BRD4 protein inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a BRD4 protein inhibitor as well as a preparation method and application thereof, and belongs to the technical field of medicines. The invention provides a BRD4 protein inhibitor, in particular a pyridone derivative with a novel structure, and the BRD4 protein inhibitor has BRD4 protein inhibition activity better than that of a positive drug JQ-1. Tests prove that the compound provided by the invention shows relatively high activity, and the prepared compound has good BRD4 protein inhibition activity, and particularly shows relatively good activity in resisting colon cancer cell proliferation. In addition, the invention also provides a method for preparing the BRD4 protein inhibitor, and the method has the advantages of short synthetic route, simple preparation process and easy operation, and can meet the needs of large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a BRD4 protein inhibitor and a preparation method and application thereof, and more particularly to a pyridinone derivative and a preparation method and application thereof. BACKGROUND

[0002] Bromodomain-containing protein 4 (BRD4) is a very important protein in the bromodomain and extraterminal domain (BET) family of proteins. The co-crystal structure of the bromodomain protein and the ligand shows that the acetyl lysine is combined near the central hydrophobic cavity of the protein, and acts on the asparagine residue (140) existing in most bromodomains through the hydrogen bond between the N and O atoms. BET proteins such as BRD4 often have high affinity for highly acetylated histones on chromatin, can stimulate transcription elongation, and thus regulate DNA replication, transcription and even repair, and affect cell cycle and differentiation.

[0003] Abnormal expression or dysfunction of BRD4 is related to the development of various diseases, mainly including hematological malignancies, solid tumors, inflammation and cardiovascular diseases, and has become an important target for the treatment of various diseases.

[0004] Currently, there is no BRD4 inhibitor successfully marketed, and the inhibitors under clinical research mainly include: Pelabresib combined with Ruxolitinib (Ruxolitinib) is being used in the treatment of myelofibrosis clinical phase III study; Mivebresib has completed the clinical phase I experiment in the treatment of patients with advanced cancer; AZD-5153 has completed the clinical phase I study for the treatment of breast cancer and non-Hodgkin's lymphoma and other tumors, and is being used in the clinical phase II study for the treatment of acute myeloid leukemia. RVX-208 has completed the clinical phase II study for the treatment of atherosclerosis-related diseases and early diabetes.

[0005] PFI-1 is a pan-BET class protein inhibitor based on fragment optimization design reported by Pfizer in 2012, and has an IC 50The bioavailability can reach 220 nM. PFI-1 exhibits good anti-proliferative activity against various leukemia cell lines, with inhibitory activity ranging from 1 to 10 μM. However, due to its small cLogP molecular weight and weak lipid solubility, its bioavailability is poor, at only 32%. GlaxoSmithKline developed GSK046, which has more than 1000 times the BD2 selectivity of BRD4 and can significantly increase the expression of inflammatory factors such as IL-17A, IL-22, and Th17, demonstrating good anti-inflammatory activity. However, clinical studies have shown a risk of genotoxicity, thus limiting its further research.

[0006] BRD4 has become a popular drug development target in the past decade due to its important role in the development and progression of diseases such as inflammation, cardiovascular disease, and tumors. Since the first BRD4 inhibitor (+)-JQ-1 was reported, various types of BRD4 inhibitors have been developed and entered clinical trials, such as the BRD4 inhibitors mentioned in invention patents with publication numbers CN113754593A and CN107739370A. However, in general, most existing BRD4 inhibitors suffer from insufficient clinical efficacy or significant toxic side effects, and no related drugs have been successfully marketed. Therefore, BRD4 inhibitors with newer structures and better efficacy are still urgently needed for development, hoping to bring hope for the treatment of diseases related to abnormal BRD4 regulation. Summary of the Invention

[0007] Based on the shortcomings of existing technologies, the applicant has designed a BRD4 protein inhibitor with BRD4 inhibitory activity, as well as its preparation method and application.

[0008] The specific technical solution of the present invention is as follows:

[0009] This invention provides a BRD4 protein inhibitor, characterized in that the BRD4 protein inhibitor is a pyridone derivative with the structural formula shown in Formula I:

[0010]

[0011] Wherein, the R1 group is at least one of the following groups: hydrogen atom, methyl, ethyl, isopropyl,

[0012] The R2 group is at least one of the following groups: hydrogen atom, methyl, methylamino, ethylamino, isopropylamino, acetamino.

[0013] The R3 group is at least one of the following groups: hydrogen atom, phenoxy, cycloalkoxy, heteroaryloxy.

[0014] Further, the R1 group is methyl, ethyl, or isopropyl, the R2 group is amino, ethylamino, isopropylamino, or acetamino, and the R3 group is a hydrogen atom, phenoxy, cycloalkoxy, or heteroaryloxy.

[0015] Furthermore, the pyridone derivative is any one of the following compounds:

[0016]

[0017]

[0018] Most preferably, the pyridone derivative is any one of the following compounds:

[0019]

[0020] Experimental results in specific embodiments of the present invention show that compounds 2, 4, 5, 6, 8, 9, 11, 14, and 15 exhibit better BRD4 protein inhibitory activity than the positive control drug JQ-1. Compounds 2, 4, 5, 8, 9, 14, and 15 also exhibit better anti-proliferative activity against HCT-116 colon cancer cells than the positive control drug JQ-1.

[0021] The present invention also provides a method for preparing the BRD4 protein inhibitor, comprising the following steps:

[0022] (1) Under the action of cesium carbonate, aminopyridone and iodoalkane are substituted, or acylation with acetyl chloride is carried out to obtain product 2a-d;

[0023] (2) Under the action of cesium carbonate, pyridones with R2 substitution and iodoalkanes are substituted to obtain product 2e-i;

[0024] (3) The products 2a-d and 2e-i were reacted with pinacol diboronic acid ester in the presence of potassium acetate and DPPF palladium dichloride to obtain product 3a-i.

