A fluorescent probe targeting BRD4 protein and its preparation method and application

Abstract

The present invention discloses a fluorescent probe targeting BRD4 protein, its preparation method, and application. The structural formula of the fluorescent probe targeting BRD4 protein of the present invention is: The fluorescent probe targeting BRD4 protein of the present invention can be used to non-invasively quantify protein degradation induced by PROTACs in vivo and to early predict the cancer treatment effect of PROTACs drugs. By combining POIs targeting ligands and environmentally sensitive fluorophores, the ESR signal shows a strong fluorescence correlation with POI levels, ultimately enabling real-time monitoring of multiple cancer-related therapeutic proteins through non-invasive fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a fluorescent probe targeting BRD4 protein, a preparation method thereof, and an application thereof. Background Art

[0002] Proteins are the basic components of organisms and regulate normal metabolism and signal transduction in cells by interacting with various biological molecules. Targeted protein degradation chimeras (PROTACs) have become a breakthrough therapeutic technology that can be used to selectively target and eliminate target proteins (POIs) closely related to the pathogenesis of diseases, especially transcription factors. To date, many PROTACs targeting key oncogenic proteins have entered the clinical trial stage for cancer treatment, such as ARV-110 (targeting androgens) and ARV-471 (targeting estrogens). However, because the structure-activity mechanism of PROTACs is still not clear enough, its efficacy is currently mainly evaluated by measuring the abundance of POIs by Western blotting (WB). This method lacks the ability to monitor protein degradation in living cells in real time and non-invasively. In addition, there are usually large differences in the protein degradation effects of PROTACs in vitro and in vivo, making it difficult to accurately detect the actual effect of PROTAC-mediated cancer treatment regimens.

[0003] Therefore, it is of great significance to develop a fluorescent probe that can non-invasively quantify PROTACs-induced protein degradation in vivo and early predict the cancer therapeutic effect of PROTACs drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a fluorescent probe targeting BRD4 protein and a preparation method and application thereof.

[0005] The technical solution adopted by the present invention is:

[0006] A fluorescent probe targeting BRD4 protein, the structural formula of which is:

[0007]

[0008] A method for preparing the fluorescent probe targeting BRD4 protein as described above comprises the following steps:

[0009] 1) reacting 3-hydroxy-N,N-diethylaniline with sodium nitrite to obtain compound 1;

[0010] 2) reacting compound 1 with 1,6-dihydroxynaphthalene to obtain compound 2;

[0011] 3) reacting compound 2 with N-Boc-3-aminopropyl bromide to obtain compound 3;

[0012] 4) reacting compound 3 with trifluoroacetic acid to obtain compound 4;

[0013] 5) Compound 4 is reacted with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid to obtain a fluorescent probe targeting the BRD4 protein.

[0014] Preferably, a method for preparing a fluorescent probe targeting BRD4 protein as described above comprises the following steps:

[0015] 1) dispersing 3-hydroxy-N,N-diethylaniline and sodium nitrite in a solvent to react, and then separating and purifying the product to obtain compound 1;

[0016] 2) dispersing compound 1 and 1,6-dihydroxynaphthalene in a solvent for reaction, and then separating and purifying the product to obtain compound 2;

[0017] 3) dispersing compound 2, N-Boc-3-aminopropyl bromide, and a catalyst in a solvent for reaction, and then isolating and purifying the product to obtain compound 3;

[0018] 4) dispersing compound 3 and trifluoroacetic acid in a solvent to react to obtain compound 4;

[0019] 5) Compound 4, (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid, and a catalyst are dispersed in a solvent for reaction, and the product is separated and purified to obtain a fluorescent probe targeting the BRD4 protein.

[0020] Preferably, in step 1), the molar ratio of 3-hydroxy-N,N-diethylaniline to sodium nitrite is 1:1-2.

[0021] Preferably, in step 1), the usage ratio of 3-hydroxy-N,N-diethylaniline to solvent is 1 g:5 mL to 15 mL.

[0022] Preferably, the solvent in step 1) is a hydrochloric acid solution.

[0023] Preferably, the reaction in step 1) is carried out at room temperature (20° C. to 25° C.) and the reaction time is 4 h to 6 h.

[0024] Preferably, the specific operation of separating and purifying the product in step 1) is as follows: the reaction product is added dropwise to a mixed solution of anhydrous ethanol and anhydrous ether for precipitation, and then filtered.

