS(ESI + )calcd.for C 41 H 40 N7O8[M+H] + :758.29,found758.60.

[0144] Example 25

[0145] N 1 -(5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-amino)pentyl)-N 4 Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butyramide (compound 2)

[0146]

[0147] Compound O-2 (0.06 mmol, 27.49 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 21.33 mg of the target compound 2 as a yellow solid, with a yield of 46.1%. Mp: 159–161 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 11.11 (s, 1H), 10.24 (d, J = 5.3Hz, 1H), 10.13 (s, 1H), 7.88 (t, J = 5.4Hz, 1H), 7.78 (d, J=2.5Hz,1H),7.59(m,2H),7.27(t,J=8.0Hz,2H),7.12–6.97(m,4H),6.84(t,J=5.8Hz,3H),6.53(t,J=5.7Hz,1H), 6.45(t,J=5.7Hz,1H),6.33(s,1H),5.06(dd,J=12.7,5.3Hz,1H),3.25(dd,J=13.0,6.6Hz,2H),3.05(dd,J=12.0,6 .2Hz,2H),2.98–2.79(m,1H),2.69–2.51(m,4H),2.43(m,2H),2.41(s,3H),2.03(m,1H),1.56(m,2H),1.36(m,4H). 13C NMR(75MHz,DMSO-d6)δ(ppm)173.21,171.43,171.05,170.51,169.43,167.76,157.64,1 57.41,149.36,146.89,136.71,136.15,132.66,130.24,125.67,123.41,123.08,122.4 5,122.41,121.30,120.90,120.78,117.88,117.61,114.89,114.42,110.85,109.55,10 6.25,49.05,42.30,38.81,32.23,31.45,30.82,29.32,28.84,24.13,22.64,12.68.HPLC purity:>95.0%.LC-MS(ESI + )calcd.forC 42 H 42 N7O8[M+H] + 772.31, found 772.49.

[0148] Example 26

[0149] N 1 -(6-(2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindoline-4-amino)hexyl)-N 4 Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 3)

[0150]

[0151] Compound O-3 (0.06 mmol, 28.33 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 18.99 mg of the target compound 3 as a yellow solid, with a yield of 40.3%. Mp: 158–160 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 10.25 (d, J = 5.3Hz, 1H), 10.14 (s, 1H), 7.86 (t, J = 5.4Hz, 1H), 7.78 (d, J = 2.5Hz,1H),7.69–7.50(m,2H),7.33–7.22(m,2H),7.12–6.97(m,4H),6.85(t,J=6.4Hz,3H),6.53(t,J=5.7Hz,1H),6. 46(t,J=5.7Hz,1H),6.34(s,1H),5.06(dd,J=12.8,5.4Hz,1H),3.26(dd,J=12.9,6.5Hz,2H),3.03(dd,J=12.0,6.2H z,2H),2.96–2.76(m,1H),2.57(m,4H),2.41(s,3H),2.38(m,2H),2.04(m,1H),1.65–1.49(m,2H),1.46–1.22(m,6H). 13 C NMR(75MHz,DMSO-d6)δ(ppm)173.30,171.37,171.03,170.59,169.40,167.76,157.64,1 57.36,149.28,146.84,136.72,136.15,132.64,130.26,125.65,123.42,123.02,122.3 8,121.25,120.84,120.78,117.87,117.60,114.89,114.43,110.82,109.44,106.28,48 .98,42.21,38.90,32.16,31.44,30.75,29.56,29.07,26.57,26.51,22.62,12.72.HPLC purity:>95.0%.LC-MS(ESI + )calcd.for C 43 H 44 N7O8[M+H] + 786.33, found 786.57.

