An aza-phenanthrene compound having brd4 protein inhibitory effect and a preparation method and application thereof

Abstract

The application relates to the technical field of pharmaceutical chemistry, in particular to a kind of azaphen compounds with BRD4 protein inhibition effect and preparation method and application thereof, these compounds can inhibit the interaction between BRD4 and p53 tumor suppressor, can be used for preparing BRD4 protein inhibitor, for preventing and treating the disease related to BRD4 protein inhibitor, good anti-tumor activity, with wide application prospect, structural formula is as shown in formula (I): wherein R1 is selected from hydrogen or methoxy;R2 is selected from hydrogen, methoxy or dimethylamino;R3 is selected from hydrogen or methoxy;R4 is selected from hydrogen or alkyl;R5 is selected from substituted benzene ring or naphthalene.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a phenanthrene compound with BRD4 protein inhibitory activity and its preparation method, pharmaceutical compositions containing these compounds and their pharmaceutical uses, particularly as BRD4 protein inhibitors. Background Technology

[0002] Bromodomains (BRDs) are a class of conserved protein domains that specifically recognize acetylated lysine residues, playing a crucial regulatory role in chromatin assembly and gene transcription. BRD4, a member of the bromodomain family, performs transcriptional regulation under physiological conditions, modulating normal cellular processes. Under abnormal conditions, they recruit various proteins to chromatin and transcription sites, regulating the transcription of genes closely related to gene expression, such as c-Myc and BCL-2.

[0003] In terms of tumor development, numerous studies have demonstrated the close link between BRD4 protein and various tumors. For example, in the testes, the fusion of nucleoprotein gene chromatin with the coding region of BRD4 forms a proto-oncogene, leading to midline carcinoma. Melanoma cells express a high amount of BRD4 protein to aid their proliferation, and inhibiting BRD4 can significantly slow down the growth of melanoma cells. Malignant peripheral nerve sheath tumor cells abnormally express BRD4 protein at high levels, and inhibiting BRD4 leads to the death of these tumor cells. Liver cancer tissue expresses higher levels of BRD4 than normal tissue, and inhibiting BRD4 can suppress the proliferation of liver cancer cells. In models of hematopoietic system tumors, including AML, Burkitt lymphoma, multiple myeloma, and B-cell acute lymphoblastic leukemia, interfering with the binding of the BRD4-related oncogene MYC can inhibit the expression of the oncogene MYC.

[0004] In the development and progression of inflammation, bromodomain proteins have been considered targets for many inflammation-related diseases because they can recruit other nuclear factors to regulate the expression of downstream genes through the action of RNA polymerase. Studies have shown that BRD4 can directly bind to transcription factors, regulate the transcription of the downstream gene NF-κB, and thus promote inflammation. Literature shows that the BRD4 inhibitor JQ1 can inhibit the expression of inflammatory cytokines related to the NF-κB pathway in macrophages, inhibit LPS-induced gingivitis, and in mouse models, JQ1 can also inhibit the expression of matrix metalloproteinases, alleviate joint swelling, and reduce joint inflammation.

[0005] The crucial role of BRD4 in tumors, particularly in refractory tumors and inflammation, has made it an increasingly important target in the field of epigenetics. The search for selective, safe, and efficient small molecule compounds targeting BRD4 protein to treat diseases such as tumors, inflammation, HIV / AIDS, and cardiovascular diseases is currently a hot research topic. Interfering with the bromodomain of the BRD4 protein using small molecule compounds to achieve targeted therapy for diseases such as tumors, inflammation, and HIV / AIDS holds broad promise for development and application. Summary of the Invention

[0006] To overcome the above problems, the present invention provides a phenanthrene compound with BRD4 protein inhibitory activity, its preparation method and application.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a phenanthrene compound having BRD4 protein inhibitory activity, the structural formula of which is shown in formula (I):

[0009] ;

[0010] Equation (I);

[0011] Wherein, R1 is selected from hydrogen or methoxy; R2 is selected from hydrogen, methoxy, or dimethylamino; R3 is selected from hydrogen or methoxy; R4 is selected from hydrogen or alkyl; and R5 is selected from substituted benzene rings or naphthalene.

[0012] Furthermore, compounds of formula (I) include:

[0013] N-(4-methoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8a);

[0014] N-(4-methoxyphenyl)-2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8b);

[0015] N-(4-methoxyphenyl)-2-((naphth-1-ylamino)methyl)benzo[h]quinoline-4-amine (compound 8c);

[0016] N-(4-methoxyphenyl)-2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8d);

[0017] N1-(2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine (compound 8e);

[0018] N1,N1-Dimethyl-N4-(2-((naphth-1-ylamino)methyl)benzo[h]quinoline-4-yl)phenyl-1,4-diamine (compound 8f);

[0019] N1-(2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine (compound 8g);

[0020] N1-(2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine (compound 8h);

[0021] N-(3,5-Dimethoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8i);

[0022] N-(3,5-dimethoxyphenyl)-2-((naphth-1-ylamino)methyl)benzo[h]quinoline-4-amine (compound 8j);

[0023] N-(3,5-dimethoxyphenyl)-2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8k);

[0024] N-(3,4-Dimethoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8l);

[0025] N-(3,5-Dimethoxyphenyl)-2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8m);

[0026] N-(3,4-Dimethoxyphenyl)-2-((naphth-1-ylamino)methyl)benzo[h]quinoline-4-amine (compound 8n);

[0027] N-(3,4-Dimethoxyphenyl)-2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8o);

[0028] N-(3,4-Dimethoxyphenyl)-2-(((3-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8p).

[0029] The specific structural formulas of compounds 8a~8p are as follows:

[0030]

[0031] The preferred compounds described above have stronger BRD4 protein inhibitory capabilities and are more suitable for preparing BRD4 protein inhibitors.

[0032] The compounds also include pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, isotope labels, solvates, polycrystalline or deuterated compounds.

[0033] Furthermore, the stereoisomers include enantiomers and diastereomers.

[0034] Furthermore, "pharmaceutically acceptable salts" refer to conventional, non-toxic salts, including inorganic acid salts such as hydrochlorides, hydrobroms, sulfates, phosphates, and nitrates; organic acid salts such as acetates, propionates, oxalates, succinates, lactates, malates, tartrates, citrates, maleates, fumarates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, and ascorbic acid salts; inorganic base salts such as sodium, potassium, calcium, zinc, magnesium, and aluminum salts; and organic base salts such as arginine salts, benzyl benzoate, choline salts, diethylamine salts, diolamine salts, glycine salts, lysine salts, meglumine salts, ethanolamine salts, and aminobutanetriol salts. In addition, those skilled in the art may choose one salt and discard another based on solubility, stability, ease of formulation, etc. The determination and optimization of these salts are within the experience of those skilled in the art.

[0035] A second aspect of the present invention provides a method for preparing the above-mentioned phenanthrene compounds with BRD4 protein inhibitory activity, comprising the following steps:

[0036] (1) Compound 1 and Compound 2 were added to polyphosphoric acid and reacted at 120~140 °C for 0.5~1.5 h. After the reaction was completed, the mixture was cooled to room temperature and saturated sodium carbonate solution was slowly added to neutralize the reaction solution to obtain solid compound 3. Solid compound 3 was filtered and dried.

