A photothermal agent for targeted degradation of BRD4 protein and its application

Abstract

The present invention belongs to the field of medicine, and specifically relates to a photothermal agent that targets and degrades BRD4 protein and its use in tumor treatment. The photothermal agent provided by the present invention specifically binds to the bromodomain of the BRD4 protein and does not exhibit protein degradation activity in a dark environment. When laser light irradiates tumor cells, the photothermal agent is activated by the laser light, generating concentrated heat near the BRD4 protein, directly destroying the BRD4 protein structure and function. This photothermal agent not only increases the retention time of the photothermal agent in cancer cells, but also can use exogenous signals to allow the targeted protein degradation process to take effect only in tumor tissue, reducing side effects on normal tissue. The photothermal agent provided by the present invention has the structural formula: #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a photothermal agent for targeted degradation of BRD4 protein and application thereof in tumor treatment. Background Art

[0002] Cancer, also known as malignant tumors, is a disease caused by malfunctioning mechanisms controlling cell growth and proliferation. It has a high incidence and mortality rate worldwide. Traditional cancer treatments, including surgery, radiotherapy, and chemotherapy, have significant limitations. For example, surgery requires precise tumor location, and chemotherapy and radiotherapy, while killing cancer cells, can also damage normal cells. Therefore, there is an urgent need to develop more effective cancer treatments.

[0003] Photothermal therapy (PTT), a non-invasive cancer treatment that has garnered significant attention in recent years, primarily relies on the localized heating effect of photothermal agents. After accumulating at the tumor site, photothermal agents, under laser irradiation, convert light energy into localized heat, generating hyperthermia and thereby destroying the tumor. However, existing molecular photothermal agents are rapidly cleared from tumor cells and have a short retention time, resulting in poor therapeutic efficacy. Proteins, as essential raw materials and participants in cellular activity, are crucial components of living organisms. Research on the structure and function of proteins has revealed that proteins participate in many important cellular physiological and pathological processes, such as antioxidant protection, cell growth regulation, apoptosis inhibition, and regulation of intracellular transport. Damage to proteins can disrupt cellular function and lead to apoptosis. Therefore, designing protein-targeted photothermal agents that can be anchored to specific proteins and then directly destroy them would be an effective therapeutic approach to kill cancer cells. Summary of the Invention

[0004] To overcome the above-mentioned problems, the present invention provides a photothermal agent for targeted degradation of BRD4 protein and its application. The photothermal agent provided by the present invention specifically binds to the bromodomain of the BRD4 protein and does not exhibit protein degradation activity in a dark environment. When laser light is irradiated on tumor cells, the photothermal agent is activated by the laser light, generating concentrated heat near the BRD4 protein, directly destroying the BRD4 protein structure and function. This photothermal agent not only increases the retention time of the photothermal agent in cancer cells, but also can use exogenous signals to limit the targeted protein degradation process to tumor tissue, reducing side effects on normal tissues.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a photothermal agent for targeted degradation of BRD4 protein, the structural formula of which is shown in Formula I;

[0007]

[0008] It should be noted that the photothermal agent for targeted degradation of BRD4 protein described in the present invention, in addition to the molecular compound shown in Formula I, the stereoisomers and pharmaceutically acceptable salts of the molecular compound shown in Formula I also belong to the technical solution under the same concept as the first aspect of the present invention, and belong to the technical content applied for protection of the present invention.

[0009] Furthermore, the stereoisomers include enantiomers and diastereomers.

[0010] Furthermore, "pharmaceutically acceptable salts" refer to conventional non-toxic salts, including inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, phosphates, and nitrates; organic acid salts such as acetates, propionates, oxalates, succinates, lactates, malates, tartrates, citrates, maleates, fumarates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, and ascorbates; inorganic base salts such as sodium, potassium, calcium, zinc, magnesium, and aluminum; and organic base salts such as arginine, benzathine, choline, diethylamine, glycolamine, glycine, lysine, meglumine, ethanolamine, and tromethamine. Furthermore, those skilled in the art may choose one salt over another based on solubility, stability, ease of formulation, and the like. The determination and optimization of these salts are within the skill of the art.