[0152] Example 27

[0153] N 1 -(8-(2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindoline-4-amino)octyl)-N 4Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butyramide (compound 4)

[0154]

[0155] Compound O-4 (0.06 mmol, 30.02 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow solid of target compound 4, 25.81 mg, in a yield of 52.9%. Mp: 157–159 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 10.25 (d, J = 5.3Hz, 1H), 10.13 (s, 1H), 7.85 (t, J = 5.4Hz, 1H), 7.78 (d, J = 2.4Hz,1H),7.69–7.47(m,2H),7.27(t,J=7.9Hz,2H),7.14–6.94(m,4H),6.85(t,J=6.5Hz,3H),6.52(t,J=5.7Hz,1H ),6.46(t,J=5.7Hz,1H),6.34(s,1H),5.06(dd,J=12.8,5.3Hz,1H),3.27(dd,J=12.8,6.4Hz,2H),3.02(dd,J=12.4, 6.4Hz,2H),2.98–2.76(m,1H),2.57(m,4H),2.41(s,3H),2.39(m,2H),2.03(m,1H),1.54(m,2H),1.46–1.20(m,10H). 13C NMR(75MHz,DMSO-d6)δ(ppm)173.30,171.35,171.03,170.59,169.41,167.77,157.65,15 7.36,149.28,146.86,136.73,136.14,132.64,130.27,125.66,123.43,123.02,122.38,1 21.25,120.85,120.77,117.88,117.62,114.89,114.43,110.83,109.43,106.28,48.99, 42.28,38.92,32.19,31.44,30.76,29.59,29.18,29.13,26.79,26.75,22.62,12.72.HPLC purity:>95.0%.LC-MS(ESI + )calcd.for C 45 H 48 N7O8[M+H] + :814.36, found 814.59.

[0156] Example 28

[0157] N 1 -(10-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-amino)decyl)-N 4 Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 5)

[0158]

[0159] Compound O-5 (0.06 mmol, 31.70 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 19.03 mg of the target compound O-5 as a yellow solid, with a yield of 37.7%. Mp: 139–141 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 10.24 (d, J = 5.3Hz, 1H), 10.12 (s, 1H), 7.84 (t, J = 5.4Hz, 1H), 7.78 (d, J = 2. 4Hz,1H),7.68–7.51(m,2H),7.27(t,J=7.9Hz,2H),7.11–6.96(m,4H),6.85(t,J=6.5Hz,3H),6.52(t,J=5.7Hz,1H),6.4 6(t,J=5.7Hz,1H),6.34(s,1H),5.06(dd,J=12.8,5.3Hz,1H),3.27(dd,J=13.0,6.5Hz,2H),3.02(dd,J=12.2,6.2Hz,2H ),2.95–2.77(m,1H),2.57(m,4H),2.41(s,3H),2.38(m,2H),2.10–2.01(m,1H),1.64–1.48(m,2H),1.43–1.20(m,14H). 13 C NMR(75MHz,DMSO-d6)δ(ppm)173.29,171.34,171.01,170.58,169.41,167.77,157.65,157.36,14 9.28,146.87,136.72,136.15,132.64,130.26,125.66,123.43,123.02,122.38,121.23,120.85,1 20.75,117.88,117.62,114.88,114.42,110.82,109.43,106.27,48.99,42.28,38.93,32.19,31.4 4,30.76,29.62,29.41,29.21,29.14,26.84,26.79,22.62,12.72.HPLCpurity:>95.0%.LC-MS(ESI + )calcd.for C 47 H 52 N7O8[M+H] + :842.39, found 842.67.

[0160] Example 29

[0161] N 1 -(2-(2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoquinoline-4-yl)amino)ethoxy)-N 4Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 6)

[0162]

[0163] Compound O-6 (0.06 mmol, 27.61 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give a yellow solid of target compound O-6, 25.84 mg, in a yield of 55.7%. Mp: 155–157 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 10.25 (d, J = 5.1Hz, 1H), 10.13 (s, 1H), 7.94 (t, J = 4.7Hz, 1H), 7.77 (s, 1 H),7.59(dd,J=16.2,8.3Hz,2H),7.27(t,J=7.6Hz,2H),7.13(d,J=8.6Hz,1H),7.03(t,J=7.2Hz,3H),6.84(d,J=7. 3Hz,3H),6.62(t,J=5.5Hz,1H),6.46(t,J=5.5Hz,1H),6.34(s,1H),5.07(dd,J=12.4,4.9Hz,1H),3.60(t,J=4.9Hz ,2H),3.54–3.41(m,4H),3.34–3.18(m,2H),2.87(m,1H),2.69–2.52(m,4H),2.45(m,2H),2.41(s,3H),2.03(m,1H). 13C NMR(75MHz,DMSO-d6)δ(ppm)173.28,171.76,171.03,170.57,169.43,167.75,157 .65,157.36,149.29,146.82,136.71,136.13,132.53,130.28,125.66,123.44,12 3.03,122.39,121.25,120.84,120.78,117.89,114.90,114.43,111.16,109.71,1 06.29,69.42,69.01,49.01,42.06,38.96,32.02,31.44,30.58,22.61,12.73.HPLC purity:>95.0%.LC-MS(ESI + )calcd.for C 41 H 40 N7O9[M+H] + 774.29, found 774.56.