[0037] (2) Add compound 3 to phosphorus oxychloride solution and stir continuously at 90~110 °C for 2~4 h. After the reaction is complete, cool to room temperature and add the reaction solution to ice water to obtain solid compound 4. Filter and dry solid compound 4.

[0038] (3) Add compound 4 and compound 5 to an ethanol solution, add two drops of concentrated hydrochloric acid, and react at 75~85 °C for 20~24 h. After the reaction is complete, cool to room temperature, concentrate and purify the reactants to obtain compound 6.

[0039] (4) Compound 6 was added to acetonitrile, compound 7 and potassium carbonate were added, and the reaction was carried out at 55~65 °C for 20~24 h. After the reaction was completed, the reaction was quenched with water, and the organic solvent was used for extraction. The extracted organic phase was washed, dried, concentrated and purified to obtain a nitrogen-phenanthroline compound (compound 8) with BRD4 protein inhibition.

[0040] .

[0041] In one or more embodiments, in step (1), the molar ratio of compound 1 to compound 2 is 0.9 to 1.1:1, preferably 1:1.

[0042] In one or more embodiments, in step (2), the molar ratio of compound 3 to phosphorus oxychloride is 1:40~55, preferably 1:50.

[0043] In one or more embodiments, in step (3), the molar ratio of compound 3 and compound 4 is 0.9 to 1.1:1, preferably 1:1.

[0044] In one or more embodiments, in step (4), the molar ratio of compound 6, compound 7 and potassium carbonate is 1:1:1.4 to 1.6, preferably 1:1:1.5.

[0045] In one or more embodiments, in step (4), after extraction with dichloromethane, the organic phase is washed with a saturated sodium chloride solution, then dried, concentrated and purified.

[0046] A third aspect of the present invention provides a pharmaceutical composition comprising a phenanthrene compound having BRD4 protein inhibitory activity as described in the first aspect.

[0047] A fourth aspect of the present invention provides a pharmaceutical formulation comprising a phenanthrene compound having BRD4 protein inhibitory activity as described in the first aspect, and a pharmaceutically acceptable carrier and / or excipients.

[0048] The formulations described in this invention are oral or parenteral preparations, and may be tablets, pills, capsules or injections.

[0049] The excipients described in this invention refer to components in a pharmaceutical composition or formulation other than the active ingredient, which are non-toxic to the subject. Commonly used excipients in the art include buffers, stabilizers, preservatives, or excipients, and commonly used excipients include binders, fillers, wetting agents, disintegrants, etc.

[0050] As an example, the excipients that can be used in the formulations of the present invention include, but are not limited to, calcium phosphate, magnesium stearate, talc, dextrin, starch, gel cellulose, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidone.

[0051] The drug carriers described in this invention can be pharmaceutically acceptable solvents, suspending agents, vesicles, nanomaterials, etc., for delivering the compounds described in the first aspect of this invention into animals or humans. The carriers can be liquid or solid, and are selected according to the planned route of administration. Proteins and liposomes are also drug carriers.

[0052] Those skilled in the art can formulate the compounds of the present invention into pharmaceutical compositions or preparations using known techniques. For example, any compound (at least one compound) disclosed in the first aspect of the present invention can be mixed with a pharmaceutical excipient, and then, if necessary, the resulting mixture can be shaped into a desired form. In addition to those mentioned in the present invention, pharmaceutical preparations can also be prepared based on known pharmaceutical formulations. Furthermore, suitable pharmaceutical excipients, other than those mentioned in the present invention, are known in the art; for example, see the 2005 edition of the Handbook of Pharmaceutical Excipients (Fourth Edition).

[0053] A fifth aspect of the invention provides the use of the phenanthrene compound having BRD4 protein inhibitory activity as described in the first aspect, or the pharmaceutical composition as described in the third aspect, or the pharmaceutical preparation as described in the fourth aspect, in the preparation of a BRD4 protein inhibitor.

[0054] This invention evaluates the BRD4 protein binding ability of the compounds and finds that the compounds provided by this invention can inhibit the BRD4 protein to varying degrees and can be used to prepare BRD4 protein inhibitors.

[0055] A sixth aspect of the invention provides the use of the phenanthrene compound having BRD4 protein inhibitory activity as described in the first aspect, or the pharmaceutical composition as described in the third aspect, or the pharmaceutical preparation as described in the fourth aspect, in the preparation of a medicament for the prevention and / or treatment of diseases related to BRD4.

[0056] Diseases associated with BRD include cancer, cardiovascular disease, inflammation, neurological disorders, and obesity.

[0057] The cancers mentioned include: melanoma, papillary thyroid tumor, cholangiocarcinoma, colon cancer, ovarian cancer, midline carcinoma, non-small cell lung cancer, malignant lymphoma, liver cancer, kidney cancer, lung cancer, pancreatic cancer, bladder cancer, prostate cancer, breast cancer, pancreatic cancer, thyroid cancer, skin cancer, colorectal cancer, pancreatic cancer, ovarian cancer, breast cancer, testicular cancer, bone cancer, brain cancer, esophageal cancer, gastrointestinal cancer, soft tissue tumor, leukemia, and lymphoma, with breast cancer being the preferred candidate.

[0058] In vitro activity studies have shown that the phenanthrene compounds with BRD4 protein inhibitory activity disclosed in this invention can inhibit the interaction between BRD4 and p53 tumor suppressor factor, and have a good inhibitory effect on the proliferation of the tested triple-negative breast cancer MDA-MB-231 cell line, inhibiting cell migration, cell invasion and cell cycle arrest, and promoting cell apoptosis.

[0059] In vivo activity studies have shown that the phenanthrene compound 8a disclosed in this invention, which has BRD4 protein inhibitory activity, can significantly control the growth of tumors in a nude mouse model of MDA-MB-231 xenograft tumors at an oral dose of 50 mg / kg, and achieves good therapeutic effects.

[0060] The beneficial effects of this invention are as follows:

[0061] This invention provides a phenanthrene compound with BRD4 protein inhibitory activity, which can inhibit the interaction between BRD4 and p53 tumor suppressor factor. It can be used to prepare BRD4 protein inhibitors for the prevention and treatment of diseases related to BRD4 protein inhibitors. It has good anti-tumor activity and has broad application prospects. Attached Figure Description

[0062] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0063] Figure 1 Figures show the results of compound 8a inhibiting the migration and invasion of MDA-MB-231 cells. A and B represent the effects of serum-induced Transwell migration assay on MDA-MB-231 cell migration; C and D represent the effects of scratch healing assay on MDA-MB-231 cell migration; and E represents the effects of Matrigel three-dimensional culture invasion assay on MDA-MB-231 cell invasion. Data are mean ± SEM values ​​from three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns, not significant compared to the control group.

