Fluorescent probe for targeted recognition of squarylium cyanine amide structure of xanthine oxidase and preparation method thereof

Abstract

The invention relates to the field of chemical analysis and testing, in particular to a fluorescent probe for targeted recognition of a squarylium cyanine amide structure of xanthine oxidase and a preparation method of the fluorescent probe. The preparation method comprises the following steps: reacting 4-hydrazinobenzoic acid with 3-methyl-2-butanone to obtain a compound 1; reacting the compound 1 with iodoethane to obtain a compound 2; the preparation method comprises the following steps: reacting 2, 2, 3, 3-trimethyl-3H-indole with iodoethane to obtain a compound 3; reacting the compound 3 with diethyl thiourea to obtain a compound 4; obtaining a compound 5 from the compound 4 in the presence of glacial acetic acid and hydrochloric acid; reacting the compound 5 with the compound 2 to generate a compound SQ; the preparation method comprises the following steps: dissolving a compound SQ in DMF, adding HATU and DIPEA in an ice bath, stirring, adding 4-(aminomethyl) pyrimidine, and stirring to obtain a product SQ-T1; the fluorescent probe disclosed by the invention has the characteristics of near-infrared emission, high selectivity and the like, can recognize xanthine oxidase in a targeted manner, and has an important application prospect in the development of medicines for treating XO-related diseases.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis and testing, and in particular to a fluorescent probe with a squaraine amide structure for targeted recognition of xanthine oxidase and a preparation method thereof. Background Art

[0002] Xanthine oxidase (XO) is a molybdenum-containing metalloenzyme that is mainly distributed in the liver of animals and plays a key role in the metabolism of purines in the body. XO catalyzes the oxidation of hypoxanthine to xanthine, ultimately generating uric acid. Excessive uric acid levels are an important factor leading to gout.

[0003] At present, the methods used for XO activity detection mainly include pressure gauge method, electrochemical method, high performance liquid chromatography and radiation method. Although these methods have made significant progress, they are complex and time-consuming, and usually require a multi-step sample preparation process. In contrast, fluorescence analysis has attracted widespread attention in recent years due to its advantages such as simple operation, rapid and sensitive detection. Therefore, the development of efficient, sensitive and real-time fluorescent probes is of great significance for the diagnosis and drug development of XO-related diseases. Summary of the invention

[0004] The present invention provides a fluorescent probe SQ-T1 based on squaryl cyanine amide, the structural formula of the fluorescent probe is as follows:

[0005]

[0006] The present invention also provides a method for preparing a fluorescent probe based on squaryl cyanine amide. The synthetic route for preparing the fluorescent probe SQ-T1 is shown in the following formula:

[0007]

[0008] The specific preparation steps are as follows:

[0009] (1) 4-Hydrazinobenzoic acid and 3-methyl-2-butanone were dissolved in glacial acetic acid, refluxed at 118° C. for 12 hours, and then the solvent was removed under reduced pressure. The mixture was purified by column chromatography to obtain a yellow solid, namely, Compound 1.

[0010] (2) Compound 1 was dissolved in acetonitrile, and then iodoethane was added. After reflux reaction at 90°C for 24 hours, heating was stopped and cooled to room temperature. When a large amount of solid precipitated, it was cooled to 0°C again. After suction filtration, the filter cake was washed with cyclohexane and dried to obtain a solid, namely compound 2.

[0011] (3) 2,3,3-Trimethyl-3H-indole was dissolved in acetonitrile, and then iodoethane was added. After reflux reaction at 90°C for 12 hours, heating was stopped and cooled to room temperature. When a large amount of solid was produced, it was cooled to 0°C again. After filtration, the filter cake was washed with cyclohexane and dried to obtain a purple solid, namely compound 3.

[0012] (4) Compound 3 and diethyl thiourea were dissolved in ethanol, triethylamine was added as alkaline conditions, and the mixture was heated under reflux at 78° C. for 6 hours. The solvent was removed under reduced pressure and purified by column chromatography to obtain a solid, namely Compound 4.