[0164] Example 30

[0165] N 1 -(2-(2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoquinoline-4-yl)amino)ethoxy)ethyl)-N 4 Preparation of 3-(8-methyl-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 7)

[0166]

[0167] Compound O-7 (0.06 mmol, 30.25 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 21.48 mg of the target compound O-7 as a yellow solid, with a yield of 43.8%. Mp: 122–124 °C. 11H NMR (300 MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 10.25 (d, J = 5.3 Hz, 1H), 10.13 (s, 1H), 7.94 (t, J = 5.4 Hz, 1H), 7.78 (d, J = 2.3 Hz, 1H), 7.68–7.51 (m, 2H), 7.27 (t, J = 7.9 Hz, 2H), 7.14 (d, J = 8.6 Hz, 1H), 7.03 (dd, J = 7.9, 4.9 Hz, 3H), 6.85 (t, J = 6.2 Hz, 3H), 6.61 (t, J = 5.5 Hz, 1H), 6.46 (t, J = 5.7 Hz, 1H), 6.34 (s, 1H), 5.07 (dd, J = 12.7, 5.3 Hz, 1H), 3.61 (t, J = 5.1 Hz, 2H), 3.54 (d, J = 5.2 Hz, 4H), 3.50–3.44 (m, 2H), 3.40 (t, J = 5.9 Hz, 2H), 3.20 (dd, J = 11.0, 5.4 Hz, 2H), 2.99–2.79 (m, 1H), 2.57 (dd, J = 15.5, 8.9 Hz, 4H), 2.43 (d, J = 7.4 Hz, 2H), 2.41 (s, 3H), 2.04 (d, J = 5.2 Hz, 1H). 13 13C NMR (75 MHz, DMSO-d6) δ (ppm) 173.30, 171.70, 171.00, 170.58, 169.40, 167.75, 157.65, 157.36, 149.29, 146.83, 136.68, 136.14, 132.54, 130.28, 125.66, 123.44, 123.03, 122.39, 121.25, 120.85, 120.78, 117.89, 114.90, 114.44, 111.14, 109.68, 1–6.29, 70.14, 70.06, 69.63, 69.33, 49.01, 42.14, 39.04, 32.03, 31.45, 30.59, 2–60, 12.72. HPLC purity: >95.0%. LC-MS (ESI + ) calcd. for C 43 H 44 N7O 10 [M + H] + : 818.32, found 818.72.

[0168] Example 31

[0169] N 1-(2-(2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoquinoline-4-yl)amino)ethoxy)ethoxy)ethoxy)-N 4 Preparation of 3-(8-methyl-1-oxy-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 8)

[0170]

[0171] Compound O-8 (0.06 mmol, 32.89 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 18.86 mg of the target compound O-8 as a yellow solid, with a yield of 36.5%. Mp: 132–135 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 11.12 (s, 1H), 10.25 (d, J = 5.3Hz, 1H), 10.13 (s, 1H), 7.95 (t, J = 5.5Hz, 1H), 7.78 (d, J = 2.5Hz, 1H) ,7.67–7.51(m,2H),7.34–7.19(m,2H),7.14(d,J=8.6Hz,1H),7.03(dd,J=8.0,5.5Hz,3H),6.85(t,J=6.2Hz,3H),6.61(t,J=5. 6Hz,1H),6.46(t,J=5.7Hz,1H),6.34(s,1H),5.07(dd,J=12.8,5.4Hz,1H),3.62(t,J=5.3Hz,2H),3.58–3.43(m,10H),3.39(d, J=6.1Hz,2H),3.19(q,J=5.8Hz,2H),2.99–2.78(m,1H),2.57(m,4H),2.45(d,J=7.0Hz,2H),2.41(s,3H),2.04(d,J=4.7Hz,1H). 13C NMR(75MHz,DMSO-d6)δ(ppm)173.30,171.70,171.00,170.56,169.39,167.75,157.65,1 57.36,149.29,146.84,136.67,136.14,132.53,130.27,125.66,123.43,123.03,122.3 9,122.36,121.25,120.85,120.78,117.89,114.90,114.44,111.13,109.67,106.29,70 .24,70.04,69.57,69.33,49.01,42.13,39.04,32.04,31.44,30.59,22.61,12.72.HPLC purity:>95.0%.LC-MS(ESI + )calcd.for C 45 H 48 N7O 11 [M+H] + :862.34,found862.60.