[0025] (4) 3-Bromo-4-fluoronitrobenzene undergoes a substitution reaction with a phenol having a corresponding M1 group under the action of cesium carbonate to give product 5a-h;

[0026] (5) Product 5a-h undergoes a reduction reaction under the action of iron powder and ammonium chloride to obtain product 6a-h;

[0027] (6) Product 6a-h was sulfonated with ethylsulfonyl chloride under pyridine catalysis to give product 7a-h;

[0028] (7) The product 7a-h and the product 3a-i in step (3) are subjected to a Suzuki coupling reaction to obtain a pyridinone derivative, namely the BRD4 protein inhibitor.

[0029] R1 is a hydrogen atom, methyl, ethyl, or isopropyl, and R2 is a hydrogen atom, methyl, methylamino, ethylamino, isopropylamino, or acetamino.

[0030] The M1 group is at least one of 2,4-difluorophenoxy, 2-methoxyphenoxy, 3,4-dimethoxyphenoxy, 4-trifluoromethylpyridine-3-oxy, 4-methylpyridine-3-oxy, 2-ethyl-4-fluorophenoxy, 2-trifluoromethyl-phenoxy, and 2-methyl-4-fluorophenoxy.

[0031] Preferably, in step (1), the molar ratio of aminopyridone to iodoalkane is 1:1.5;

[0032] In step (3), the molar ratios of products 2a-d and 2e-i with pinacol diborate, DPPF palladium dichloride, and potassium acetate are 1:1.2:0.1:3, respectively. Products 2a-d and 2e-i are first reacted with pinacol diborate in a solvent containing potassium acetate and DPPF palladium dichloride. The solvent used is anhydrous dioxane.

[0033] In step (4), the molar ratio of 3-bromo-4-fluoronitrobenzene to phenol having the corresponding M1 group is 1:1.2;

[0034] In step (5), the molar ratio of iron powder to product 5a-h is at least 4:1, and 1M ammonium chloride aqueous solution needs to be added as a reaction condition.

[0035] In step (6), the molar ratio of product 6a-h to ethylsulfonyl chloride and pyridine is 1:1.2:2.2;

[0036] In step (7), products 7a-h and 3a-i are dissolved in a solvent, and then a catalyst and a base are added to carry out the reaction.

[0037] The molar ratio of product 7a-h to product 3a-i is 1:1.2;

[0038] The solvent is a mixed solution of dioxane and water.

[0039] The catalyst is tetra(triphenylphosphine)palladium.

[0040] The alkali is potassium carbonate;

[0041] The reaction conditions are: under nitrogen protection, at a reaction temperature of 95°C for 12-18 hours.

[0042] The present invention also provides the use of the BRD4 protein inhibitor in the preparation of medicaments for the treatment and / or prevention of diseases associated with abnormal BRD4 activity.

[0043] Furthermore, the diseases associated with abnormal BRD4 activity are selected from at least one of cancer, inflammatory and immune diseases, nervous system diseases, and viral diseases;

[0044] The cancer is selected from one of the following: acute myeloid leukemia, non-small cell lung cancer, colon cancer, and breast cancer;

[0045] The inflammatory and immune diseases mentioned are at least one of atherosclerosis, inflammatory bowel disease, psoriasis, and cardiovascular disease;

[0046] The viral disease mentioned is AIDS.

[0047] Most preferably, the disease associated with abnormal BRD4 activity is colon cancer.

[0048] In cancer, inhibiting BRD4 can suppress tumor cell proliferation, invasion, and migration, thereby effectively inhibiting tumor growth and spread. In inflammatory diseases, it can regulate transcription factors, inflammasomes, and immune cell function, inhibiting key inflammatory pathways and thus alleviating inflammatory responses. Although the examples only demonstrate the therapeutic effect on colon cancer, based on the mechanism of action of BRD4 in various diseases, it is speculated that this pyridone derivative can also be used to treat other diseases related to abnormal BRD4 activity, such as breast cancer, acute myeloid leukemia, inflammatory bowel disease, and psoriasis.

[0049] Experimental results in specific embodiments of the present invention show that compounds 2, 4, 5, 6, 8, 9, 11, 14, and 15 exhibit better BRD4 protein inhibitory activity than the positive control drug JQ-1. Compounds 2, 4, 5, 8, 9, 14, and 15 also exhibit better anti-proliferative activity against HCT-116 colon cancer cells than the positive control drug JQ-1.

[0050] The present invention also provides a pharmaceutical composition comprising the BRD4 protein inhibitor or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0051] The active compounds can be formulated into forms suitable for administration via any suitable route, using one or more pharmaceutically acceptable carriers through conventional methods. Therefore, the active compounds of the present invention can be formulated into various dosage forms for oral administration, injection (e.g., intravenous, intramuscular, or subcutaneous), inhalation, or insufflation. The compounds of the present invention can also be formulated into dosage forms such as tablets, hard or soft capsules, aqueous or oily suspensions, emulsions, injections, dispersible powders or granules, suppositories, lozenges, or syrups.

[0052] As a general guideline, the active compound is preferably administered in unit dose form or by self-administration as a single dose by the patient. The unit dose of the compound or composition of the present invention may be expressed as a tablet, capsule, sachet, bottled liquid, powder, granule, lozenge, suppository, regenerated powder, or liquid formulation. A suitable unit dose may be 0.1–1000 mg.