[0025] Preferably, in step 2), the molar ratio of the compound 1 to 1,6-dihydroxynaphthalene is 1:1-2.

[0026] Preferably, in step 2), the amount ratio of compound 1 to solvent is 1 g: 5 mL to 10 mL.

[0027] Preferably, the solvent in step 2) is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran.

[0028] Preferably, the reaction in step 2) is carried out at a temperature of 140° C. to 160° C., and the reaction time is 4 h to 6 h.

[0029] Preferably, the specific operation of separating and purifying the product in step 2) is as follows: extracting the reaction product with dichloromethane, and then taking the organic phase for purification by column chromatography.

[0030] Preferably, in step 3), the molar ratio of compound 2 to N-Boc-3-aminopropyl bromide is 1:1-2.

[0031] Preferably, in step 3), the molar ratio of the compound 2 to the catalyst is 1:2-4.

[0032] Preferably, the catalyst in step 3) is at least one of potassium carbonate, triethylamine, and N,N-diisopropylethylamine.

[0033] Preferably, in step 3), the usage ratio of the compound 2 to the solvent is 1 g:5 mL to 10 mL.

[0034] Preferably, the solvent in step 3) is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran.

[0035] Preferably, the reaction in step 3) is carried out at a temperature of 60° C. to 80° C., and the reaction time is 7 h to 24 h.

[0036] Preferably, the specific operation of separating and purifying the product in step 3) is as follows: extracting the reaction product with dichloromethane, and then taking the organic phase for purification by column chromatography.

[0037] Preferably, in step 4), the usage ratio of compound 3 to trifluoroacetic acid is 1 g: 2 mL to 4 mL.

[0038] Preferably, in step 4), the usage ratio of compound 3 to solvent is 1 g:4 mL to 10 mL.

[0039] Preferably, the solvent in step 4) is dichloromethane.

[0040] Preferably, the reaction in step 4) is carried out at room temperature (20° C. to 25° C.) and the reaction time is 1 h to 2 h.

[0041] Preferably, in step 5), the molar ratio of compound 4 to (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid is 1:1-2.

[0042] Preferably, in step 5), the molar ratio of the compound 4 to the catalyst is 1:4-8.

[0043] Preferably, the catalyst in step 5) is at least one of N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0044] Preferably, in step 5), the ratio of compound 4 to solvent is 1 g: 10 mL to 50 mL.

[0045] Preferably, the solvent in step 5) is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane.

[0046] Preferably, the reaction in step 5) is carried out at room temperature (20° C. to 25° C.) and the reaction time is 12 h to 24 h.

[0047] Preferably, the specific operation of separating and purifying the product in step 5) is as follows: extracting the reaction product with dichloromethane, and then taking the organic phase for purification by column chromatography.

[0048] A use of the fluorescent probe targeting the BRD4 protein as described above in the preparation of a detection reagent for quantitatively detecting the expression effect of the protein BRD4 in vivo.

[0049] A use of the fluorescent probe targeting BRD4 protein as described above in the preparation of a detection reagent for detecting the effect of cancer treatment.

[0050] The beneficial effects of the present invention are as follows: the fluorescent probe targeting BRD4 protein of the present invention can be used to non-invasively quantify protein degradation induced by PROTACs in vivo and to early predict the cancer treatment effect of PROTACs drugs. By combining POIs targeting ligands and environmentally sensitive fluorophores, the ESR signal shows a strong fluorescence correlation with the POIs level, and ultimately real-time monitoring of various cancer-related therapeutic proteins can be achieved through non-invasive fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Fluorescence spectra of methylene blue, fluorescein, rhodamine B and Nile red in different solvent environments.

[0052] Figure 2 is the H NMR spectrum of compound 2.

[0053] Figure 3 is the H NMR spectrum of compound 3.

[0054] Figure 4 This is the H NMR spectrum of JQ1-NR.

[0055] Figure 5 Fluorescence spectra of JQ1-NR and different concentrations of BRD4 protein.

[0056] Figure 6 for Figure 5 Corresponding linear analysis result graph.

[0057] Figure 7 This is the selectivity test result diagram of JQ1-NR.

[0058] Figure 8 This is the test result of protein degradation agent JV8 inducing BRD4 protein degradation.