[0172] Example 32

[0173] N 1 -(5-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoquinoline-5-yl)amino)pentyl)-N 4 Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 9)

[0174]

[0175] Compound O-9 (0.06 mmol, 27.49 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 22.17 mg of the target compound O-9 as a yellow solid, with a yield of 47.9%. Mp: 175–177 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.08 (s, 1H), 10.24 (d, J = 5.3Hz, 1H), 10.13 (s, 1H), 7.88 (t, J = 5.5Hz, 1H), 7.78 (d, J = 2.5Hz, 1H), 7 .61(dd,J=8.9,2.6Hz,1H),7.55(d,J=8.4Hz,1H),7.33–7.20(m,2H),7.09(t,J=5.0Hz,1H),7.02(dd,J=8.1,6.2Hz,2H),6.93(d, J=1.6Hz,1H),6.84(t,J=5.6Hz,4H),6.45(t,J=5.7Hz,1H),6.33(s,1H),5.04(dd,J=12.7,5.4Hz,1H),3.19–3.03(m,4H),2.97–2 .80(m,1H),2.56(dd,J=14.5,7.4Hz,4H),2.46–2.35(m,2H),2.41(s,3H),2.00(m,1H),1.56(dt,J=13.6,6.9Hz,2H),1.33(m,4H). 13 C NMR(75MHz,DMSO-d6)δ(ppm)173.30,171.42,171.03,170.66,168.16,167.61,157.64,157 .35,154.86,149.29,136.12,134.64,130.27,125.65,125.57,123.43,123.02,122.38,121 .25,121.24,120.84,120.77,117.88,116.22,114.88,114.42,106.27,49.05,42.89,38.8 1,32.14,31.45,30.74,29.38,28.35,24.33,22.70,12.72.HPLCpurity:>95.0%.LC-MS(ESI + )calcd.for C 42 H 42 N7O8[M+H] + 772.31, found 772.49.

[0176] Example 33

[0177] N 1 -(8-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoquinoline-5-yl)amino)octyl)-N 4Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 10)

[0178]

[0179] Compound O-10 (0.06 mmol, 30.02 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 23.25 mg of the target compound O-10 as a yellow solid, with a yield of 47.6%. Mp: 143–145 °C. 1 H NMR (300MHz, DMSO-d6) δ (ppm) 11.08 (s, 1H), 10.25 (d, J = 5.2Hz, 1H), 10.13 (s, 1H), 7.85 (t, J = 5.4Hz, 1H), 7.78 (d, J = 2.4Hz, 1H), 7.6 1(dd,J=8.9,2.4Hz,1H),7.56(d,J=8.4Hz,1H),7.27(t,J=7.9Hz,2H),7.11(t,J=4.8Hz,1H),7.03(dd,J=8.0,5.2Hz,2H),6.94(s,1H ),6.85(t,J=6.9Hz,4H),6.46(t,J=5.7Hz,1H),6.34(s,1H),5.04(dd,J=12.7,5.3Hz,1H),3.13(dd,J=12.2,6.3Hz,2H),3.02(dd,J= 12.2, 6.3Hz, 2H), 2.87 (m, 1H), 2.57 (m, 4H), 2.41 (s, 3H), 2.39 (d, J = 7.2Hz, 2H), 1.99 (m, 1H), 1.63–1.48 (m, 2H), 1.45–1.24 (m, 10H). 13C NMR(75MHz,DMSO-d6)δ(ppm)173.31,171.36,171.03,170.67,168.17,167.62,157.66, 157.35,154.90,149.29,136.13,134.65,130.27,125.67,125.57,123.44,123.02,122. 39,121.26,120.84,120.78,117.88,116.19,114.89,114.43,106.29,49.06,42.93,38 .93,32.17,31.45,30.74,29.60,29.22,29.21,28.68,26.96,26.81,22.70,12.72.HPLC purity:>95.0%.LC-MS(ESI + )calcd.for C 45 H 48 N7O8[M+H] + :814.36, found 814.66.