[0053] In addition to the active compound, the pharmaceutical composition of the present invention may contain one or more excipients, wherein the excipients are selected from the following components: fillers (diluents), binders, wetting agents, disintegrants, or excipients, etc. Depending on the method of administration, the composition may contain 0.1% to 99% by weight of the active compound.

[0054] As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to: the activity of the specific compound used, the severity of the disease, the patient's age, the patient's weight, the patient's health status, the patient's behavior, the patient's diet, the timing of administration, the route of administration, the rate of excretion, and the combination of drugs; in addition, the optimal treatment mode, such as the treatment pattern, daily dosage, or the type of medicinal salt, can be validated based on conventional treatment protocols. Attached Figure Description

[0055] Figure 1 This is a synthetic route diagram for the pyridone derivatives of the present invention. Detailed Implementation

[0056] The present invention will be further illustrated 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. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and biological materials described are commercially available unless otherwise specified.

[0057] Example 1

[0058] The synthetic route of the pyridone derivatives of the present invention is as follows: Figure 1 As shown, the specific preparation method is as follows:

[0059] Step 1: Synthesize compound 2a-d:

[0060] Accurately weigh 3-amino-5-bromo-1-methylpyridine-2(1H)-one (i.e. Figure 11a) (203.4 mg, 1.00 mmol), iodomethane (or iodoethane, iodopropyl ether) (283.88 mg, 2 mmol), and cesium carbonate (651.64 mg, 2 mmol) were mixed in N,N-dimethylformamide (2.5 mL), and the mixture was heated to 60 °C and stirred for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, the solvent was removed by evaporation under reduced pressure, and the mixture was extracted with ethyl acetate and water, repeated three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation under reduced pressure. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (2:1, v / v) as the eluent to give products 2a-d in yields of 100%, 55%, 36%, and 23%, respectively.

[0061] The second step is to synthesize compound 2e-i:

[0062] Accurately weigh 5-bromopyridin-2-one (i.e. Figure 1 1b)(174.00 mg, 1 mmol) or 3-methyl-5-bromopyridin-2-one (i.e. Figure 1 1c (188.00 mg, 1 mmol) was mixed with iodomethane (or iodoethane, iodopropyl ether) (283.88 mg, 2 mmol) and cesium carbonate (651.64 mg, 2 mmol) in N,N-dimethylformamide (2.5 mL), and the mixture was heated to 60 °C and stirred for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, the solvent was removed by evaporation under reduced pressure, and the mixture was extracted with ethyl acetate and water, repeated three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation under reduced pressure. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (2:1, v / v) as the eluent to give products 2e-i in yields of 75%, 47%, 33%, 83%, and 55%, respectively.

[0063] The third step is to synthesize compound 3a-i:

[0064] Accurately weigh the compound bromopyridone (i.e. Figure 12a-i (0.5 mmol), bis(pinacolyl)diboron (152.4 mg, 0.6 mmol), 1,1′-bis(diphenylphosphine)ferrocene palladium dichloride (36.6 mg, 0.05 mmol), and potassium acetate (147.2 mg, 1.5 mmol) were mixed and dissolved in 1,4-dioxane (2.0 mL). The mixture was heated to 90 °C and stirred for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and extracted with ethyl acetate and water, repeated three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation under reduced pressure. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (3:1, v / v) as the eluent to give product 3a-i in yields of 26%, 32%, 56%, 48%, 52%, 46%, 65%, 49%, and 55%, respectively.

[0065] Step 4: Synthesize compound 5a-h:

[0066] Accurately weigh 440.0 mg (2 mmol) of 3-bromo-4-fluoronitrobenzene and arylphenol (i.e., Figure 1 4a-g (2.4 mmol) and cesium carbonate (781.97 mg, 2.4 mmol) were mixed in N,N-dimethylformamide (4.0 mL) and heated to 110 °C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, the solvent was removed by evaporation under reduced pressure, and the mixture was extracted with ethyl acetate and water, repeated three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation under reduced pressure. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (3:1, v / v) as the eluent, giving products 5a-h with yields of 100%, 88%, 92%, 72%, 80%, 93%, 65%, and 92%, respectively.

[0067] Step 5: Synthesize compound 6a-h:

[0068] Accurately weigh the nitrobenzene compound (i.e. Figure 1 5a-h (1.0 mmol) and reduced iron powder (446.4 mg, 8.0 mmol) were mixed in a mixture of ethanol (20.0 mL) and 1M ammonium chloride aqueous solution (8.0 mL), and the mixture was heated to 85 °C and reacted for 2 hours. After the reaction was completed, the mixture was filtered while hot, and the solvent was removed by evaporation under reduced pressure. The product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (2:1, v / v) as the eluent, yielding product 6a-h in yields of 66%, 57%, 62%, 60%, 62%, 53%, 68%, and 49%, respectively.

[0069] Step 6: Synthesize compound 7a-h:

[0070] Accurately weigh the aminobenzene compound (i.e. Figure 1Dissolve 6a-h (1.0 mmol) and pyridine (174.02 mg, 2.2 mmol) in dichloromethane (2.0 mL), in an ice bath at 0 °C for 15 minutes, then add ethylsulfonyl chloride (154.30 mg, 1.2 mmol), and react at room temperature for 12 hours. Quench the reaction with saturated ammonium chloride solution, and extract the mixture with dichloromethane, repeating the process three times. Combine the organic phases, dry with anhydrous sodium sulfate, filter, and evaporate under reduced pressure to remove the solvent. Purify the product by silica gel column chromatography using dichloromethane / methanol (20:1, v / v) as the eluent to give product 7a-h in yields of 72%, 68%, 88%, 54%, 75%, 82%, 56%, and 80%, respectively.