[0064] Figure 2The results show the effects of compound 8a on apoptosis and cell cycle in MDA-MB-231 cells. Figure A shows the apoptosis detection results of MDA-MB-231 cells treated with different concentrations of compound 8a (0, 2.5, 5, and 10 μM); Figure B shows the percentage of cells in each phase; Figure C shows the effect of different concentrations of compound 8a (0, 2.5, 5, and 10 μM) on the cell cycle progression of MDA-MB-231 cells; Figure D shows the percentage of cells in each phase in group B. Data are mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001, compared with the control group.

[0065] Figure 3 To verify that compound 8a targets BRD4, the results are as follows: A shows the BRD4 knockout efficiency in MDA-MB-231 cells determined by Western blotting, with GAPDH as a control; B shows the cell growth inhibition curves after transfecting MDA-MB-231 cells with BRD4 siRNA or control siRNA and then treating them with different concentrations of compound 8a for 72 hours; C and D show the results of the CETSA assay.

[0066] Figure 4 The acute toxicity test of compound 8a is shown in Figure A, where A is the weight change graph of mice in each group; and B is the survival curve of mice in each group.

[0067] Figure 5 The in vivo antitumor effect of compound 8a on MDA-MB-231 tumors is shown in Figure 1. A represents the body weight of mice in each group; B represents the tumor growth curve of mice in each group; C represents the final tumor volume of mice; D represents the final tumor weight of mice; E represents the tumor image; and F represents the H&E and IHC analysis (200×) of mouse tumor tissue sections. Detailed Implementation

[0068] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0069] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0070] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0071] Example 1: Preparation of Compound 3

[0072] 1-Naphthylamine (10 mmol) and ethyl 4-chloro-3-oxobutyrate (10 mmol) were added to 20 mL of polyphosphoric acid. The mixture was heated at 130 °C for 1 hour. After cooling to room temperature, saturated sodium carbonate solution was slowly added to neutralize the reaction solution, yielding solid compound 3. The obtained solid compound 3 was filtered and dried, and used directly in the next step without purification.

[0073] Example 2 Preparation of Compound 4

[0074] Compound 3 was added to a phosphorus oxychloride solution (0.2 M, 50 mL) and stirred continuously at 100 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature and added to ice water to obtain solid compound 4. Solid compound 4 was filtered and dried and used directly in the next step without purification.

[0075] Example 3 Preparation of 2-(chloromethyl)-N-(4-methoxyphenyl)benzo[h]quinoline-4-amine (compound 6a)

[0076] Compound 4 (10 mmol) and 4-methoxyaniline (10 mmol) were added to an ethanol solution, and two drops of concentrated hydrochloric acid were added. The mixture was reacted at 80 °C for 24 h. After the reaction was completed, the mixture was cooled to room temperature, concentrated under vacuum, and then purified by column chromatography using a mixture of petroleum ether and ethyl acetate as the eluent to obtain compound 6a.

[0077] Example 4 Preparation of N-(4-methoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (compound 8a)

[0078] Compound 6a (10 mmol) was added to acetonitrile to prepare a 0.2 mol / L solution. 4-methoxyaniline (10 mmol) and potassium carbonate (15 mmol) were added, and the mixture was reacted at 60 °C for 24 h. After the reaction was completed, the reaction was quenched with water, extracted with dichloromethane, and the organic phase was washed with saturated sodium chloride solution, dried, concentrated under vacuum, and purified by column chromatography using a mixture of petroleum ether and ethyl acetate as eluent to obtain compound 8a, with an overall yield of 65%.

[0079] Compound 8a obtained in this example was characterized, with the following melting points: mp: 244-247°C; and its 1H NMR spectrum: 1H-NMR (400 MHz; DMSO-d6): δ 9.24 (dd, J = 6.1, 3.5 Hz, 1H), 8.81 (s, 1H), 8.27 (d, J =9.2 Hz, 1H), 7.98 (dd, J = 6.0, 3.3 Hz, 1H), 7.82 (d, J = 9.1 Hz, 1H), 7.70 (dq, J = 6.3, 3.3 Hz, 2H), 7.13-7.09 (m, 2H), 6.89-6.86 (m, 2H), 6.71-6.68 (m, 2H), 6.57-6.54 (m, 2H), 5.97-5.95 (m, 1H), 4.39 (d, J = 4.7 Hz, 2H), 3.76 (s, 3H), 3.61 (s, 3H). Carbon NMR spectrum: 13 C-NMR (101 MHz; DMSO-d6): δ 159.6, 155.8, 150.8, 149.1, 145.9, 142.7, 133.21, 133.19, 131.0, 127.8, 127.5, 126.4, 124.7, 124.36, 124.23, 119.6, 114.62, 114.51, 114.46, 113.5, 100.5, 55.29, 55.21, 50.2. High-resolution mass spectrometry (ESI-HRMS) m / z: calcd for [M+H] + 436.2020, found 436.2023. Its purity was determined to be 99.5% by high performance liquid chromatography (HPLC).

[0080] N-(4-methoxyphenyl)-2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine (8b)

[0081] mp: 154-155 ℃; 1H NMR (DMSO-d6, 400 MHz) δ 9.21 – 9.11 (1H, m), 8.81 (1H, s), 8.26 (1H, d, J=9.2 Hz), 8.02 – 7.95 (1H, m), 7.83 (1H, d, J=9.1 Hz), 7.77 – 7.62 (2H, m), 7.13 – 7.03 (2H, m), 6.91 – 6.84 (2H, m), 6.81 – 6.74(3H, m), 6.73 – 6.66 (2H, m), 4.65 (2H, s), 3.77 (3H, s), 3.65 (3H, s), 2.99(3H, s); 13C NMR (DMSO-d6, 101 MHz) δ 151.45, 144.17, 133.72, 128.07, 126.92,124.98, 124.75, 120.04, 114.98, 114.90, 114.35, 101.01, 55.72, 55.67; HRMS (ESI-HRMS) m / z Calcd for [M+H]+ :450.2181. Found: 450.2164; HPLC purity 99.1%.

[0082] N-(4-methoxyphenyl)-2-((naphthyl-1-amino)methyl)benzo[h]quinoline-4-amine (8c)

[0083] mp: 165-166 ℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.30 – 9.22 (1H, m), 8.77(1H, s), 8.28 – 8.18 (2H, m), 8.03 – 7.96 (1H, m), 7.82 (2H, dd, J=8.4, 3.2Hz), 7.76 – 7.68 (2H, m), 7.53 – 7.41 (2H, m), 7.21 – 7.11 (2H, m), 7.08 (1H,t, J=5.9 Hz), 7.05 (1H, s), 6.93 – 6.84 (2H, m), 6.55 – 6.47 (2H, m), 6.41(1H, dd, J=7.5, 1.2 Hz), 4.67 (2H, d, J=5.9 Hz), 3.62 (3H, s); 13C NMR (DMSO-d6, 101 MHz) δ 159.47, 155.94, 149.36, 143.98, 133.71, 133.49, 131.42,128.51, 128.25, 128.05, 127.12, 126.92, 126.11, 125.03, 124.73, 124.61,124.30, 123.61, 121.96, 120.09, 116.06, 114.57, 104.70, 100.66, 55.58, 49.81;HRMS (ESI-HRMS) m / z Calcd for [M+H] + :456.2076. Found: 456.2065; HPLC purity99.8%.