[0180] Example 34

[0181] N 1 -(10-((2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoquinoline-5-yl)amino)decyl)-N 4 Preparation of 3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl)butadiamide (compound 11)

[0182]

[0183] Compound O-11 (0.06 mmol, 31.70 mg) was dissolved in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (1 mL). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure and set aside. Then, intermediate L (0.06 mmol, 25.87 mg), DIPEA (0.24 mmol, 42 μL), and HATU (0.15 mmol, 57.03 mg) were dissolved in DMF (3 mL), and the reaction was carried out at room temperature for 12 h. After the reaction was completed by TLC, the mixture was extracted with water and ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography to give 24.58 mg of the target compound O-11 as a yellow solid, with a yield of 48.7%. Mp: 132–134 °C. 1H NMR (300MHz, DMSO-d6) δ (ppm) 11.08 (s, 1H), 10.25 (d, J = 5.0Hz, 1H), 10.13 (s, 1H), 7.84 (t, J = 4.6Hz, 1H), 7.78 (s, 1H),7.59(dd,J=17.5,8.6Hz,2H),7.27(t,J=7.6Hz,2H),7.11(s,1H),7.07–6.98(m,2H),6.94(s,1H),6.84(d,J= 7.8Hz,4H),6.46(t,J=5.5Hz,1H),6.34(s,1H),5.04(dd,J=12.5,5.0Hz,1H),3.07(dd,J=34.7,5.5Hz,4H),2.95– 2.77(m,1H),2.55(m,4H),2.41(s,3H),2.40–2.20(m,2H),1.95(m,1H),1.54(t,J=5.3Hz,2H),1.44–1.25(m,14H). 13 C NMR(75MHz,DMSO-d6)δ(ppm)173.30,171.34,171.02,170.66,168.17,167.61,157.65,157 .36,154.90,149.28,136.14,134.65,130.26,125.66,125.56,123.43,123.02,122.38,121 .24,120.85,120.76,117.88,116.19,114.89,114.42,106.28,49.06,42.94,38.94,32.17 ,31.46,30.76,29.62,29.46,29.43,29.27,29.23,28.70,27.01,26.85,22.70,12.72.HPLC purity:>95.0%.LC-MS(ESI + )calcd.for C 47 H 52 N7O8[M+H] + :842.39, found 842.71.

[0184] Example 35

[0185] 1. CCK-8 cytotoxicity assay

[0186] The antiproliferative effects of the compound on human myeloid monocytic leukemia cell line MV4-11, human acute monocytic leukemia cell line THP-1, and human promyelocytic leukemia cell line HL-60 were detected using the CCK-8 assay.

[0187] Experimental Procedure: MV4-11, THP-1, and HL-60 cell lines were cultured to the logarithmic growth phase. Cells were collected, centrifuged, and the culture medium was discarded. Serum-free culture medium was added, and the cells were resuspended by pipetting. Cell counting was performed under a microscope using a cell counting chamber. The cell suspension concentration was then adjusted to 2 × 10⁻⁶ cells / mL. 5 Cells were seeded into 96-well plates, with 50 μL of cell suspension added to each well and sterile PBS solution added to the edge wells. Then, 50 μL of complete culture medium containing different concentrations of the drug was added to each well. The initial drug concentration was set at 10 μM, and serial dilutions were performed three-fold, with three replicates for each concentration. The 96-well plates were placed in a cell culture incubator and incubated at 37°C with 5% CO2 for 72 hours. After incubation, 10 μL of CCK-8 assay reagent was added to each well, and incubation continued for another 3 hours. After incubation, the OD value at 450 nm absorption wavelength was measured using a microplate reader. Finally, the IC50 values ​​of each compound were obtained using GraphPad Prism 8.0 software. 50 The values ​​are shown in Table 1 below.