[0013] (5) Compound 4 was dissolved in acetonitrile, glacial acetic acid and hydrochloric acid were added, and the mixture was heated at 60° C. for 8 hours under reflux conditions. The pH was adjusted to 5-7 with aqueous sodium bicarbonate solution, and the mixture was extracted with saturated brine and ethyl acetate. Finally, the mixture was purified by column chromatography to obtain a yellow solid, namely, Compound 5.

[0014] (6) Compound 5 and Compound 2 were dissolved in a mixed solvent of toluene and n-butanol in a volume ratio of 1:1, and heated under reflux at 120° C. for 2 hours. The solvent was removed under reduced pressure and purified by column chromatography to obtain a blue solid, namely, Compound SQ.

[0015] (7) Compound SQ was dissolved in DMF, HATU and DIPEA were added under ice bath conditions, and the mixture was stirred for 0.5-2 hours. After removing the ice bath, 4-(aminomethyl)pyrimidine was added, and the mixture was stirred at room temperature for 6-8 hours. The mixture was extracted with saturated brine and dichloromethane, dried, and purified by column chromatography to obtain product SQ-T1.

[0016] The molar ratio of compound SQ, HATU and DIPEA is 1:1:3 to 1:2:5, and the molar ratio of added 4-(aminomethyl)pyrimidine to compound SQ is 1:1 to 1.2:1.

[0017] The present invention also provides an application of the fluorescent probe: the prepared fluorescent probe is applied to detect the fluorescence imaging of endogenous XO in liver cells.

[0018] The fluorescent probe detects XO by fluorescence enhancement in a 10 mM HEPES buffer solution.

[0019] Beneficial Effects

[0020] The fluorescent probe of the present invention uses a cyanine dye as a fluorescent group, has simple synthesis steps and low cost. Its emission wavelength is in the near-infrared region, has low fluorescence background interference, and thus significantly improves the clarity and accuracy of fluorescence imaging. Its long-wavelength infrared emission characteristics are less harmful to biological cells, and imaging can be performed without affecting the normal physiological functions of cells. The probe shows high selectivity for XO and is suitable for living cell detection, such as fluorescence imaging of endogenous XO in liver cells (LO2). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the hydrogen spectrum of the probe SQ-T1 prepared in Example 1.

[0022] Figure 2 This is the carbon spectrum of the probe SQ-T1 prepared in Example 1.

[0023] Figure 3 The probe SQ-T1 prepared in Example 1 was 2×10 -3 mol / L in HEPES buffer with different concentrations of XO after its action.

[0024] Figure 4 The probe SQ-T1 prepared in Example 1 was 2×10 -3 mol / L in HEPES buffer with different concentrations of XO.

[0025] Figure 5 The probe SQ-T1 prepared in Example 1 was 2×10 -3 mol / L concentration of different analytes in HEPES buffer.

[0026] Figure 6 The probe SQ-T1 prepared in Example 1 was 2×10 -3 mol / L concentration in HEPES buffer for XO (100U / mL) versus time.

[0027] Figure 7 The probe SQ-T1 prepared in Example 1 was 1×10 -3 Confocal fluorescence imaging of human skin fibroblasts (HSF) and normal human hepatocytes (LO2) after incubation with 1 mol / L for 1 hour. DETAILED DESCRIPTION

[0028] The present invention will be specifically described below in conjunction with the accompanying drawings:

[0029] Example 1

[0030] (1) Preparation of Compound 1

[0031] 4-Hydrazinobenzoic acid (2 g, 0.013 mol) and 3-methyl-2-butanone (1.6 g, 0.018 mol) were added to a round-bottom flask, followed by 8 mL of glacial acetic acid, and refluxed at 118° C. for 12 hours. After the reaction, the solvent was removed under low pressure and purified by column chromatography to obtain 1.53 g of a yellow solid with a yield of 57%.

[0032] (2) Preparation of Compound 2

[0033] Compound 1 (500 mg, 2.46 mmol) was added to a round-bottom flask containing 5 mL of acetonitrile, followed by iodoethane (763 mg, 4.93 mmol), and the mixture was refluxed at 90° C. for 24 hours. After a large amount of solid was formed in the reaction flask, the reaction was cooled to 0° C., filtered under low pressure, and the filter cake was washed with cyclohexane to obtain 394 mg of the product with a yield of 45%.