[0011] The second aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned photothermal agent for targeted degradation of BRD4 protein or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0012] The third aspect of the present invention provides a pharmaceutical preparation comprising the above-mentioned photothermal agent for targeted degradation of BRD4 protein or its stereoisomers, a pharmaceutically acceptable salt and at least one pharmaceutically acceptable excipient and / or carrier.

[0013] The excipients of the present invention refer to ingredients other than the active ingredient in a pharmaceutical composition or pharmaceutical preparation that are non-toxic to the subject. Common excipients in the art include buffers, stabilizers, preservatives, or excipients, and common excipients include binders, fillers, wetting agents, disintegrants, and the like.

[0014] As an example, the excipients that can be used in the preparation of the present invention include but are not limited to: the excipient is selected from calcium phosphate, magnesium stearate, talc, dextrin, starch, gel cellulose, methyl cellulose, carboxymethyl cellulose sodium salt and polyvinyl pyrrolidone.

[0015] The drug carrier of the present invention can be a pharmaceutically acceptable solvent, suspending agent, vesicle, nanomaterial, etc., used to deliver the compound described in the first aspect of the present invention to an animal or human. The carrier can be liquid or solid and is selected based on the intended route of administration. Proteins and liposomes are also drug carriers.

[0016] Those skilled in the art can use known technology to prepare the compound of the present invention into a pharmaceutical composition or preparation. For example, any compound (at least one compound) disclosed in the above-mentioned first aspect of the present invention is mixed with a pharmaceutical excipient, and then, if necessary, the resulting mixture is formed into a desired shape. Except for the exceptions mentioned in the present invention, the preparation of pharmaceutical preparations can also be carried out according to known pharmaceutical preparations. And, except for the exceptions mentioned in the present invention, suitable pharmaceutical excipients are known in the art, for example, referring to the 2005 version of the Handbook of Pharmaceutical Excipients (4th edition).

[0017] The fourth aspect of the present invention provides the use of the photothermal agent for targeted degradation of BRD4 protein or its stereoisomers, pharmaceutically acceptable salts, or the pharmaceutical composition of the second aspect, or the pharmaceutical preparation of the third aspect in the preparation of drugs for treating tumors.

[0018] Furthermore, the tumor includes benign tumors and / or malignant tumors; the malignant tumors include solid tumors and hematological tumors, wherein solid tumors include breast cancer.

[0019] It should be noted that the term "tumor" as used herein, as known to those skilled in the art, includes benign and / or malignant tumors. Benign tumors are defined as excessive cell proliferation that is incapable of forming aggressive, metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular and biochemical abnormalities that are capable of developing systemic diseases (e.g., metastasis to distant organs).

[0020] Solid tumors such as breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx tumors, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testicle, stomach, skin (such as melanoma), ureter, vagina and vulva tumors. Malignant tumors include hereditary cancers, such as retinoblastoma and Wilms tumor (Wilms tumor). In addition, malignant tumors include primary tumors in the organs and corresponding secondary tumors (tumor metastasis) in distal organs. Hematologic malignancies such as aggressive and indolent forms of leukemia and lymphoma, i.e. non-Hodgkin's disease, chronic and acute myeloid leukemia (CML / AML), acute lymphocytic leukemia (ALL), Hodgkin's disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, tumor-like syndromes, cancers of unknown primary site, and AIDS-related malignancies.

[0021] The beneficial effects of the present invention are:

[0022] Studies have shown that BRD4 protein is one of the core driver genes for cancer development. BRD4 protein can regulate and activate the expression of its downstream oncogenes MYC and Bcl-2, thereby promoting the proliferation of breast cancer cells. At the same time, studies have shown that inactivation of BRD4 protein or downregulation of its expression can inhibit the development of cancer. Therefore, taking BRD4 protein as the target protein, a photothermal agent that can target and degrade BRD4 protein is provided, directly destroying the structure and function of BRD4 protein, and thus effectively inhibiting the growth of tumor cells, which is of great significance for improving the effect of cancer treatment. The photothermal agent provided by the present invention can specifically bind to the bromodomain of BRD4 protein and does not show protein degradation activity in a dark environment. When the laser is irradiated to the tumor cell, the photothermal agent will be activated by the laser, and heat will be concentrated near the BRD4 protein, directly destroying the structure and function of the BRD4 protein. The photothermal agent will not only increase the retention time of the molecular photothermal agent in the cancer cell, but also can use exogenous signals to make the targeted protein degradation process only take effect in the tumor tissue, reducing the side effects on normal tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 This is the synthetic route of the compounds in Examples 1 to 3 of the present invention;