[0188] Table 1. Antitumor proliferation activity of compounds 1-14 in MV4-11, THP-1, and HL-60 cell lines.

[0189]

[0190] a All experiments were conducted in triplicate.

[0191] The antitumor activity of compound 7 in various solid tumor cell lines is shown in Table 2.

[0192] Table 2. Antitumor proliferation activity of compound 7 in various solid tumor cell lines.

[0193]

[0194]

[0195] a All experiments were conducted in triplicate.

[0196] The following experiments use compound 7 and compound (+)-JQ-1 as examples.

[0197] 2. Western Blot Protein Immunoblotting Assay

[0198] The effects of compound 7 and reference compound (+)-JQ-1 on the expression level of BRD4 protein in tumor cells were investigated using Western blotting.

[0199] Experimental procedure: After culturing MV4-11 cells to the logarithmic growth phase, the cells were collected and the cell culture concentration was adjusted to 2 × 10⁻⁶. 6 / mL. Add 1mL of cell suspension to each well of a 6-well plate and incubate for 12 hours until cells adhere. Add 1mL of complete culture medium containing different concentrations of drug and incubate for another 24 hours. After incubation, collect the cell slurry, centrifuge, and discard the supernatant. Wash 2-3 times with pre-chilled PBS, centrifuge, and discard the supernatant. Add 100μL of RIPA lysis buffer containing 1mM PMSF to each sample, mix by pipetting, and place on ice for 30min of lysis. Centrifuge at 15,000rpm for 15min at 4℃, and transfer the supernatant to a new 1.5mL EP tube for storage at -20℃. Subsequently, the concentration of the protein extract was determined using a BCA protein quantification kit, and the corresponding loading volume for 20μg of protein was calculated. The protein was separated by SDS-PAGE electrophoresis, and the gel containing the target protein was cut according to the protein marker position after electrophoresis. Wet transfer was used to transfer protein bands onto a PVDF membrane. After simple washing with TBST, the membrane was blocked for 1 hour on a shaker with blocking buffer containing 5% skim milk powder. After blocking, the PVDF membrane was washed 4-5 times with TBST for 10 minutes each time, followed by overnight incubation of the bands at 4°C with the appropriate primary antibody dilution buffer. The PVDF membrane was then washed 4-5 times with TBST for 10 minutes each time, followed by incubation of the bands at room temperature for 2 hours with secondary antibody dilution buffer. The protein bands were developed in ECL chemiluminescence solution, observed, and photographed. Finally, grayscale analysis of the protein bands was performed using ImageJ and GraphPad Prism 8.0 software.

[0200] The degradation effects of compound 7 and reference compound (+)-JQ-1 on BRD4 protein in MV4-11 cells are shown in the figure. Figure 1

[0201] The above pharmacological data show that the compound of general formula (I) of this invention can effectively reduce the abundance of BRD4 protein and block downstream signal transduction of BRD4, and has excellent anti-tumor proliferative activity.

[0202] Example 36

[0203] Tablets containing compound 7:

[0204]

[0205]

[0206] The raw materials and excipients are mixed, granulated, dried, and tableted using conventional methods.

[0207] Although the invention has been described with reference to specific embodiments, modifications and equivalent variations will be apparent to those skilled in the art, and are all included within the scope of the invention.

Claims

1. A class of PROTAC molecules targeting degradation of BRD4, characterized in that, The targeted BRD4 degradation PROTAC molecule is selected from the compounds shown in the general formula (I) or stereoisomers, tautomers and pharmaceutically acceptable salts thereof: Wherein: R 1 selected from C 1-5 alkylene, R 2 selected from C 2-l2 alkylene, -CH2(CH2OCH2) n CH2-, n = an integer from 1 to 5; R 3 selected from C l-5 alkyl.

2. The targeted BRD4 degradation PROTAC molecule according to claim 1, wherein: R 1 selected from C 2-4 alkylene; R 2 selected from C 4-10 alkylene, -CH2(CH2OCH2) n CH2-, n = an integer from 2 to 4; R 3 selected from C l-3 alkyl.