[0071] Step 7: Synthesize compound 8a-p:

[0072] Accurately weigh the sulfonamide compound (i.e. Figure 1 7a-h)(0.05mmol), arylboronic acid ester compounds ( Figure 1 3a-i (0.06 mmol), tetra(triphenylphosphine)palladium (1.9 mg, 0.0018 mmol), and potassium carbonate (21 mg, 0.15 mmol) were dissolved in 2.0 mL of 1,4-dioxane and 0.5 mL of water, and the mixture was heated to 95 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and extracted with ethyl acetate and water, repeated three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation under reduced pressure. The product was purified by silica gel column chromatography using dichloromethane / methanol (40:1, v / v) as the eluent to give products 8a-p, i.e., compounds 1-16, with yields of 65%, 56%, 62%, 54%, 54%, 58%, 63%, 56%, 55%, 56%, 60%, 56%, 58%, 48%, 66%, and 56%, respectively.

[0073] Compound 1: 1 H NMR (600MHz, DMSO-d6) δ9.78 (s, 1H), 7.35-7.31 (m, 2H), 7.27 (dt, J=7.8, 1.2Hz, 1H), 7.20 (d, J=2.3Hz, 1H), 7.13 (ddd, J=8.0, 2.1, 0.9 Hz, 1H), 6.30 (d, J=2.3Hz, 2H), 5.72 (q, J=5.1Hz, 1H), 3.52 (s, 3H), 3.10 (q, J=7.3Hz, 2H), 2.74 (d, J=5.2Hz, 3H), 1.19 (t, J=7.3Hz, 3H). 13C NMR (150MHz, DMSO-d6) δ156.57, 139.24, 138.99, 138.92, 129.71, 121.44, 121.14, 118.87, 117.61, 116.69, 103.62, 45.15, 36.87, 29.43, 8.06. ESI-MS m / z: 322.1224[M+H] + .

[0074] Compound 2: 1 H NMR (600MHz, DMSO-d6) δ9.65 (s, 1H), 7.23 (d, J=2.7Hz, 1H), 7.13-7.05 (m, 4H), 6.88 (td, J=7.4, 6.6, 2.2Hz, 1H), 6.70 (d, J=8. 8Hz, 1H), 6.37 (d, J=2.1Hz, 1H), 3.75 (s, 3H), 3.47 (s, 3H), 3.07 (q, J=7.3Hz, 2H), 2.60 (d, J=5.2Hz, 3H), 1.21 (t, J=7.3Hz, 3H). 13 C NMR (150MHz, DMSO-d6) δ156.38, 150.45, 150.40, 144.66, 138.31, 133.42, 129.28, 124.51, 123.13, 1 22.26, 120.89, 120.53, 119.46, 118.15, 116.12, 113.15, 106.51, 55.61, 44.99, 36.79, 29.22, 8.03. ESI-MS m / z: 444.1586[M+H] + .

[0075] Compound 3: 1 H NMR (600MHz, DMSO-d6) δ9.71 (s, 1H), 7.19 (d, J = 2.7Hz, 1H), 7.11 (dd, J = 8.8, 2.7Hz, 1H), 7.08 (d, J = 2.3Hz, 1H), 6.86 (t, J = 8.9Hz, 2H), 6. 66-6.64 (m, 2H), 6.33 (dd, J=8.7, 2.8Hz, 1H), 5.17 (s, 2H), 3.70-3.68 (m, 6H), 3.45 (s, 3H), 3.08 (q, J=7.3Hz, 2H), 1.20 (t, J=7.3Hz, 3H). 13C NMR (150MHz, DMSO-d6) δ156.50, 150.72, 150.33, 149.74, 144.76, 137.39, 133.87, 129.97, 124.55, 1 21.82, 120.39, 119.96, 115.55, 112.48, 111.13, 108.79, 103.74, 55.87, 55.57, 44.99, 36.95, 8.04. ESI-MS m / z: 460.1537[M+H] + .

[0076] Compound 4: 1 H NMR (600MHz, DMSO-d6) δ9.72 (s, 1H), 7.25 (d, J=2.7Hz, 1H), 7.14 (dd, J=8.7, 2.7Hz, 1H ), 7.06 (d, J = 2.2Hz, 1H), 6.91 (d, J = 8.7Hz, 1H), 6.85 (d, J = 8.8Hz, 1H), 6.65 (d, J = 2.8Hz , 1H), 6.33 (dd, J=8.7, 2.8Hz, 1H), 6.23 (d, J=2.1Hz, 1H), 5.57 (q, J=5.1Hz, 1H), 3.68 (m , 6H), 3.46 (s, 3H), 3.09 (q, J=7.3Hz, 2H), 2.57 (d, J=5.2Hz, 3H), 1.21 (t, J=7.3Hz, 3H). 13 C NMR (150MHz, DMSO-d6) δ156.36, 151.12, 149.97, 149.79, 144.60, 138.37, 134.07, 130.56, 123.16, 121. 89, 120.50, 120.34, 115.98, 112.55, 108.21, 106.31, 103.33, 55.92, 55.59, 45.03, 36.78, 29.25, 8.04. ESI-MS m / z: 474.1694[M+H] + .