[0034] (3) Preparation of Compound 3

[0035] 2,3,3-Trimethyl-3H-indole (2 g, 0.013 mol) was added to a round-bottom flask containing 10 mL of acetonitrile, followed by iodoethane (2.9 g, 0.019 mol), and the mixture was refluxed at 90°C for 12 hours. After a large amount of solid was generated in the reaction flask, the reaction was cooled to 0°C, filtered under low pressure, and the filter cake was washed with cyclohexane to obtain 3.29 g of purple solid with a yield of 83%.

[0036] (4) Preparation of Compound 4

[0037] Compound 3 (870 mg, 2.76 mmol), diethyl thiourea (469 mg, 2.76 mmol) and 4 mL of ethanol were added to a round-bottom flask in sequence, and then 0.5 mL of triethylamine was added, and the mixture was heated to 78° C. and refluxed for 6 hours. After the reaction was completed, the solvent was removed under reduced pressure, and 345 mg of a solid was obtained by column chromatography purification with a yield of 40%.

[0038] (5) Preparation of Compound 5

[0039] Compound 4 (200 mg, 0.64 mmol) was added to a round-bottom flask containing 4 mL of acetonitrile, and 0.9 mL of glacial acetic acid and 0.3 mL of hydrochloric acid were added in sequence, and heated at 60 ° C for 8 hours under reflux conditions. After the reaction was completed, the pH was adjusted to weak acidity with sodium bicarbonate aqueous solution, followed by extraction with saturated brine and ethyl acetate. 98 mg of yellow solid was obtained by column chromatography purification with a yield of 54%.

[0040] (6) Preparation of compound SQ

[0041] Compound 5 (250 mg, 0.88 mmol) and compound 2 (348 mg, 0.94 mmol) were dissolved in a total of 8 mL of a mixed solvent of PhMe (toluene) and n-BuOH (n-butanol) in a volume ratio of 1:1, and then the reaction solution was heated to 120° C. and refluxed for 2 hours. After the reaction was completed, the solvent was removed under reduced pressure, and 268 mg of a blue solid was obtained by column chromatography purification with a yield of 61%.

[0042] (7) Preparation of compound SQ-T1

[0043] Compound SQ (50 mg, 0.1 mmol) was dissolved in 2 mL DMF, and HATU (38 mg, 0.1 mmol) and DIPEA (38.7 mg, 0.3 mmol) were added in turn under ice bath conditions, and stirred for 0.5 hours. After removing the ice bath, 4-(aminomethyl)pyrimidine (10.9 mg, 0.1 mmol) was added and stirred at room temperature for 6 hours. After the reaction was completed, it was extracted with saturated brine and dichloromethane, the organic phase was collected, dried, and purified by column chromatography to obtain the product. The final product was 21.9 mg, and the yield was 37.3%. The H NMR spectrum of the product SQ-T1 is shown as follows: Figure 1 As shown, the NMR C spectrum is Figure 2 shown.

[0044] Figure 1 This is the hydrogen spectrum of the fluorescent probe SQ-T1 based on the squaryl cyanine amide structure.

[0045] 1 H NMR(400MHz, DMSO-d6)δ9.21-9.09(m,2H),8.74(d,J=5.3Hz,1H),8.05(d,J= 1.7Hz,1H),7.95(dd,J=8.4,1.7Hz,1H),7.57(d,J=7.4Hz,1H),7.48-7.35(m, 4H),7.23(td,J=7.2,1.6Hz,1H),5.87(s,1H),5.81(s,1H),4.57(d,J=5.8Hz ,2H),4.25-4.06(m,4H),1.70(d,J=2.6Hz,12H),1.29(dt,J=9.0,7.2Hz,6H).

[0046] Figure 2 This is the carbon spectrum of the fluorescent probe SQ-T1 based on the squaryl cyanine amide structure.

[0047] 13C NMR(101MHz,DMSO-d6)δ170.24,167.73,167.22,166.13,158.07,157.30,144.58,141.80,141.45,128.30,128.07,122.38,121.31, 118.58,110.62,109.18,86.32,49.12,48.10,44.22,40.09,39.89,39.68,39.47,39.26,39.05,38.84,26.63,26.17,11.85,11.63.