[0059] Figure 9 Confocal imaging of the fluorescence signal of JQ1-NR after 4T1 cells were treated with different concentrations of JV8.

[0060] Figure 10 This is the correlation test result between JQ1-NR fluorescence signal and BRD4 protein level after 4T1 cells were treated with different concentrations of JV8.

[0061] Figure 11 This is the tumor volume-time relationship curve of mice.

[0062] Figure 12 This is the body weight-time relationship curve of mice.

[0063] Figure 13 Fluorescence imaging of JQ1-NR signals in mouse tumors.

[0064] Figure 14 This is a graph showing the correlation test results between JQ1-NR signals and tumor volume changes in mouse tumors. DETAILED DESCRIPTION

[0065] The present invention will be further explained and illustrated below with reference to specific embodiments.

[0066] Example:

[0067] A fluorescent probe targeting BRD4 protein, the preparation method of which is as follows:

[0068] 1) 1.65 g (10 mmol) of 3-hydroxy-N,N-diethylaniline was dispersed in a hydrochloric acid solution prepared from 10 mL of hydrochloric acid (36%, v / v) and 10 mL of pure water. A sodium nitrite solution prepared from 1.38 g (20 mmol) of sodium nitrite and 5 mL of pure water was then added dropwise. After the addition, the mixture was stirred at room temperature for 4 h. The reaction product was then added to a 200 mL anhydrous ethanol-anhydrous ether mixed solution (anhydrous ethanol:anhydrous ether volume ratio of 1:1) for precipitation. The solid was filtered and vacuum dried to obtain 971 mg of compound 1 (yield 50%).

[0069] 2) 2.72 g (14 mmol) of compound 1 and 2.24 g (14 mmol) of 1,6-dihydroxynaphthalene were dispersed in 20 mL of N,N-dimethylformamide (DMF), stirred at 150 ° C in the dark for 4 h, cooled naturally to room temperature, and extracted with dichloromethane (DCM) (3 × 50 mL). The organic phase was collected and dried over anhydrous magnesium sulfate to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol = 60 / 1) and dried in vacuo to obtain 2.67 g of compound 2 (dark red solid, yield 57%);

[0070] 3) 500 mg (1.5 mmol) of compound 2, 714 mg (3 mmol) of N-Boc-3-aminopropyl bromide, and 420 mg (3 mmol) of potassium carbonate were dispersed in 5 mL of N,N-dimethylformamide, stirred in the dark at 65 ° C for 12 h, and cooled naturally to room temperature. The reaction product was added to ice water and continued to stir, and then extracted with dichloromethane (3×50 mL). The organic phase was collected and dried over anhydrous magnesium sulfate to obtain a crude product. The crude product was purified by column chromatography (dichloromethane / methanol = 70 / 1) and dried in vacuo to obtain 427 mg of compound 3 (dark purple solid, yield 58%);

[0071] 4) Disperse 500 mg (1 mmol) of compound 3 and 2 mL of trifluoroacetic acid (TFA) in 2 mL of dichloromethane, stir at room temperature for 2 h, remove the solvent under vacuum, and dry in vacuo to obtain 460 mg of compound 4 (yield 90%);

[0072] 5) 245 mg (0.5 mmol) of compound 4, 200 mg (0.5 mmol) of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (JQ1-carboxyl), 258 mg (2 mmol) of N,N-diisopropylethylamine (DIPEA) and 760 mg (2 mmol) of 2-(7-azabenzotriazole)-N,N , N',N'-tetramethyluronium hexafluorophosphate (HATU) was dispersed in 10 mL of N,N-dimethylformamide and stirred at room temperature for 24 h. The reaction product was added to ice water and continued to stir. It was then extracted with dichloromethane (3×50 mL). The organic phase was collected and dried over anhydrous magnesium sulfate to obtain a crude product. The crude product was purified by column chromatography (dichloromethane / methanol = 30 / 1) and vacuum dried to obtain 116 mg of a fluorescent probe targeting BRD4 protein (denoted as JQ1-NR; dark purple solid, yield 30%).

[0073] The synthesis reaction of the fluorescent probe targeting BRD4 protein is as follows:

[0074]

[0075] Performance testing:

[0076] 1) Screening of environmentally sensitive fluorophores:

[0077] Fluorescence spectra of methylene blue (MB), fluorescein (FL), rhodamine B (RB) and Nile red (NR) in different solvent environments Figure 1 shown.