[0077] Compound 5: 11H NMR (600 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.14 (d, J = 4.8 Hz, 1H), 7.86 (s, 1H), 7.29 - 7.26 (m, 2H), 7.17 (dd, J = 8.7, 2.7 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 6.20 (d, J = 2.2 Hz, 1H), 5.60 (q, J = 5.2 Hz, 1H), 3.45 (s, 3H), 3.11 (q, J = 7.3 Hz, 2H), 2.58 (d, J = 5.2 Hz, 3H), 2.22 (s, 3H), 1.21 (t, J = 7.3 Hz, 3H). 13 13C NMR (150 MHz, DMSO-d6) δ 156.36, 152.14, 148.76, 144.04, 138.44, 137.90, 136.33, 134.88, 130.65, 126.04, 123.14, 122.03, 120.38, 120.33, 115.78, 105.90, 45.09, 36.79, 29.26, 15.25, 8.07. ESI-MS m / z: 429.1594 [M+H] + .

[0078] Compound 6: 1 1H NMR (600 MHz, DMSO-d6) δ 9.74 (s, 1H), 7.26 (d, J = 2.6 Hz, 1H), 7.14 (dd, J = 8.8, 2.7 Hz, 1H), 7.12 (dd, J = 9.4, 3.2 Hz, 1H), 7.03 (d, J = 2.3 Hz, 1H), 6.94 (td, J = 8.5, 3.2 Hz, 1H), 6.83 (d, J = 8.7 Hz, 1H), 6.68 (dd, J = 8.9, 4.8 Hz, 1H), 6.19 (d, J = 2.3 Hz, 1H), 5.63 - 5.55 (m, 1H), 3.46 (s, 3H), 3.09 (q, J = 7.3 Hz, 2H), 2.60 - 2.55 (m, 5H), 1.21 (t, J = 7.3 Hz, 3H), 1.09 (t, J = 7.6 Hz, 3H). 13C NMR (150MHz, DMSO-d6) δ158.56, 156.98, 156.36, 150.82, 149.75, 138.32, 135.76, 134.14, 130.58, 123. 05, 122.13, 120.51, 119.90, 118.00, 115.97, 113.45, 106.13, 45.03, 36.77, 29.15, 22.62, 14.07, 8.03. ESI-MS m / z: 460.1701[M+H] + .

[0079] Compound 7: 1 H NMR (600MHz, DMSO-d6) δ9.88 (s, 1H), 7.69 (dd, J=7.9, 1.7Hz, 1H), 7.52 (dt, J=7.5, 1 .7Hz, 1H), 7.32 (d, J=2.8Hz, 1H), 7.22 (dd, J=8.8, 2.7Hz, 1H), 7.17 (t, J=7.6Hz, 1H) , 7.05 (s, 1H), 7.01 (d, J = 2.3Hz, 1H), 6.74 (d, J = 8.4Hz, 1H), 6.14 (d, J = 2.3Hz, 1H), 5 .58 (s, 1H), 3.42 (s, 3H), 3.14 (q, J=7.4Hz, 2H), 2.53 (s, 3H), 1.22 (t, J=7.3Hz, 3H). 13 C NMR (150MHz, DMSO-d6) δ156.31, 155.20, 147.22, 138.40, 135.78, 134.33, 131.87, 127.12, 123.17, 1 22.27, 122.19, 121.91, 121.54, 120.25, 118.66, 116.38, 115.40, 105.61, 45.16, 36.72, 28.93, 8.04. ESI-MS m / z: 482.1356[M+H] + .

[0080] Compound 8: 11H NMR (600 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.45 (d, J = 4.7 Hz, 1H), 8.10 (s, 1H), 7.73 (d, J = 5.0 Hz, 1H), 7.32 (d, J = 2.6 Hz, 1H), 7.24 (dd, J = 8.7, 2.8 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H), 7.03 (d, J = 2.3 Hz, 1H), 6.13 (d, J = 2.3 Hz, 1H), 5.60 (q, J = 5.3 Hz, 1H), 3.42 (s, 3H), 3.16 (q, J = 7.3 Hz, 2H), 2.55 (d, J = 5.2 Hz, 3H), 1.22 (t, J = 7.3 Hz, 3H). 13 13C NMR (150 MHz, DMSO-d6) δ 156.31, 150.14, 146.83, 144.13, 138.97, 138.49, 136.26, 131.69, 128.17, 124.77, 123.24, 121.83, 121.59, 120.51, 120.23, 115.14, 105.40, 45.16, 36.72, 28.97, 8.04. ESI-MS m / z: 483.1309 [M+H] + .

[0081] Compound 9: 1 1H NMR (600 MHz, DMSO-d6) δ 9.73 (s, 1H), 7.26 (d, J = 2.6 Hz, 1H), 7.16 - 7.10 (m, 2H), 7.05 (d, J = 2.2 Hz, 1H), 6.93 (td, J = 8.9, 3.7 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 6.68 (dd, J = 8.9, 4.9 Hz, 1H), 6.21 (d, J = 2.3 Hz, 1H), 5.59 (q, J = 5.2 Hz, 1H), 3.46 (s, 3H), 3.09 (q, J = 7.3 Hz, 2H), 2.58 (d, J = 5.3 Hz, 3H), 2.18 (s, 3H), 1.21 (t, J = 7.3 Hz, 3H). 13C NMR (150MHz, DMSO-d6) δ158.35, 156.77, 156.35, 151.12, 149.60, 138.34, 134.12, 130.33, 129.92, 1 23.08, 122.16, 120.47, 119.73, 117.98, 115.94, 113.45, 106.13, 45.03, 36.78, 29.22, 15.89, 8.03. ESI-MS m / z: 446.1545[M+H] + .