[0084] N-(4-methoxyphenyl)-2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (8d)

[0085] mp: 131-132 ℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.30 – 9.18 (1H, m), 8.83 (1H, s), 8.28 (1H, d, J=9.2 Hz), 8.06 – 7.95 (1H, m), 7.84 (1H, d, J=9.1 Hz), 7.77 – 7.66 (2H, m), 7.24 – 7.12 (2H, m), 7.01 – 6.87 (3H, m), 6.73 (1H, d, J=8.6 Hz), 6.14 (1H, d, J=2.8 Hz), 6.11 – 6.04 (2H, m), 4.42 (2H, d, J=5.7Hz), 3.78 (5H, d, J = 4.0 Hz), 3.56 (2H, s); 13C NMR (DMSO-d6, 101 MHz) δ 158.69,156.52, 154.81, 149.94, 141.64, 139.47, 133.74, 133.56, 131.38, 128.27,128.08, 126.95, 125.39, 124.85, 120.12, 115.15, 115.05, 111.09, 100.84,98.86, 98.46, 56.57, 55.74, 55.38, 49.38; HRMS (ESI-HRMS) m / z Calcd for [M+H] + :466.2130. Found: 466.2120; HPLC purity 95.1 %.

[0086] N1-(2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine(8e)

[0087] mp: 143-145℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.23 (1H, dd, J=6.2, 3.5Hz), 8.68 (1H, s), 8.27 (1H, d, J=9.2 Hz), 7.97 (1H, dd, J=6.2, 3.2 Hz), 7.80(1H, d, J=9.1 Hz), 7.69 (2H, dt, J=6.2, 3.5 Hz), 7.03 (2H, d, J=8.8 Hz), 6.93(1H, s), 6.74 – 6.65 (4H, m), 6.59 – 6.53 (2H, m), 5.91 (1H, s), 4.35 (2H, d,J=5.6 Hz), 3.62 (3H, s), 2.90 (6H, s); 13C NMR (DMSO-d6, 101 MHz) δ 159.82,151.26, 150.08, 148.13, 143.30, 133.68, 131.49, 129.72, 128.11, 127.95,126.76, 125.16, 125.12, 124.46, 120.14, 114.98, 114.88, 113.96, 113.56,100.66, 55.77, 50.80, 40.88; HRMS (ESI-HRMS) m / z Calcd for [M+H] + :449.2341. Found: 449.2330; HPLC purity 99.8 %.

[0088] N1,N1-Dimethyl-N4(2-((naphth-1-ylamino)methyl)benzo[h]quinoline-4-yl)phenyl-1,4-diamine (8f)

[0089] mp:232-233 ℃, 1 H NMR (DMSO-d6, 400 MHz) δ 9.29 – 9.22 (1H, m), 8.67(1H, s), 8.24 (1H, s), 8.21 (1H, dd, J=7.4, 2.2 Hz), 7.98 (1H, dt, J=7.6, 2.6Hz), 7.85 – 7.75 (2H, m), 7.75 – 7.67 (2H, m), 7.51 – 7.41 (2H, m), 7.23 –7.12 (2H, m), 7.08 (1H, d, J=5.9 Hz), 7.00 (1H, s), 6.83 – 6.75 (2H, m), 6.41(1H, dd, J=7.5, 1.2 Hz), 6.34 – 6.24 (2H, m), 4.65 (2H, d, J=5.8 Hz), 2.76 (6H, s); 13C NMR (DMSO-d6, 101 MHz) δ 159.28, 149.77, 147.80, 146.50, 143.99,134.56, 133.69, 131.46, 129.57, 128.48, 128.15, 128.02, 127.11, 126.82,126.07, 125.05, 124.56, 124.47, 122.01, 120.11, 116.00, 114.93, 113.70,113.22, 104.71, 100.27, 49.82, 40.83; HRMS (ESI-HRMS) m / z Calcd for [M+H] + :469.2392. Found: 469.2380; HPLC purity 96.5 %.

[0090] N1-(2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine (8g)

[0091] mp: 168-170 ℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.14 (1H, dt, J=7.7, 2.8Hz), 8.67 (1H, s), 8.26 (1H, d, J=9.2 Hz), 7.99 – 7.92 (1H, m), 7.78 (1H, d,J=9.1 Hz), 7.67 (2H, tt, J=6.3, 4.7 Hz), 7.03 – 6.97 (2H, m), 6.79 – 6.73(3H, m), 6.72 – 6.64 (4H, m), 4.61 (2H, s), 3.64 (3H, s), 2.99 (3H, s), 2.90(6H, s); 13C NMR (DMSO-d6, 101 MHz) δ 158.99, 151.40, 149.91, 148.03, 146.61,144.32, 133.65, 129.65, 128.12, 127.95, 126.74, 125.07, 124.81, 124.46,120.11, 114.95, 114.80, 114.35, 113.47, 100.69, 59.74, 55.73, 40.87; HRMS(ESI-HRMS) m / z Calcd for [M+H] + :463.2498. Found: 463.2484; HPLC purity 95.3%.

[0092] N1-(2-(((2,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine (8h)

[0093] mp: 179-180 ℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.25 – 9.18 (1H, m), 8.73 (1H, s), 8.29 (1H, d, J=9.1 Hz), 8.02 – 7.95 (1H, m), 7.82 (1H, d, J=9.1 Hz), 7.71 (2H, dd, J=6.6, 2.9 Hz), 7.14 – 7.05 (2H, m), 6.83 (1H, s), 6.73 (3H, dd, J=8.8, 6.3 Hz), 6.15 (1H, d, J=2.9 Hz), 6.11 – 6.04 (2H, m), 4.39 (2H, d,J=5.5 Hz), 3.78 (3H, s), 3.57 (3H, s), 2.92 (6H, s); 13C NMR (DMSO-d6, 101 MHz)δ 158.47, 150.50, 148.36, 146.00, 141.63, 139.52, 129.52, 128.18, 128.06,126.87, 125.67, 124.87, 124.63, 120.14, 114.93, 113.61, 111.06, 100.45,98.86, 98.45, 56.57, 55.38, 49.40, 40.89; HRMS (ESI-HRMS) m / z Calcd for [M+H] + :479.2447. Found: 479.2437; HPLC purity 98.0 %.

[0094] N-(3,5-Dimethoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine(12)amine(8i)

[0095] mp: 69-70 ℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.26 (1H, dt, J=6.1, 3.5Hz), 8.91 (1H, s), 8.23 ​​(1H, d, J=9.2 Hz), 8.04 – 7.96 (1H, m), 7.86 (1H, d,J=9.1 Hz), 7.72 (2H, dt, J=6.1, 3.5 Hz), 7.44 (1H, s), 6.73 – 6.63 (2H, m), 6.59 – 6.52 (2H, m), 6.44 (2H, d, J=2.2 Hz), 6.19 (1H, t, J=2.2 Hz), 6.07(1H, t, J=5.9 Hz), 4.44 (2H, d, J=5.8 Hz), 3.63 (6H, s), 3.59 (3H, s); 13C NMR(DMSO-d6, 101 MHz) δ 161.44, 160.63, 151.28, 148.23, 146.62, 143.35, 133.71,131.38, 128.37, 128.05, 127.00, 125.08, 120.29, 116.17, 115.08, 113.69,103.15, 99.31, 95.89, 55.72, 55.48, 51.05; HRMS (ESI-HRMS) m / z Calcd for [M+H] + :466.2130. Found: 466.2117, HPLC purity 98.2 %.