[0025] Figure 2 This is the synthetic route of the compounds in Examples 4 to 11 of the present invention;

[0026] Figure 3 This is the synthetic route of the compound in Example 12 of the present invention;

[0027] Figure 4 is the mass spectrum of compound PTJQ in Example 12 of the present invention;

[0028] Figure 5 is the NMR spectrum of compound PTJQ in Example 12 of the present invention;

[0029] Figure 6 is the ultraviolet absorption spectrum of compound PTJQ in Example 12 of the present invention;

[0030] Figure 7 1 is the fluorescence spectrum of the compound PTJQ in Example 12 of the present invention;

[0031] Figure 8 The PTJQ solutions with different concentrations in Experimental Example 1 are at 0.35W / cm 2 Temperature variation with time under laser irradiation;

[0032] Figure 9 This is a graph showing the temperature change over time of the PTJQ solution under laser irradiation of different powers in Experimental Example 1;

[0033] Figure 10 The heating and cooling curves of the PTJQ solution in Experimental Example 1 under 635nm laser irradiation;

[0034] Figure 11 is the linear relationship curve between time and -ln(θ) in Experimental Example 1;

[0035] Figure 12 This is a gel imaging image of PTJQ binding to BRD4 protein in Experimental Example 2;

[0036] Figure 13 This is a diagram showing the co-localization of PTJQ and intracellular BRD4 protein in Experimental Example 3;

[0037] Figure 14 This is a graph showing the expression of BRD4 protein, MYC, and Bcl-2 under laser irradiation in Experimental Example 4;

[0038] Figure 15 Figure 5 is a graph showing the cytotoxicity of PTJQ in Experimental Example 5; a is the cell survival rate of 4T1 cells after incubation with different concentrations of PTJQ materials for 12 hours; b is the cell survival rate of 4T1 cells after incubation with 20 μM PTJQ materials and laser irradiation at different power densities; c is the cell survival rate of 4T1 cells after different treatments; d is a confocal image of live / dead cell staining of 4T1 cells under different treatments;

[0039] Figure 16 Figure 6 is a graph showing changes in tumor volume and mouse body weight during in vivo treatment, where a is a graph showing changes in body weight and b is a graph showing changes in tumor volume.

[0040] Figure 17 These are H&E staining images of the main organs (heart, liver, spleen, lung, and kidney) in mice during in vivo treatment in Experimental Example 6. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0043] Experimental materials and instruments:

[0044] Materials: Diphenyl phosphoryl chloride, acetic hydrazide, p-chlorobenzoylacetonitrile, N-tert-butyloxycarbonyl-1,2-butanediamine, 4-tert-butyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-aspartate (Fmoc-Asp-OtBu), 2,3,3-trimethyl-4,5-benzo-3H-indole, and trifluoroacetic acid were purchased from Tianjin Xiens Biochemical Technology Co., Ltd. N,N-diisopropylethylamine (DIPEA), morpholine, potassium tert-butoxide, and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Adamas Reagent Co., Ltd. Anhydrous methanol, acetonitrile, ethanol, toluene, acetic anhydride, 2-butanone, piperidine, N,N-dimethylformamide (DMF), 1-butanol, dimethyl sulfoxide (DMSO), and triethylamine (TEA) were purchased from Sinopharm Chemical Reagent Co., Ltd. Petroleum ether, ethyl acetate, dichloromethane, and methanol were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Tris(hydroxymethyl)aminomethane and bovine serum albumin (BSA) were purchased from Beijing Solebao Technology Co., Ltd. Calcein / PI cell viability and cytotoxicity assay kit, Coomassie blue staining solution, and SDS-PAGE loading buffer were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Annexin V-FITC / PI cell apoptosis detection kit was purchased from Booster Biochemical Technology Co., Ltd. All chemical reagents were of analytical grade and used directly in experiments without purification. All aqueous solutions were prepared using deionized water (18.2 MΩ·cm -1 Mouse breast cancer cell line (4T1) was purchased from Shanghai Aolu Biotechnology Co., Ltd. Balb / C mice (4-6 weeks old, female, weighing 18-21 g) were used in the experiment.