[0082] Compound 10: 1 H NMR (600MHz, DMSO-d6) δ9.79 (s, 1H), 8.15 (d, J=4.8Hz, 1H), 7.88 (s, 1H), 7.29-7. 26 (m, 2H), 7.17 (dd, J=8.7, 2.7Hz, 1H), 7.08 (d, J=2.2Hz, 1H), 6.91 (d, J=8.7Hz, 1 H), 6.28 (d, J=2.2Hz, 1H), 5.34 (t, J=5.8Hz, 1H), 3.46 (s, 3H), 3.12 (q, J=7.5Hz, 2 H), 2.98-2.92 (m, 2H), 2.21 (s, 3H), 1.21 (t, J=7.3Hz, 3H), 1.05 (t, J=7.1Hz, 3H). 13 C NMR (150MHz, DMSO-d6) δ156.40, 152.14, 148.95, 144.12, 138.15, 137.10, 136.46, 134.75, 130.56, 126.06, 123.19, 122.02, 120.41, 120.15, 115.76, 106.43, 45.11, 36.88, 36.79, 15.22, 13.65, 8.07. ESI-MS m / z: 443.1748[M+H] + .

[0083] Compound 11: 11H NMR (600 MHz, DMSO-d6) δ 9.81 (s, 1H), 8.21 (d, J = 4.8 Hz, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.90 (s, 1H), 7.60 (d, J = 9.4 Hz, 1H), 7.36 (d, J = 4.3 Hz, 1H), 7.25 (s, 1H), 7.16 (d, J = 8.8 Hz, 1H), 6.90 (dd, J = 8.7, 2.2 Hz, 1H), 6.39 (d, J = 9.4 Hz, 1H), 3.44 (s, 3H), 3.12 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.21 (t, J = 8.4 Hz, 3H). 13 13C NMR (150 MHz, DMSO-d6) δ 161.00, 151.87, 149.02, 143.82, 140.79, 139.15, 138.06, 134.75, 128.66, 126.42, 125.02, 121.86, 120.89, 119.58, 118.54, 114.22, 45.15, 37.01, 15.35, 8.08. ESI-MS m / z: 400.1324 [M+H] + .

[0084] Compound 12: 1 1H NMR (600 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.84 - 8.80 (m, 1H), 8.28 (d, J = 5.1 Hz, 1H), 8.03 (s, 1H), 7.53 (d, J = 5.1 Hz, 1H), 7.28 (d, J = 2.7 Hz, 1H), 7.20 (dd, J = 8.8, 2.7 Hz, 1H), 7.08 (d, J = 2.2 Hz, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 3.45 (s, 3H), 3.37 - 3.32 (m, 1H), 3.13 (q, J = 7.3 Hz, 2H), 2.31 (s, 3H), 1.22 (t, J = 7.3 Hz, 3H), 1.07 - 1.04 (m, 6H). 13C NMR (150MHz, DMSO-d6) δ158.32, 158.09, 156.42, 152.87, 148.32, 144.77, 141.44, 135.95, 135.31, 130. 72, 127.02, 126.12, 123.35, 121.91, 120.58, 120.31, 115.49, 45.19, 43.09, 36.96, 21.67, 15.59, 8.07. ESI-MS m / z: 457.1901[M+H] + .

[0085] Compound 13: 1 H NMR (600MHz, DMSO-d6) δ9.80 (s, 1H), 8.17 (d, J = 4.8Hz, 1H), 7.91 (s, 1H), 7.89 (d, J = 2. 5Hz, 1H), 7.58 (dd, J=9.4, 2.5Hz, 1H), 7.29 (d, J=4.8Hz, 1H), 7.25 (d, J=2.6Hz, 1H), 7.1 7 (dd, J=8.7, 2.6Hz, 1H), 6.90 (d, J=8.7Hz, 1H), 6.38 (d, J=9.4Hz, 1H), 3.90 (q, J=7.1Hz , 2H), 3.12 (q, J=7.3Hz, 2H), 2.21 (s, 3H), 1.21 (t, J=7.3Hz, 3H), 1.16 (t, J=7.1Hz, 3H). 13 C NMR (150MHz, DMSO-d6) δ160.30, 151.70, 149.01, 144.41, 140.66, 138.64, 137.99, 136.81, 134. 71, 128.79, 126.13, 121.89, 120.82, 119.67, 119.01, 114.54, 45.13, 43.99, 15.26, 14.49, 8.08. ESI-MS m / z: 414.1485[M+H] + .

[0086] Compound 14: 1H NMR (600MHz, DMSO-d6) δ9.79 (s, 1H), 8.15 (d, J = 4.7Hz, 1H), 7.91 (s, 1H), 7.64 (s, 1H), 7.45 (s, 1H), 7.28 (d, J = 4.8Hz, 1H), 7.26 (d, J = 2.6Hz, 1 H), 7.18 (dd, J=8.7, 2.6Hz, 1H), 6.92 (d, J=8.7Hz, 1H), 5.08-5.00 (m, 1H), 3.12 (q, J=7.3Hz, 2H), 2.21 (s, 3H), 2.00 (s, 3H), 1.24-1.17 (m, 9H). 13 C NMR (150MHz, DMSO-d6) δ160.44, 151.89, 148.70, 144.19, 138.29, 136.87, 136.43, 134.89, 130.97, 129.51, 127.35, 126.03, 121.92, 120.46, 119.99, 114.07, 46.30, 45.08, 21.25, 17.20, 15.15, 8.05. ESI-MS m / z: 428.1641[M+H] + .