[0096] N-(3,5-Dimethoxyphenyl)-2-((naphthyl-1-amino)methyl)benzo[h]quinoline-4-amine (8j)

[0097] mp: 87.2-89.0 ℃; 1 H NMR (DMSOd6, 400 MHz) δ 9.32 – 9.25 (1H, m), 8.89 (1H, s), 8.23 ​​(2H, dd, J=9.3, 3.2 Hz), 8.04 – 7.99 (1H, m), 7.87 (1H, d, J=9.1 Hz), 7.75 (3H, tdd, J=10.2, 6.7, 4.5 Hz), 7.52 – 7.42 (3H, m), 7.19 –7.11 (2H, m), 7.07 (1H, d, J=8.1 Hz), 6.47 (1H, d, J=7.5 Hz), 6.36 (2H, d, J=2.2 Hz), 6.08 (1H, t, J=2.2 Hz), 4.70 (2H, d, J=5.6 Hz), 3.42 (6H, s); 13C NMR(DMSO-d6, 101 MHz) δ 161.32, 160.17, 148.27, 144.24, 143.25, 134.54, 133.74,131.37, 128.50, 128.11, 127.17, 127.08, HRMS (ESI-HRMS) m / z Calcd for [M+H] + :486.2181. Found: 486.2168; HPLC purity 96.5 %.

[0098] N-(3,5-dimethoxyphenyl)-2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (8k)

[0099] mp: 77.8-80.4 ℃; 1H NMR (DMSO-d6, 400 MHz) δ 9.26 (1H, dd, J=6.2, 3.5Hz), 8.90 (1H, s), 8.23 ​​(1H, d, J=9.2 Hz), 8.00 (1H, dt, J=6.0, 3.4 Hz), 7.86(1H, d, J=9.1 Hz), 7.72 (2H, dt, J=6.1, 3.5 Hz), 7.41 (1H, s), 6.50 – 6.44(3H, m), 6.20 (1H, t, J=2.2 Hz), 5.83 (2H, d, J=2.2 Hz), 5.69 (1H, t, J=2.1Hz), 4.44 (2H, d, J=5.9 Hz), 3.66 (6H, s), 3.58 (6H, s); 13C NMR (DMSO-d6,101 MHz) δ 161.59, 161.46, 160.25, 150.92, 148.29, 146.50, 143.33, 133.72,128.40, 128.07, 127.01, 125.18, 125.03, 120.28, 116.17, 103.16, 99.37, 95.96,91.58, 89.10, 55.48, 55.11, 50.37; HRMS (ESI-HRMS) m / z Calculated for [M+H] + :496.2236. Found: 496.2220; HPLC purity 95.9 %.

[0100] N-(3,4-Dimethoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (8l)

[0101] mp: 182-183 ℃; 1H NMR (DMSO-d6, 400 MHz) δ 9.24 (1H, dd, J=6.2, 3.5Hz), 8.79 (1H, s), 8.26 (1H, d, J=9.1 Hz), 7.98 (1H, dd, J=6.1, 3.2 Hz), 7.82(1H, d, J=9.1 Hz), 7.70 (2H, dt, J=6.2, 3.4 Hz), 7.13 (1H, s), 6.90 – 6.83(2H, m), 6.74 – 6.66 (3H, m), 6.59 – 6.52 (2H, m), 5.97 (1H, t, J=6.0 Hz),4.38 (2H, d, J=5.9 Hz), 3.76 (3H, s), 3.60 (6H, d, J=4.2 Hz); 13 C NMR (DMSO-d6,101 MHz) δ 160.31, 151.25, 149.63, 149.53, 146.43, 145.87, 143.32, 134.08,133.70, 131.43, 128.23, 128.00, 126.86, 125.13, 124.72, 120.12, 115.20,115.04, 113.76, 112.86, 108.10, 101.06, 56.21, 55.75, 55.71, 50.87; HRMS(ESI-HRMS) m / z Calcd for [M+H] + : 466.2130. Found: 466.2117; HPLC purity 97.1%.

[0102] N-(3,5-Dimethoxyphenyl)-2-(((4-((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine(8m)

[0103] mp: 127-129 ℃; 1H NMR (DMSO-d6, 400 MHz) δ 8.90 (0H, s), 8.21 (0H, d,J=9.2 Hz), 7.85 (0H, d, J=9.2 Hz), 7.73 – 7.67 (1H, m), 7.25 (0H, s), 6.77 –6.67 (2H, m), 6.43 (1H, d, J=2.2 Hz), 6.18 (1H, t, J=2.2 Hz), 4.67 (1H, s), 3.63 (4H, d, J=9.5 Hz), 3.07 (1H, s); 13 C NMR (DMSO-d6, 101 MHz) δ 161.42,159.86, 151.39, 148.22, 144.32, 143.30, 133.70, 128.41, 128.06, 127.01,125.12, 124.99, 120.26, 116.10, 115.03, 113.90, 103.21, 99.21, 95.93, 60.05,55.68, 55.50; HRMS (ESI-HRMS) m / z Calcd for [M+H] + :480.2287. Found: 480.2274; HPLC purity 99.4 %.

[0104] N-(3,4-Dimethoxyphenyl)-2-((naphthyl-1-amino)methyl)benzo[h]quinoline-4-amine (8n)

[0105] mp: 213-214 ℃; 1 H NMR (DMSOd6, 400 MHz) δ 9.30 – 9.24 (1H, m), 8.79 (1H, s), 8.28 – 8.19 (2H, m), 8.02 – 7.98 (1H, m), 7.86 – 7.78 (2H, m), 7.75– 7.68 (2H, m), 7.47 (2H, tt, J=7.1, 5.5 Hz), 7.23 – 7.07 (4H, m), 6.73 (1H,s), 6.44 (3H, q, J=7.2 Hz), 4.67 (2H, d, J=5.8 Hz), 3.63 (3H, s), 3.36 (3H,s); 13C NMR (DMSO-d6, 101 MHz) δ 159.78, 149.45, 149.33, 146.56, 145.56,144.09, 134.55, 133.92, 133.71, 128.50, 128.27, 128.05, 127.15, 126.93,126.12, 125.04, 124.77, 124.62, 123.54, 121.92, 120.09, 116.04, 114.49,112.42, 107.69, 104.42, 100.71, 56.08, 55.45, 50.10; HRMS (ESI-HRMS) m / zCalcd for [M+H] + :486.2181. Found: 486.2169; HPLC purity 97.0 %.