[0045] Instruments: high-resolution mass spectrometer (Bruker Daltonics maXis UHR-TOF, Germany); nuclear magnetic resonance spectrometer (Bruker, Switzerland); FLS-980 fluorometer (Edinburgh Instruments Ltd, UK); UV-1700 ultraviolet-visible spectrophotometer (Shimadzu, Japan); Laser scanning Confocal Microscopy SP8 confocal fluorescence microscope (Leica, Germany); Synergy 2 multi-function microplate reader (Biotek, USA); high-speed refrigerated centrifuge (Sigma 3K 15, USA).

[0046] In order to enable those skilled in the art to more clearly 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.

[0047] See also Figures 1 to 3 , the synthetic routes of the compounds in Examples 1 to 12 of the present invention.

[0048] Example 1: Synthesis of Compound 1

[0049]

[0050] 4-Methylquinoline (12 mmol, 1.6 mL) and benzyl bromide (10 mmol, 1.2 ml) were dissolved in 30 mL of acetonitrile, heated to 110°C under argon and refluxed for 6 h. After the reaction, the solvent was dried and the product was dissolved in 3 mL of methanol. 30 mL of ether was slowly added dropwise thereto. The precipitate was collected by filtration, washed with ether, and dried to obtain compound 1 as a white solid.

[0051] Example 2: Synthesis of Compound 2

[0052]

[0053] Compound 1 (5 mmol, 1.57 g) and N,N-dibenzamide (10 mmol, 1.96 g) were dissolved in acetic anhydride (6 mL), heated to 150 ° C under argon and refluxed for 1 h. The reaction mixture was added dropwise to 200-250 mL of continuously stirred ether, filtered, and the precipitate was dried to obtain brown-red compound 2.

[0054] Example 3: Synthesis of Compound 3 (PT)

[0055]

[0056] Compound 2 (2 mmol, 587 mg) and 6-(2,3,3-trimethylindol-1-ium-1-yl)hexanoic acid bromide (2 mmol, 1 g) were dissolved in dichloromethane (10 mL), 1.2 mL of triethylamine (TEA) was added, and the mixture was stirred at room temperature for 24 h; the resulting mixture was distilled under reduced pressure to remove the solvent, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol (v / v, 5:1)) to obtain compound PT.

[0057] Example 4: Synthesis of Compound 4

[0058]

[0059] Sulfur (17 mmol, 0.56 g) was added to an ethanol solution (30 mL) containing p-chlorobenzoylacetonitrile (17 mmol, 3 g), 2-butanone (34 mmol, 3 mL), and morpholine (68 mmol, 5.9 mL) at 25°C. The mixture was heated to 70°C for 3.5 h, cooled to room temperature, poured into 100 mL of water, mixed, and extracted with ethyl acetate (50 mL × 3). The organic layers were combined and washed with 50 mL of saturated brine. The organic layers were collected and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v, 30:1 to 5:1)) to obtain Compound 4 as a yellow solid.

[0060] Example 5: Synthesis of Compound 5

[0061]

[0062] 4-tert-Butyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-aspartate (20 mmol, 8.32 g), benzotriazole-1-tetramethyl hexafluorophosphate (HBTU) (20 mmol, 7.59 g), and N,N-diisopropylethylamine (DIPEA) (40 mmol, 7.2 mL) were dissolved in DMF (5 mL), and compound 4 (10 mmol, 2.66 g) was added. The resulting mixed solution was stirred at room temperature for 24 h, and then water (50 mL) was added. The aqueous layer was extracted with ethyl acetate (20 mL×3). The organic layers were combined and washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to a brown oil. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v, 30:1 to 5:1)) to obtain compound 5 as a yellow solid.

[0063] Example 6: Synthesis of Compound 6

[0064]

[0065] To a solution of compound 5 (3.7 mmol, 2.5 g) in DMF (50 mL) was slowly added piperidine (5 mL). The reaction mixture was stirred at room temperature for 3 h. When TLC indicated completion of the reaction, the mixture was diluted with ethyl acetate (30 mL × 3), washed with water (200 mL) and saturated NaCl (30 mL) solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v, 30:1 to 5:1)) to afford compound 6 as a white solid.