[0087] Compound 15: 1 H NMR (600MHz, DMSO-d6) δ9.79 (s, 1H), 8.16 (d, J = 4.8Hz, 1H), 7.90 (s, 1H), 7.76 (d, J = 2.5Hz, 1H), 7.51-7.46 (m, 1H), 7.28 (d, J = 4.8Hz, 1H), 7.24 (d, J = 2.7Hz, 1H), 7.16 (dd, J=8.7, 2.7Hz, 1H), 6.89 (d, J=8.7Hz, 1H), 3.44 (s, 3H ), 3.11 (q, J=7.3Hz, 2H), 2.20 (s, 3H), 1.98 (s, 3H), 1.21 (t, J=7.3Hz, 3H). 13 C NMR (150MHz, DMSO-d6) δ161.45, 151.75, 149.04, 144.40, 138.62, 137.73, 136.79, 136.37, 134. 67, 129.00, 127.02, 126.11, 121.96, 120.75, 119.72, 113.80, 45.13, 37.20, 16.92, 15.18, 8.07. ESI-MS m / z: 414.1483[M+H] + .

[0088] Compound 16:1 H NMR (600MHz, DMSO-d6) δ9.79 (s, 1H), 8.15 (d, J = 4.7Hz, 1H), 7.88 (s, 1H), 7.73 (s, 1H), 7.47 (s, 1H), 7.27 (d, J=4.8Hz, 1H), 7.25 (d, J=2.6Hz, 1H), 7.17 (dd, J= 8.7, 2.6Hz, 1H), 6.92 (d, J=8.7Hz, 1H), 3.90 (q, J=7.1Hz, 2H), 3.12 (q, J=7.3H z, 2H), 2.21 (s, 3H), 1.98 (s, 3H), 1.21 (t, J=7.3Hz, 3H), 1.14 (t, J=7.1Hz, 3H). 13 C NMR (150MHz, DMSO-d6) δ160.73, 151.78, 148.84, 144.28, 138.35, 137.60, 136.57, 135.22, 134.80, 129.20, 127.42, 126.07, 121.95, 120.65, 119.95, 114.03, 45.12, 44.15, 16.98, 15.21, 14.48, 8.08. ESI-MS m / z: 428.1636[M+H] + .

[0089] Example 1: Colon cancer inhibitory activity

[0090] HCT-116 colon cancer cells in the logarithmic growth phase were collected and 8 × 10⁻⁶ were used. 3 Cells were seeded at a density of 1:1 in 96-well plates and cultured overnight. Afterward, the positive control drug JQ-1 was added, with three auxiliary wells for each concentration. The plates were cultured for another 72 hours. After 72 hours, the 96-well plates were removed, the culture medium was discarded, and 100 μL of 10% trichloroacetic acid aqueous solution was added to each well. The plates were fixed at 4°C for at least 2 hours. After fixation, the 96-well plates were removed, the trichloroacetic acid solution was discarded, and the plates were rinsed at least four times with slowly flowing water and dried. Then, 80 μL of 0.4% SRB solution prepared with 1% acetic acid was added for staining for 20 minutes. During staining, a 1% acetic acid aqueous solution was prepared. After staining, the plates were rinsed four times with 1% acetic acid aqueous solution to remove unbound dye and dried again. Once the 96-well plates were completely dry, 100 μL of 10 mM Tris solution was added to each well, and the plates were shaken to completely dissolve the dye bound to the proteins. The absorbance was measured at 515 nm using a microplate reader. Inhibition rate = (OD value of control group - OD value of treated group) / OD value of control group × 100%. The IC50 of the compound's inhibitory activity is calculated using Graghpad Prism 9 software. 50 The values ​​are shown in Table 1.

[0091] Table 1

[0092] Compound IC 50 (μM) Compound IC 50 (μM) 1 ＞10.00 10 ＞10.00 2 0.73±0.01 11 4.16±0.06 3 ＞10.00 12 ＞10.00 4 0.18±0.01 13 3.78±0.05 5 0.84±0.02 14 0.43±0.01 6 ＞10.00 15 0.88±0.04 7 5.96±0.09 16 3.06±0.03 8 0.80±0.03 JQ-1 0.973±0.02 9 0.56±0.01 - -

[0093] Conclusion: Compounds 2, 4, 5, 8, 9, 14, and 15 in this study exhibit better anti-proliferative activity against HCT-116 colon cancer cells than the positive control drug JQ-1.

[0094] Example 2: BRD4 activity test

[0095] The following method was used to determine the inhibitory effect of the compounds prepared in this invention on the activity of BRD4 protein.

[0096] The experimental methods are briefly described below: BRD4 protein inhibitory activity was measured using the BRD4 bromodomain 1 TR-FRETAssay Kit (Cayman, catalog number: 600520) based on time-resolved fluorescence technology. JQ-1 inhibitor was used as a positive control, and three replicates were set up. 5 μL of the test compound (prepared in Examples 1-3) or buffer and 10 μL of europium(III)-chelated BRD4 bromodomain 1 were added to each well of a white 384-well plate. The plate was incubated at 20°C in the dark for 15 min. Then, 5 μL of a BRD4 bromodomain 1 ligand / APC receptor mixture was added, and the plate was incubated at room temperature in the dark for 60 min. Fluorescence signals were detected using a Tecan multi-mode microplate reader. The fluorescence intensity value measured at λ = 665 nm was F1, and the fluorescence intensity value at λ = 620 nm was F2. The signal ratio was calculated using the formula: Signal ratio = F1 / F2 × 10⁻¹⁰. 4 The inhibition rate of the test compound against protein kinase was calculated using the formula: Inhibition rate (%) = [1 - (Signal ratio of test compound - Minimum signal ratio) / (Maximum signal ratio - Minimum signal ratio)] × 100. Where the maximum signal ratio is the solvent control, and the minimum signal ratio is the signal ratio at a test concentration of 10 μM JQ⁻¹. The IC₀ of the inhibitory activity of the compound was calculated using Graghpad Prism 9 software. 50 The values ​​are shown in Table 2.