[0106] N-(3,4-Dimethoxyphenyl)-2-(((4-((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine(8o)

[0107] mp: 72.7-75.7 ℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.20 – 9.14 (1H, m), 8.81 (1H, s), 8.26 (1H, d, J=9.1 Hz), 8.02 – 7.94 (1H, m), 7.82 (1H, d, J=9.1Hz), 7.74 – 7.65 (2H, m), 6.93 (1H, s), 6.88 – 6.83 (2H, m), 6.78 – 6.72 (2H,m), 6.71 – 6.65 (3H, m), 4.64 (2H, s), 3.76 (3H, s), 3.63 (3H, s), 3.62 (3H,s), 3.01 (3H,s); 13C NMR (DMSO-d6, 101 MHz) δ 159.47, 151.37, 149.59, 149.46,146.72, 145.82, 144.30, 134.01, 133.68, 131.43, 128.25, 128.00, HRMS (ESI-HRMS) m / z Calcd for [M+H] + :480.2287. Found: 480.2276; HPLC purity 97.9 %.

[0108] N-(3,4-Dimethoxyphenyl)-2-(((3-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine (8p)

[0109] mp: 153-154 ℃; 1 H NMR (DMSO-d6, 400 MHz) δ 9.25 (1H, dt, J=6.2, 3.5Hz), 8.82 (1H, s), 8.27 (1H, d, J=9.3 Hz), 8.03 – 7.94 (1H, m), 7.83 (1H, d,J=9.1 Hz), 7.71 (2H, dt, J=6.2, 3.4 Hz), 7.13 (1H, s), 6.96 – 6.84 (3H, m), 6.72 (1H, dd, J=8.5, 2.5 Hz), 6.41 (1H, t, J=6.0 Hz), 6.19 (2H, dh, J=3.2,1.5 Hz), 6.11 (1H, ddd, J=8.1, 2.4, 0.9 Hz), 4.42 (2H, d, J=5.9 Hz), 3.76 (3H, s), 3.61 (6H, s); 13C NMR (DMSO-d6, 101 MHz) δ 160.77, 159.99, 150.44,149.63, 149.55, 146.41, 145.89, 133.71, 131.43, 130.00, 128.24, 128.01,126.87, 125.11, 124.78, 120.11, 115.20, 115.06, 112.82, 108.11, 105.91,101.84, 101.01, 98.63, 56.19, 55.72, 55.03, 50.17; HRMS (ESI-HRMS) m / z Calcd for [M+H] + : 465.2052. Found: 466.2119; HPLC purity 95.3 %.

[0110] Experimental Example 1

[0111] The AlphaScreen (Amplified Luminous Proximity Homogeneous Assay) is a widely used drug screening method for detecting protein-protein interactions. 25 nM FLAG(f:)-labeled BRD4 phosphorylation-dependent interaction domain (pPDID; aa 287-530) or 150 nM FLAG(f:)-labeled full-length (FL)f:BRD4 is immobilized on a recipient bead and interacts with 150 nM GST-labeled p53 REG (aa 309-393) or GST-labeled FL p53 immobilized on a donor bead. In this experiment, the FLAG-labeled pPDID and FL BRD4 used were expressed and purified in insect Sf9 cells, while the GST-labeled p53 REG and FL p53 were expressed and purified in bacteria. The reaction was carried out in 20 μL of detection buffer containing 20 mM Tris-HCl (pH 7.3), 100 mM NaCl, 0.2 mM EDTA (pH 8.0), 0.01% NP40 (or IGEPEAL), and 1% BSA (Sigma, A7030). Alpha GSH donor beads (final 5.0 µg / mL, PerkinElmer, Cat# 6765300) and AlphaLISA anti-FLAG receptor beads (final 2.5 µg / mL, PerkinElmer, Cat# AL112) were added to a PROXIPLATE-384-PLUS plate (PerkinElmer, Cat# 6008280). After the plate was gently shaken overnight at room temperature, the interaction signal was detected using a PerkinElmer Envision multiplate reader with excitation and emission wavelengths set to 680 nm and 615 nm, respectively. Starting at 40 µM (final DMSO concentration of 5%), eight doses were prepared for each compound using 2-fold serial dilutions: 40 µM, 20 µM, 10 µM, 5 µM, 2.5 µM, 1.25 µM, 0.625 µM, and 0.3125 µM, with three replicates for each dose. Inhibitory activity (IC50) (i.e., the concentration of the compound that inhibits 50% of protein-protein interactions) was calculated using GraphPad Prism 8.0.

[0112] The inhibitory activity (IC50) of the synthesized compound against the interaction between BRD4 and p53 proteins was determined using the AlphaScreen method at eight concentration gradients. The results are shown in Table 1 below:

[0113] Table 1. Inhibitory activity (IC50) of compounds 8a-8p against the interaction between BRD4 and p53 proteins.

[0114]

[0115] Compounds 8a-8p in this invention all exhibit good activity in inhibiting the interaction between the BRD4 phosphorylation-dependent interaction domain pPDID and the p53 regulatory domain p53REG, as well as the interaction between BRD4 protein and p53 protein, with IC50 values ​​of 0.06-4.05 μM and 0.43-8.54 μM, respectively.

[0116] Experimental Example 2: Determination of the inhibitory activity of compound 8a on the proliferation of MDA-MB-231 cells.

[0117] Cells were seeded at a concentration of 3000 cells / well in 96-well clear plates, 100 μL per well. They were cultured for 24 h at 37 °C with 5% CO2. Then, starting from 160 µM (DMSO final concentration 0.5%), seven doses of each compound were prepared by serial dilution (2-fold): 160 µM, 80 µM, 40 µM, 20 µM, 10 µM, 5 µM, and 2.5 µM, with three replicates for each dose. The serially diluted compound solutions were added to the wells, and the cells were cultured for another 72 h or 96 h. Cell viability was evaluated using the CCK8 assay, and absorbance at 450 nm was recorded using a BMG CLARIOstar microplate reader. Finally, the IC50 value was calculated using GraphPad Prism 8.0. The IC50 values ​​of the tested compound 8a in inhibiting tumor cell proliferation are shown in Table 2.

[0118] Table 2. IC50 of compound 8a in inhibiting tumor cell proliferation.

[0119]

[0120] In vitro cell proliferation studies of compound 8a in this invention showed that compound 8a had an IC50 of 10.5 μM in MDA-MB-231 cells after 72 h of treatment and an IC50 of 12.6 μM after 96 h.