[0066] Example 7: Synthesis of Compound 7

[0067]

[0068] To a toluene solution (100 mL) of compound 6 (3.5 mmol, 1.5 g) was slowly added SiO2 (50 mmol, 3 g), and the mixture was heated at 160°C for 6 h using a two-necked flask and a water separator, maintaining a constant volume of solvent. After the reaction was completed, the solvent was evaporated to obtain a crude product, which was purified by silica gel column chromatography (dichloromethanol (v / v, 300:1)) to obtain compound 7 as a yellow solid.

[0069] Example 8: Synthesis of Compound 8

[0070]

[0071] Under a low temperature environment of -80°C, compound 7 (1.5 mmol, 600 mg) was dissolved in THF solution (15 mL), and potassium tert-butoxide (1.5 mmol, 175 mg) was added dropwise, stirred for 30 min, and then diphenyl phosphoryl chloride (1.2 mmol, 245 μL) was added, stirred for 45 min, and then acetic acid hydrazide (4 mmol, 322 mg) was added, stirred at room temperature for 1 h, and finally 1-butanol (12 mL) was added. The mixed solution was heated to 90°C and reacted for 2 h, cooled to room temperature, and the crude product was purified by silica gel column chromatography (the eluent was first petroleum ether / ethyl acetate (v / v, 5:1), and then changed to dichloromethane / petroleum ether / methanol (v / v / v, 300:50:1)) to obtain compound 8.

[0072] Example 9: Synthesis of Compound 9

[0073]

[0074] Compound 8 (1.09 mmol, 0.5 g) and trifluoroacetic acid (TFA) (33.15 mmol, 2.5 mL) were added to 2.5 mL of DCM and stirred at room temperature for 2 h. The solvent and TFA were evaporated and the residue was washed twice with toluene and twice with dichloromethane. The solvent was removed under reduced pressure to obtain compound 9.

[0075] Example 10: Synthesis of Compound 10

[0076]

[0077] Compound 9 (1 mmol, 400 mg), HATU (1 mmol, 380 mg), and DIPEA (3 mmol, 522 μL) were added to DMF (5 mL) and activated for half an hour. N-tert-Butyloxycarbonyl-1,2-butanediamine and TEA (1 mmol, 139 μL) were then added and stirred overnight. After removing the solvent under reduced pressure, the crude product was purified by silica gel column chromatography using dichloromethane / methanol (v / v, 10:1) as the eluent to obtain Compound 10.

[0078] Example 11: Synthesis of Compound 11

[0079]

[0080] Compound 10 (1.09 mmol, 0.5 g) and TFA (33.15 mmol, 2.5 mL) were added to 2.5 mL of DCM and stirred at room temperature for 2 h. The solvent TFA was evaporated and the residue was washed twice with toluene and twice with dichloromethane. The solvent was removed under reduced pressure to obtain compound 11.

[0081] Example 12: Synthesis of Compound 12 (PTJQ)

[0082]

[0083] Compound 3 (0.1 mmol, 60 mg), HBTU (0.12 mmol, 47 mg), and DIPEA (0.2 mmol, 50 μL) were dissolved in DCM and stirred at room temperature for 15 min. Compound 11 (0.1 mmol, 47 mg) was then dissolved in a small amount of DCM (4 mL) and added dropwise to the mixed solution. TEA (0.1 mmol, 17 μL) was added and stirred at room temperature for 12 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol (v / v, 10:1)) to obtain the compound PTJQ represented by Formula I. The mass spectrum of PTJQ is shown in FIG. Figure 4 As shown, the NMR spectrum is Figure 5 As shown in the UV absorption spectrum Figure 6 The fluorescence spectrum is shown in Figure 7 shown.

[0084] Experimental Example 1

[0085] Measure and calculate the PTJQ photothermal conversion efficiency (η):

[0086] The calculation formula for photothermal conversion efficiency (η) includes:

[0087]

[0088] Where ΔT max is the temperature change of the sample at the highest steady-state temperature, is the temperature change of the solvent (e.g., water) at the highest steady-state temperature. h is the heat transfer coefficient, s is the surface area of ​​the container, and the value of hs is determined by formula (2). I is the laser power density, and A is the absorbance at 635 nm.