[0097] Table 2

[0098] Compound IC 50 (nM) Compound IC 50 (nM) 1 ＞100.0 10 52.8±0.9 2 12.4±0.7 11 28.3±0.9 3 66.8±0.9 12 124.6±0.8 4 10.5±0.6 13 245.9±0.8 5 7.9±0.5 14 14.1±0.6 6 22.2±0.7 15 8.9±0.6 7 73.1±0.6 16 40.3±0.7 8 27.4±0.9 JQ-1 33.4±1.0 9 16.8±0.8 - -

[0099] Conclusion: Compounds 2, 4, 5, 6, 8, 9, 11, 14, and 15 of this disclosure exhibit better BRD4 protein inhibitory activity than the positive control drug JQ-1. Among compounds 2, 4, 5, 8, 9, 14, and 15 that show better anti-HCT-116 colon cancer cell proliferation activity than the positive control drug JQ-1, compound 5 exhibits highly efficient BRD4 protein inhibitory activity.

Claims

1. A BRD4 protein inhibitor, characterized in that, The BRD4 protein inhibitor is a pyridinone derivative, with the structural formula shown in Formula I: Wherein, the R1 group is at least one of the following groups: hydrogen atom, methyl, ethyl, isopropyl, The R2 group is at least one of the following groups: hydrogen atom, methyl, methylamino, ethylamino, isopropylamino, acetamino. The R3 group is at least one of the following groups: hydrogen atom, phenoxy, cycloalkoxy, heteroaryloxy.

2. The BRD4 protein inhibitor according to claim 1, characterized in that, The R1 group is methyl, ethyl, or isopropyl; the R2 group is amino, ethylamino, isopropylamino, or acetamino; and the R3 group is a hydrogen atom, phenoxy, cycloalkoxy, or heteroaryloxy.

3. The BRD4 protein inhibitor according to claim 2, characterized in that, The pyridone derivative is any one of the following compounds:

4. The method for preparing the BRD4 protein inhibitor according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Under the action of cesium carbonate, aminopyridone and iodoalkane are substituted, or acylation with acetyl chloride is carried out to obtain product 2a-d; (2) Under the action of cesium carbonate, pyridones with R2 substitution and iodoalkanes are substituted to obtain product 2e-i; (3) The products 2a-d and 2e-i were reacted with pinacol diboronic acid ester in the presence of potassium acetate and DPPF palladium dichloride to obtain product 3a-i. (4) 3-Bromo-4-fluoronitrobenzene undergoes a substitution reaction with a phenol having a corresponding M1 group under the action of cesium carbonate to give product 5a-h; (5) Product 5a-h undergoes a reduction reaction under the action of iron powder and ammonium chloride to obtain product 6a-h; (6) Product 6a-h was sulfonated with ethylsulfonyl chloride under pyridine catalysis to give product 7a-h; (7) The product 7a-h and the product 3a-i in step (3) are subjected to a Suzuki coupling reaction to obtain a pyridinone derivative, namely the BRD4 protein inhibitor. R1 is a hydrogen atom, methyl, ethyl, or isopropyl, and R2 is a hydrogen atom, methyl, methylamino, ethylamino, isopropylamino, or acetamino. The M1 group is at least one of 2,4-difluorophenoxy, 2-methoxyphenoxy, 3,4-dimethoxyphenoxy, 4-trifluoromethylpyridine-3-oxy, 4-methylpyridine-3-oxy, 2-ethyl-4-fluorophenoxy, 2-trifluoromethyl-phenoxy, and 2-methyl-4-fluorophenoxy.

5. The method for preparing the BRD4 protein inhibitor according to claim 4, characterized in that, In step (1), the molar ratio of aminopyridone to iodoalkane is 1:1.5; In step (3), the molar ratios of products 2a-d and 2e-i with pinacol diborate, DPPF palladium dichloride, and potassium acetate are 1:1.2:0.1:3, respectively. Products 2a-d and 2e-i are first reacted with pinacol diborate in a solvent containing potassium acetate and DPPF palladium dichloride. The solvent used is anhydrous dioxane. In step (4), the molar ratio of 3-bromo-4-fluoronitrobenzene to phenol having the corresponding M1 group is 1:1.2; In step (5), the molar ratio of iron powder to product 5a-h is at least 4:1, and 1M ammonium chloride aqueous solution needs to be added as a reaction condition. In step (6), the molar ratio of product 6a-h to ethylsulfonyl chloride and pyridine is 1:1.2:2.2; In step (7), products 7a-h and 3a-i are dissolved in a solvent, and then a catalyst and a base are added to carry out the reaction. The molar ratio of product 7a-h to product 3a-i is 1:1.2; The solvent is a mixed solution of dioxane and water. The catalyst is tetra(triphenylphosphine)palladium. The alkali is potassium carbonate; The reaction conditions are: under nitrogen protection, at a reaction temperature of 95°C for 12-18 hours.

6. Use of the BRD4 protein inhibitor according to any one of claims 1 to 3 in the preparation of a medicament for treating and / or preventing diseases associated with abnormal BRD4 activity.

7. The application according to claim 6, characterized in that, The diseases associated with abnormal BRD4 activity are selected from at least one of cancer, inflammatory and immune diseases, nervous system diseases, and viral diseases; The cancer is selected from one of the following: acute myeloid leukemia, non-small cell lung cancer, colon cancer, and breast cancer; The inflammatory and immune diseases mentioned are at least one of atherosclerosis, inflammatory bowel disease, psoriasis, and cardiovascular disease; The viral disease mentioned is AIDS.

8. The application according to claim 7, characterized in that, The disease associated with abnormal BRD4 activity is colon cancer.

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