[0121] Experimental Example 3: Determination of the effect of compound 8a on cell migration and cell invasion

[0122] Transwell migration assay: MDA-MB-231 cells were collected and adjusted to 1 × 10⁻⁶ cells per 0.1 mL serum-free DMEM. 6Cells were injected with different concentrations of the test compound at ratios of 0, 2.5 μM, 5 μM, and 10 μM, then placed in Transwell chambers (Corning 3422) and in receiving wells containing 0.6 mL of DMEM containing 10% FBS. The chambers were incubated at 37 °C in a 5% CO2 incubator for 24–40 hours. The receiving wells were washed once with PBS and fixed with 4% paraformaldehyde solution for 10 min at room temperature. After washing twice with PBS, the cells were stained with 0.1% crystal violet solution for 10 min, washed, inverted, and air-dried. Images were taken using an inverted fluorescence microscope (Olympus). Scratch assay and cell migration measurement: MDA-MB-231 cells were seeded in 6-well plates at 1 × 10⁶ cells per well. 6 Cells were cultured for 24 h, and scratches were created using 10 μL pipette tips. Cells were washed three times with 1× PBS, then added to serum-free medium containing the compound. After 24 h of culture, the scratches were photographed using an inverted fluorescence microscope (Olympus). The percentage of scratch healing was calculated compared to the control group. Cell invasion assay: MDA-MB-231 cells were collected in DMEM containing 1% FBS, 2% Matrigel (Corning Matrigel membrane matrix GFR), and different concentrations of the test compound, and then injected at 2×10⁻⁶ cells / cm². 4 Cells were added to Matrigel-pre-coated (120 μL) 24-well plates, and images of the cells were taken using an inverted fluorescence microscope (Olympus) after 3–5 days.

[0123] from Figure 1 As can be seen from the Transwell migration assay results, compound 8a can inhibit the migration of MDA-MB-231 cells in a dose-dependent manner compared with the control group. Figure 1 (A and B). Scratch test results showed that compound 8a significantly reduced scratch healing rate in a dose-dependent manner (A and B). Figure 1 Compound 8a (C and D) further confirmed its ability to inhibit cell migration. In cell invasion assays, compound 8a was able to inhibit the invasion of MDA-MB-231 cells in a dose-dependent manner. Figure 1 (E). In summary, compound 8a can effectively inhibit the migration and invasion of tumor cells.

[0124] Experimental Example 4: Determination of the effects of compound 8a on apoptosis and cell cycle.

[0125] Apoptosis assay: MDA-MB-231 cells were seeded in 6-well plates at 300,000 cells per well. After 24 h, cells were treated with different concentrations (0, 2.5 μM, 5 μM, 10 μM) of compound solutions for 24 h, and then collected. Cells were washed twice with PBS, and then resuspended in 100 μL of binding buffer. 5 μL of Annexin V, FITC, and 5 μL of PI staining solution were added, and the cells were incubated at room temperature in the dark for 15 min. 400 μL of binding buffer was added, and the staining results were detected by flow cytometry (Beckman CytoFLEX S). The staining results were analyzed using CytExpert. Cell cycle assay: MDA-MB-231 cells were seeded in 6-well plates at 300,000 cells per well. After 24 h, cells were treated with a compound solution prepared in serum-free medium for 24 h, and then collected. Cells were washed once with PBS and fixed overnight with 70% ethanol. Wash once with PBS, add 0.5 mL of propidium iodide staining solution (containing 10 μL RNase A solution and 25 μL PI staining solution), slowly and thoroughly resuspend the cell pellet, incubate at 37 ºC in the dark for 30 minutes, then store at 4 ºC or on ice in the dark. Flow cytometry (Beckman CytoFLEX S) was used to detect the staining results, and CytExpert was used to analyze the results.

[0126] from Figure 2 It can be seen that, compared with the control group, compound 8a effectively induced apoptosis in cells after treatment for 24 h, and the proportion of apoptotic cells increased with increasing concentration. Figure 2 (A and B). Furthermore, the effects of the compounds on the cell cycle were evaluated. Compound 8a treatment increased the number of cells in the G1 phase but decreased the number of cells in the S and G2 / M phases. Clearly, compound 8a arrested the cell cycle in the G1 phase. Figure 2 (C and D). In summary, compound 8a can effectively induce apoptosis in MDA-MB-231 cells and arrest the cell cycle in the G1 phase, thereby inhibiting the pathological process of tumors.

[0127] Experimental Example 5 verifies that the target site of compound 8a is BRD4.

[0128] To verify whether BRD4 is indeed the target of compound 8a in cells, the expression of BRD4 in MDA-MB-231 cells was silenced. MDA-MB-231 cells were first treated with BRD4 siRNA or a control siRNA, and then re-seeded into 96-well plates at a density of 5000 cells per well. After treatment with compound 8a for 72 hours, cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) method. Cell thermal transfer assay (CETSA): MDA-MB-231 cells were harvested, washed with 1× PBS, and then resuspended in 1× PBS containing a protease inhibitor. The cell suspension was repeatedly freeze-thawed three times with liquid nitrogen, and centrifuged at 20000g for 20 minutes at 4°C to separate the soluble fraction from cell debris. To generate the CETSA melting curve, the cell extract was divided into smaller aliquots and treated with DMSO (1% vol / vol) or 1 (10 μM). After incubation at room temperature for 10 minutes, each cell extract was individually heated for 3 minutes at different temperatures in a thermal cycler (Applied Biosystems Life Technologies), followed by cooling at room temperature for 3 minutes. The heated and cooled cell extracts were then centrifuged at 20,000g for 20 minutes at 4°C to separate soluble components from the precipitate. The supernatant was transferred to new microtubes for analysis using Western blotting.

[0129] The results showed that in MDA-MB-231 cells treated with BRD4 siRNA, the level of BRD4 protein decreased sharply. Figure 3 (A) demonstrates that BRD4 siRNA can knock out BRD4 in MDA-MB-231 cells. Compared with MDA-MB-231 cells treated with control siRNA, the inhibitory effect of compound 8a in MDA-MB-231 cells treated with BRD4 siRNA was significantly weakened ( Figure 3 (B). These data indicate that BRD4 is the intended target of compound 8a. Cell heat transfer assays (CETSA) were performed on MDA-MB-231 cell extracts, and the thermostability of BRD4 protein was detected by Western blotting to further demonstrate whether compound 8a can directly bind to BRD4. The results show that compound 8a directly binds to BRD4 in cells to enhance its thermostability. Figure 3 (C and D in the middle). These results indicate that compound 8a targets BRD4.

[0130] Experimental Example 6: Acute toxicity test of compound 8a to SPF Kunming rats

[0131] After acclimatization, mice were randomly divided into three groups of six each. The compound was prepared as a suspension using 0.5% sodium carboxymethyl cellulose solution and administered by gavage at a rate of 0.1 mL / 10 g. Mice were fasted for 6 hours before gavage but allowed free access to water, and fasted for 2 hours after gavage. Mice were observed, and their weight and survival status were recorded.

[0132] Conclusion: The acute toxicity of compound 8a was assessed by gavage administration to mice (50 mg / kg and 200 mg / kg), with measurements of body weight and mouse survival. Figure 4 It can be seen that mice given 50 mg / kg or 200 mg / kg of compound 8a did not show any adverse reactions, remained active until the end of the experiment, did not die, and did not show significant changes in body weight compared with the solvent control group.

[0133] Experimental Example 7: Determination of the therapeutic effect of compound 8a on nude mice with orthotopic xenograft tumor model.