[0089]

[0090] m is the mass of the solvent containing the photothermal agent, C is the specific heat capacity of the solvent, τ sis the relevant time constant, which can be determined by formula (3).

[0091] t=-τ s ln (θ) (3)

[0092] θ is a dimensionless parameter called the driving force temperature and is calculated by formula (4).

[0093]

[0094] T max and T surr are the maximum steady-state temperature and the ambient temperature, respectively.

[0095] The measurement results are as follows Figures 8 to 11 As shown in Figure 1, the temperature of PTJQ solution changes with time under the irradiation of 635nm laser. Figure 8 PTJQ solutions with different concentrations at 0.35W / cm 2 Temperature variation with time under laser irradiation; Figure 9 This is a graph showing the temperature change of PTJQ solution over time under laser irradiation of different powers; Figure 10 The heating and cooling curves of PTJQ solution under 635nm laser irradiation; Figure 11 is the linear relationship curve between time and -ln(θ); Figures 10 and 11 The result is brought into the above formula, and it is calculated that the light-to-heat conversion efficiency of the PTJQ of the present invention is about 38%.

[0096] Experimental Example 2: Binding of PTJQ to BRD4 protein

[0097] Compounds PT and PTJQ were reacted with BRD4 protein at 37°C, and then the free photothermal agent was removed by dialysis, and the fluorescence signal of the photothermal agent on the protein band was detected by gel imaging. Figure 12 It was shown that the fluorescence intensity of the photothermal agent PTJQ on the BRD4 protein band was higher than that of the PT group, indicating that the photothermal agent PTJQ can stably bind to the BRD4 protein.

[0098] Experimental Example 3: Co-localization experiment of PTJQ and BRD4 protein

[0099] Immunofluorescence staining was used to detect the colocalization of the photothermal agent PTJQ and BRD4 protein. 4T1 cells were incubated with the photothermal agent PT and PTJQ for 3 hours, then fixed with 4% PFA at 4°C for 20 minutes, treated with 0.2% titan X-100-PBS for 10 minutes, and blocked with BAS-PBS at 37°C for 1 hour. The cells were then stained with primary and secondary antibodies related to the BRD4 protein. Finally, fluorescence imaging of the BRD4 protein and photothermal agent in the cells was performed using a confocal fluorescence microscope. The results are shown in Figure 2. Figure 13 It was shown that in 4T1 cells, the fluorescence of BRD4 protein overlapped well with the fluorescence of PTJQ, indicating that PTJQ had a good localization effect on BRD4.

[0100] Experimental Example 4: Effect of PTJQ on Protein Activity

[0101] Immunofluorescence staining experiments were used to evaluate the expression of intracellular proteins under the synergistic effect of laser irradiation and PTJQ. Figure 14 The results showed that under laser irradiation, the expression of BRD4 in mouse breast cancer (4T1) cells incubated with PTJQ was significantly reduced, and the expression of BRD4-mediated MYC and Blc-2 oncogenes was also downregulated, indicating that the heat generated by PTJQ can effectively destroy BRD4 protein and thus inhibit tumor cell growth.

[0102] Experimental Example 5: Cytotoxicity Experiment

[0103] The potential cytotoxicity of PTJQ at the cellular level was further investigated by measuring the cell viability of mouse breast cancer (4T1) cells using the MTT assay. Cells were first incubated with different concentrations (0, 5, 10, 15, 20, 25 μM) of PTJQ for 12 h. Figure 15 It was observed in a that when the cell concentration was 20 μM, the cell viability was still greater than 90%, indicating that PTJQ1 itself had no toxicity to cells. In addition, in order to study the photothermal effect of PTJQ in vitro, the cytotoxicity of 4T1 cells under laser irradiation with different laser power densities was evaluated. It was found that cells incubated with the photothermal agent PTJQ had a negative effect on the cell viability at 0.35 W·cm -2 After 5 minutes of 635 nm laser irradiation, cell viability decreased by 86% ( Figure 15 Therefore, a photothermal agent concentration of 20 μM and a power of 0.35 W·cm were selected. -2 The laser power density was used as the optimization parameter. Subsequently, the cell killing ability of different treatment groups was tested by MTT method ( Figure 15 The results showed that the survival rate of 4T1 cells in the PBS, PTJQ and laser irradiation groups did not change significantly, while the cell viability in the PTJQ+laser group was the lowest, indicating that PTJQ can effectively damage tumor cells. In addition, the live-dead cell staining experiment ( Figure 15 Middle (d) further verified that PTJQ has a good killing effect on tumor cells under laser irradiation.