[0078] Depend on Figure 1 It can be seen that the fluorescence changes of MB, FL and RB in different solvents are chaotic, while the fluorescence intensity of NR decreases with the increase of solvent polarity; the polarity of different solvents is quantified by the Lippert-Mataga polarity parameter Δf, and a linear analysis is performed between the fluorescence intensity of different fluorophores and solvent polarity. It is found that only the fluorescence intensity of NR has a good correlation with the solvent polarity Δf (r=-0.8515, P=0.0073), indicating that NR is an environmentally sensitive fluorophore and is suitable for constructing an environmentally sensitive fluorescent probe (ESR) system.

[0079] 2) Structural characterization of a fluorescent probe (JQ1-NR) targeting BRD4 protein:

[0080] The H NMR spectrum of compound 2 ( 1 H NMR) Figure 2 As shown, the H NMR spectrum of compound 3 is as follows Figure 3As shown, the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe (JQ1-NR) targeting BRD4 protein is as follows Figure 4 shown.

[0081] Depend on Figures 2-4 It can be seen that the present invention has indeed synthesized a fluorescent probe targeting BRD4 protein with the expected structure.

[0082] 3) Fluorescence spectra of the fluorescent probe (JQ1-NR) targeting BRD4 protein and different concentrations of BRD4 protein:

[0083] JQ1-NR was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 2 mmol / L. JQ1-NR (2 μmol / L) was then incubated with different concentrations of BRD4 protein (0-10 μg / mL) in PBS buffer solution (10 mmol / L, pH = 7.4) at 37°C for 1 h. The fluorescence changes of JQ1-NR were tested. The fluorescence spectra of JQ1-NR and different concentrations of BRD4 protein were shown in Figure 2. Figure 5 The corresponding linear analysis results are shown in Figure 6 shown.

[0084] Depend on Figure 5 and Figure 6 It can be seen that: with the increase of BRD4 protein concentration (0-10 μg / mL), the JQ1-NR signal showed a 2.5-fold enhancement, showing a positive linear correlation (r=0.9871, P=0.0002).

[0085] 4) Selective testing of the fluorescent probe (JQ1-NR) targeting BRD4 protein:

[0086] JQ1-NR was mixed with different interfering substances and then the fluorescence spectrum was tested. The selectivity test results of JQ1-NR were as follows: Figure 7 shown.

[0087] Depend on Figure 7 It was found that, except for BRD4, all other substances failed to cause changes in the fluorescence intensity of JQ1-NR.

[0088] 5) Protein degradation agent JV8 induced BRD4 protein degradation test:

[0089] 4T1 cells were incubated with different concentrations of protein degrader JV8 (0-100 nmol / L) for 24 h, and the changes in BRD4 protein levels were observed by immunoblotting. The test results are as follows: Figure 8 shown.

[0090] Depend on Figure 8It can be seen that the BRD4 protein in the whole cell and cytoplasm gradually decreased with the increase of JV8 drug concentration, showing a dose-dependent manner.

[0091] 6) Confocal imaging of the fluorescence signal of a fluorescent probe (JQ1-NR) targeting BRD4 protein after treating 4T1 cells with different concentrations of JV8:

[0092] 4T1 cells were incubated with different concentrations of protein degrader JV8 (0-100 nmol / L) for 24 h, and then incubated with JQ1-NR (2 μmol / L) for 1 h. The fluorescence imaging of the cells was observed by laser confocal microscopy. The test results are as follows: Figure 9 shown.

[0093] Depend on Figure 9 It can be seen that the fluorescence signal of JQ1-NR gradually decreases with the increase of JV8 drug concentration, showing a dose-dependency.

[0094] 7) Correlation analysis between the fluorescence signal of the fluorescent probe targeting BRD4 protein (JQ1-NR) and BRD4 protein levels:

[0095] Linear analysis was performed between the JQ1-NR fluorescence signal and the BRD4 protein level in cells treated with JV8. Figure 10 shown.

[0096] Depend on Figure 10 It can be seen that there is a better linear relationship between the fluorescence signal of JQ1-NR and the BRD4 protein level in the cytoplasm, which proves the potential of JQ1-NR signal as an alternative strategy to western blotting and can be used for non-invasive quantification of BRD4 degradation induced by the drug JV8.