[0134] Five-week-old female nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After 7 days of acclimatization feeding, 1 million MDA-MB-231 tumor cells (25 μL 1× PBS plus 25 μL Matrigel) were subcutaneously injected into the mammary pads to allow them to grow to an appropriate size. When the tumor volume reached 100 mm², [the tumor was targeted]. 3 Mice were randomly divided into a solvent group and a compound 8a 50 mg / kg group, with 7 mice in each group. Mice were administered the compound via gavage once daily for 3 weeks. Tumor size (volume = length × width²) was measured twice weekly using calipers, and mouse weight was also measured. All procedures were performed in accordance with the laboratory animal care and usage guidelines of the laboratory animal research institution.

[0135] Compared with the solvent control group, the growth of tumors in the MDA-MB-231 orthotopic xenograft breast cancer model was significantly inhibited by gavage administration of compound 8a (50 mg / kg) for 21 days. Figure 5 At the end of the experiment, compared with the control group, compound 8a (50 mg / kg) significantly reduced tumor volume and weight. Figure 5 (C~E). Compound 8a exhibited a tumor growth inhibition rate (TGI) of 49.1%. No substantial weight loss or death was observed in the mice used in this experiment during the study. Figure 5 (A). These data indicate that compound 8a has a good therapeutic effect on orthotopic xenograft tumor models without causing serious side effects. Immunohistochemical (IHC) analysis was performed on tumor tissues to further examine the mechanism of the anticancer effect of compound 8a in vivo. In the compound 8a (50 mg / kg) group, the protein levels of BRD4, Mucin 5AC, and c-Myc were decreased, while p21 was upregulated ( Figure 5This indicates that compound 8a exerts its antitumor effect by downregulating BRD4 and its downstream related signaling pathway proteins Mucin 5AC and c-Myc, and upregulating p21.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A phenanthrene compound with BRD4 protein inhibitory activity, characterized in that, Its structural formula is shown in equation (Ⅰ): ； Equation (I); The phenanthrene compounds that have BRD4 protein inhibitory activity also include their pharmaceutically acceptable salts; The phenanthrene compounds with BRD4 protein inhibitory activity are N-(4-methoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine, N-(4-methoxyphenyl)-2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine, N-(4-methoxyphenyl)-2-((naphth-1-ylamino)methyl)benzo[h]quinoline-4-amine, N-(4-methoxyphenyl) -2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine, N1-(2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine, N1,N1-dimethyl-N4-(2-((naphthyl-1-ylamino)methyl)benzo[h]quinoline-4-yl)phenyl-1,4-diamine, N1-(2-(((4-methoxyphenyl)) (methyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine, N1-(2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-yl)-N4,N4-dimethylphenyl-1,4-diamine, N-(3,5-dimethoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine, N-(3,5-dimethoxyphenyl) N-(3,5-dimethoxyphenyl)-2-(((3,5-dimethoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine, N-(3,4-dimethoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine, N-(3,5-dimethoxyphenyl)-2-(((4-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine, N-(3,5-dimethoxyphenyl)-2-(((4-methoxyphenyl)(methyl) )amino)methyl)benzo[h]quinoline-4-amine, N-(3,4-dimethoxyphenyl)-2-((naphth-1-ylamino)methyl)benzo[h]quinoline-4-amine, N-(3,4-dimethoxyphenyl)-2-(((4-methoxyphenyl)(methyl)amino)methyl)benzo[h]quinoline-4-amine, N-(3,4-dimethoxyphenyl)-2-(((3-methoxyphenyl)amino)methyl)benzo[h]quinoline-4-amine.

2. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 1, characterized in that, Includes the following steps: (1) Compound 1 and Compound 2 were added to polyphosphoric acid and reacted at 120~140 °C for 0.5~1.5 h. After the reaction was completed, the mixture was cooled to room temperature and saturated sodium carbonate solution was slowly added to neutralize the reaction solution to obtain solid compound 3. Solid compound 3 was filtered and dried. (2) Add compound 3 to phosphorus oxychloride solution and stir continuously at 90~110 °C for 2~4 h. After the reaction is complete, cool to room temperature and add the reaction solution to ice water to obtain solid compound 4. Filter and dry solid compound 4. (3) Add compound 4 and compound 5 to an ethanol solution, add two drops of concentrated hydrochloric acid, and react at 75~85 °C for 20~24 h. After the reaction is complete, cool to room temperature, concentrate and purify the reactants to obtain compound 6. (4) Add compound 6 to acetonitrile, add compound 7 and potassium carbonate, and react at 55~65 °C for 20~24 h. After the reaction is completed, quench the reaction with water, extract with organic solvent, wash, dry, concentrate and purify the extracted organic phase to obtain phenanthrene compound 8 with BRD4 protein inhibition. 。 3. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 2, characterized in that, In step (1), the molar ratio of compound 1 to compound 2 is 0.9~1.1:

1.

4. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 3, characterized in that, In step (1), the molar ratio of compound 1 to compound 2 is 1:

1.

5. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 2, characterized in that, In step (2), the molar ratio of compound 3 to phosphorus oxychloride is 1:40~55.

6. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 5, characterized in that, In step (2), the molar ratio of compound 3 and phosphorus oxychloride is 1:

50.

7. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 2, characterized in that, In step (3), the molar ratio of compound 3 to compound 4 is 0.9~1.1:

1.

8. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 7, characterized in that, In step (3), the molar ratio of compound 3 and compound 4 is 1:

1.

9. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 2, characterized in that, In step (4), the molar ratio of compound 6, compound 7 and potassium carbonate is 1:1:1.4~1.

6.

10. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 9, characterized in that, In step (4), the molar ratio of compound 6, compound 7 and potassium carbonate is 1:1:1.

5.

11. The method for preparing the phenanthrene compound with BRD4 protein inhibitory activity as described in claim 2, characterized in that, In step (4), after extraction with dichloromethane, the organic phase is washed with a saturated sodium chloride solution, then dried, concentrated and purified.

12. A pharmaceutical composition, characterized in that, This includes the phenanthrene compounds with BRD4 protein inhibitory activity as described in claim 1.

13. A pharmaceutical preparation, characterized in that, It includes the phenanthrene compounds with BRD4 protein inhibitory activity as described in claim 1, as well as pharmaceutically acceptable carriers and / or excipients.

14. The use of the phenanthrene compound of claim 1 with BRD4 protein inhibitory activity, the pharmaceutical composition of claim 12, or the pharmaceutical formulation of claim 13 in the preparation of a BRD4 protein inhibitor.

15. The use of the phenanthrene compound of claim 1 with BRD4 protein inhibitory activity, the pharmaceutical composition of claim 12, or the pharmaceutical preparation of claim 13 in the preparation of a medicament for the prevention and / or treatment of diseases related to BRD4.

16. The application as described in claim 15, characterized in that, The diseases associated with BRD4 include cancer, cardiovascular disease, inflammation, neurological disorders, and obesity.

17. The application as described in claim 16, characterized in that, The cancers mentioned are: bile duct cancer, ovarian cancer, midline cancer, liver cancer, kidney cancer, lung cancer, bladder cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, breast cancer, testicular cancer, bone cancer, brain cancer, gastrointestinal cancer, soft tissue tumor, leukemia, and lymphoma.

18. The application as described in claim 17, characterized in that, The cancer in question is breast cancer.

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