[0104] Experimental Example 6: In vivo experiment

[0105] Female Balb / c mice were selected as research subjects. Tumor-bearing mouse models were established by subcutaneous injection of mouse breast cancer cells. 3 Start drug administration. The mice were divided into different groups, and each group was given a different treatment method: (1) PBS group; (2) laser group; (3) PTJQ group; (4) PT+laser group (5) PTJQ+laser group (experimental group). "Laser" means that the mouse tumor site was irradiated with 635nm near-infrared light; and the drug administration method was intratumoral injection. During the experimental period, the tumor volume and mouse weight were observed every other day. The experimental results showed that the tumors of the mice in the PBS group grew very fast, and the tumor cells treated with only laser and only PTJQ also grew very fast, while the tumor growth rate of the mice treated with PTJQ+laser group was significantly inhibited, indicating that the present invention can effectively inhibit tumor growth in mouse models. On the contrary, compared with the experimental group, the tumor volume of the mice in the PT+laser group was higher than that of the experimental group. This result shows that the group treated with the present invention showed the best tumor treatment effect ( Figure 16 The weight of mice is an important parameter for evaluating the systemic toxicity of materials to the body. The weight of mice in all groups did not change significantly over time, which means that the treatment with the present invention did not produce obvious side effects on mice and was suitable for in vivo tumor treatment ( Figure 16 In addition, after seven groups of mice were treated with different methods, the tissue sections of major organs (heart, liver, spleen, lung and kidney) were sliced ​​and stained with H&E. Figure 17 This shows that there is no obvious damage to the organs of the mice.

[0106] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A photothermal agent for targeted degradation of BRD4 protein or its stereoisomers or pharmaceutically acceptable salts, characterized in that: The structural formula of the photothermal agent targeting degradation of BRD4 protein is shown in Formula I; ； Formula I.

2. The photothermal agent for targeted degradation of BRD4 protein according to claim 1, or its stereoisomers or pharmaceutically acceptable salts, is characterized in that: Said stereoisomers are enantiomers and diastereomers.

3. The photothermal agent for targeted degradation of BRD4 protein according to claim 1, or its stereoisomers or pharmaceutically acceptable salts, is characterized in that: The pharmaceutically acceptable salts include inorganic acid salts, organic acid salts, inorganic base salts and organic base salts.

4. The photothermal agent for targeted degradation of BRD4 protein according to claim 3, or its stereoisomers or pharmaceutically acceptable salts, is characterized in that: The inorganic acid salts are: hydrochloride, hydrobromide, sulfate, phosphate and nitrate; the organic acid salts are: acetate, propionate, oxalate, succinate, lactate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, benzenesulfonate, p-toluenesulfonate and ascorbate; the inorganic base salts are: sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt and aluminum salt; the organic base salts are: arginine salt, benzathine salt, choline salt, diethylamine salt, glycolamine salt, glycine salt, lysine salt, meglumine salt, ethanolamine salt and tromethamine salt.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the photothermal agent for targeted degradation of BRD4 protein according to claim 1 or a stereoisomer or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the photothermal agent for targeted degradation of BRD4 protein according to claim 1 or a stereoisomer thereof, a pharmaceutically acceptable salt and at least one pharmaceutically acceptable excipient.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the photothermal agent for targeted degradation of BRD4 protein according to claim 1 or a stereoisomer thereof, a pharmaceutically acceptable salt and at least one pharmaceutically acceptable carrier.

8. The pharmaceutical preparation according to claim 6, wherein Excipients are buffers, stabilizers, preservatives or excipients.

9. The pharmaceutical preparation according to claim 7, wherein The carrier is a pharmaceutically acceptable solvent, suspending agent, vesicle, or nanomaterial.

10. Use of the photothermal agent for targeted degradation of BRD4 protein according to claim 1 or its stereoisomers, pharmaceutically acceptable salts, or the pharmaceutical composition according to claim 5, or the pharmaceutical preparation according to any one of claims 6 to 9 in the preparation of a drug for treating tumors; The tumor is breast cancer.

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