[0097] 8) Fluorescence imaging of the BRD4 protein-targeting fluorescent probe (JQ1-NR) in live mouse tumors:

[0098] Fifteen mice with 4T1 tumors were selected and the tumor volume reached 100 mm. 3 Afterwards, the mice were randomly divided into three groups, with 5 mice in each group. Different concentrations of JV8 (0 mg / kg, 5 mg / kg and 10 mg / kg) were intraperitoneally administered once every other day for a total of five times. The mice were treated for 15 days. During the entire treatment process, the tumor volume was measured with a vernier caliper every two days, and the weight changes of the mice in each experimental group were detected. The tumor volume-time relationship curve of the mice obtained by the test is shown in Figure 2. Figure 11 The mouse weight-time relationship curve is shown in Figure 12 The formula for calculating tumor volume is as follows: Volume (mm 3 )=0.5×length×width 2To evaluate the potential of using POI-labeled probes to predict the therapeutic efficacy of RROTACs at an early stage, mice were injected with JQ1-NR intratumorally after the second administration, and quantitative imaging was performed 4 h after injection. Figure 13 The signal changes of JQ1-NR were linearly fitted with the changes in tumor volume, and the results were shown in Figure 14 shown.

[0099] Depend on Figure 11 It can be seen that compared with the control group, high concentration of JV8 showed better tumor inhibitory effect.

[0100] Depend on Figure 12 It can be seen that after administration of different concentrations of JV8, the body weight of mice did not decrease significantly.

[0101] Depend on Figure 13 It can be seen that after the administration of high concentration of JV8, the fluorescence signal change of JQ1-NR is more obvious than that of the control group.

[0102] Depend on Figure 14 It can be seen that the change in tumor volume was negatively correlated with the change in probe fluorescence intensity (r=-0.9688, P<0.0001), indicating that the higher the ΔFL intensity in the tumor, the smaller the relative tumor volume of the mice 14 days after treatment.

[0103] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fluorescent probe targeting BRD4 protein, characterized in that: The structural formula is: 。 2. A method for preparing a fluorescent probe targeting BRD4 protein according to claim 1, characterized in that: The following steps are involved: 1) 3-hydroxy-N,N-diethylaniline and sodium nitrite are reacted to obtain compound 1, the structural formula of compound 1 is ; 2) Compound 1 is reacted with 1,6-dihydroxynaphthalene to obtain compound 2, the structural formula of which is ; 3) Compound 2 is reacted with N-Boc-3-aminopropyl bromide to obtain compound 3, the structural formula of which is ; 4) Compound 3 is reacted with trifluoroacetic acid to obtain compound 4, the structural formula of which is ; 5) Compound 4 is reacted with (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid to obtain a fluorescent probe targeting the BRD4 protein.

3. The preparation method according to claim 2, wherein: In step 1), the molar ratio of 3-hydroxy-N,N-diethylaniline to sodium nitrite is 1:1-2.

4. The preparation method according to claim 2, wherein: Step 2) The molar ratio of the compound 1 to 1,6-dihydroxynaphthalene is 1:1-2.

5. The preparation method according to claim 2, wherein: Step 3) The molar ratio of compound 2 to N-Boc-3-aminopropyl bromide is 1:1-2.

6. The preparation method according to claim 2, wherein: Step 4) The usage ratio of compound 3 and trifluoroacetic acid is 1 g: 2 mL to 4 mL.

7. The preparation method according to claim 2, characterized in that: Step 5) The molar ratio of compound 4 to (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid is 1:1-2.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The reaction in step 1) is carried out at room temperature for 4 to 6 hours; the reaction in step 2) is carried out at a temperature of 140° C. to 160° C. for 4 to 6 hours; the reaction in step 3) is carried out at a temperature of 60° C. to 80° C. for 7 to 24 hours; the reaction in step 4) is carried out at room temperature for 1 to 2 hours; and the reaction in step 5) is carried out at room temperature for 12 to 24 hours.

9. Use of the fluorescent probe targeting BRD4 protein according to claim 1 in preparing a detection reagent for quantitatively detecting the expression effect of protein BRD4 in vivo.

10. Use of the fluorescent probe targeting BRD4 protein according to claim 1 in preparing a detection reagent for detecting the effect of cancer treatment.

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