3. The class of PROTAC molecules targeted to degrade BRD4 according to claim 1, characterized in that: The targeted BRD4 degradation PROTAC molecule is selected from the compounds shown in the following formula or stereoisomers, tautomers and pharmaceutically acceptable salts thereof: R 1 selected from -(CH2)2-, -(CH2)3-; R 2 selected from C 4-l0 alkylene, -CH2(CH2OCH2) n CH2-, n = an integer from 1 to 4; R 3 selected from methyl.

4. The class of PROTAC molecules targeted to degrade BRD4 according to claim 1, characterized in that, The targeted BRD4 degradation PROTAC molecule is selected from the following compounds or stereoisomers, tautomers and pharmaceutically acceptable salts thereof:

5. The PROTAC molecule for targeted degradation of BRD4 according to any one of claims 1-4, characterized in that, The pharmaceutically acceptable salt is selected from acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hydrochloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthoate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / diphosphate / bisphosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinafoate, aluminum salt, arginate, benzathine salt, calcium salt, choline salt, diethylamine salt, diethanolamine salt, glycine salt, lysine salt, magnesium salt, meglumine salt, ethanolamine salt, sodium salt, potassium salt, ammonium salt, atropine sulfate salt or zinc salt.

6. Use of the targeted BRD4 degradation PROTAC molecule according to any one of claims 1-4 in the preparation of a medicament for the treatment and / or prevention of a tumor; the tumor is selected from any one of lung cancer, breast cancer, acute myelocytic leukemia, chronic leukemia, prostate cancer, hepatoma, cervical cancer, colon cancer.

7. Use according to claim 6, characterized in that, Use of the targeted BRD4 degradation PROTAC molecule according to any one of claims 1-4 as the only active ingredient, or together with other anti-tumor compounds as the active ingredient in the preparation of a medicament for the treatment and / or prevention of a tumor.

8. A pharmaceutical formulation composition, characterized by, A pharmaceutical composition containing a therapeutically effective amount of the targeted BRD4 degradation PROTAC molecule according to any one of claims 1-4 and a pharmaceutically acceptable adjuvant.

9. The pharmaceutical formulation composition of claim 8, wherein, The dosage form of the pharmaceutical composition is selected from tablets, capsules, pills, suppositories, soft capsules, oral liquids, suspensions or injection solutions.

10. A method of preparing the PROTAC molecule for targeted degradation of BRD4 as described in claim 4, characterized in that, The method is as follows according to the reaction route: The method comprises the following steps: The final product compound 1-11 is obtained by a 13-step synthesis reaction using 3-methyl-1H-pyrrole-2-carboxylic acid ethyl ester, 2-bromo-1-fluoro-4-nitrobenzene, 3-fluorophthalimide and 4-fluorophthalimide as raw materials; (1) Using 3-methyl-1H-pyrrole-2-carboxylic acid ethyl ester a and bromoacetaldehyde diethyl acetal as raw materials, a substitution reaction is carried out under the action of NaH to obtain b; (2) Compound b is subjected to ester hydrolysis under alkaline conditions to obtain c; (3) Compound c is subjected to condensation under organic alkaline conditions with ammonium chloride catalyzed by a condensation agent to obtain amide d; (4) Compound d is subjected to self-cyclization under acidic conditions to obtain e; (5) Compound e is subjected to bromination to obtain f; (6) 2-Bromo-1-fluoro-4-nitrobenzene g is used as a reactant to react with phenol to obtain h; (7) Compound h is subjected to reduction with iron powder to obtain i; (8) Compound i is subjected to catalysis with pinacol diboron catalyzed by a palladium catalyst to obtain j; (9) Compound j is subjected to Suzuki coupling reaction with compound f to obtain k; (10) Compound k is subjected to condensation with succinic anhydride to obtain amide, and l is obtained respectively; (11) 3-Fluoro-phthalimide m-1 is used to react with 3-amino-2,6-piperidinedione hydrochloride to obtain n-1; (12) Compound n-1 is reacted with N-Boc-diamine to obtain compounds o-1 to o-8; (13) Compounds o-1 to o-8 are subjected to amide condensation with compound l to obtain final products 1-8. The preparation of compounds 9-11 is different from that of compound 1-8 in that 3-fluoro-phthalimide m-1 is replaced by 4-fluoro-phthalimide m-2 in (11).

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