[0048] Example 2

[0049] The preparation of compound SQ-T1 comprises the following specific steps:

[0050] Compound SQ (50 mg, 0.1 mmol) was dissolved in 2 mL DMF, and HATU (57 mg, 0.15 mmol) and DIPEA (51.6 mg, 0.4 mmol) were added in turn under ice bath conditions, and stirred for 1 hour. After removing the ice bath, 4-(aminomethyl)pyrimidine (12 mg, 0.11 mmol) was added and stirred at room temperature for 7 hours. After the reaction was completed, it was extracted with saturated brine and dichloromethane, the organic phase was collected, dried, and purified by column chromatography to obtain the product. The final product was 28.8 mg, and the yield was 49%.

[0051] Example 3

[0052] Preparation of compound SQ-T1

[0053] Compound SQ (50 mg, 0.1 mmol) was dissolved in 2 mL DMF, and HATU (76 mg, 0.2 mmol) and DIPEA (64.5 mg, 0.5 mmol) were added in turn under ice bath conditions and stirred for 2 hours. After removing the ice bath, 4-(aminomethyl)pyrimidine (14 mg, 0.12 mmol) was added and stirred at room temperature for 8 hours. After the reaction was completed, it was extracted with saturated brine and dichloromethane, the organic phase was collected, dried and purified by column chromatography to obtain the product. The final product was 32 mg with a yield of 54%.

[0054] Example 4

[0055] The changes in the absorption spectrum and fluorescence spectrum of the probe SQ-T1 prepared in Example 1 before and after reacting with different concentrations of XO are detected. The specific steps include:

[0056] 196 μL of 10 mM HEPES buffer solution was added to a 96-well plate. Eleven groups were set up in parallel, with 2 μL of XO aqueous solution (0 U / mL to 250 U / mL) and 2 μL of 2.0×10 -3 mol / L fluorescent probe SQ-T1 DMSO solution, after mixing evenly, use a microplate reader to measure the absorbance of each well. The results are plotted with XO concentration as the horizontal axis and absorbance as the vertical axis. Figure 3 . Figure 3 As shown in Figure 3, no significant absorption change was observed after SQ-T1 interacted with XO in HEPES buffer solution.

[0057] Subsequently, under the same conditions, the fluorescence intensity was measured using an excitation wavelength of 560 nm, and the results were plotted with the emission wavelength as the abscissa and the fluorescence intensity as the ordinate. Figure 4 The figure shows that when the excitation wavelength is 560nm, the fluorescence intensity of SQ-T1 at 655nm gradually increases with the increase of XO concentration, and a 15nm red shift occurs (from 640nm to 655nm), while SQ-T1 itself does not show obvious fluorescence in HEPES buffer.

[0058] Example 5

[0059] The selectivity of the fluorescent probe SQ-T1 prepared in Example 1 was detected, and the specific steps included:

[0060] 2 μL of 100 U / mL XO aqueous solution and other analytes (hemoglobin (Hb), xanthine oxidase (XO), nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), oxidized glutathione (GSSH), reduced glutathione (GSH), insulin (Insulin), pepsin (Pepsin), catalase (CAT), blank (Blank)) were added to a 96-well plate, followed by 196 μL of 10 mM HEPES buffer and 2 μL of 2 × 10 -3 mol / L probe SQ-T1 DMSO solution, mix the solution in each well evenly, and detect the fluorescence intensity of each well by microplate reader. Figure 5 As shown in the figure, when 100U / mL XO solution was added, the fluorescence intensity of the probe solution at 655nm was significantly enhanced. In contrast, the fluorescence intensity of the probe for other analytes in HEPES solution did not change significantly. This indicates that the probe has a highly selective recognition effect on XO in the HEPES solution system.

[0061] Example 6

[0062] The fluorescence intensity of the fluorescent probe SQ-T1 prepared in Example 1 is detected to change with time after adding XO. The specific steps include:

[0063] Two groups of experiments were set up in a 96-well plate: one group was added with 2 μL of 100 U / mL XO aqueous solution, 196 μL of 10 mM HEPES buffer solution, and 2 μL of 2×10 -3 mol / L probe SQ-T1 DMSO solution; the other group was a blank control, in which only 198 μL of 10 mM HEPES buffer solution and 2 μL of 2×10 -3 mol / L probe SQ-T1 DMSO solution. After mixing the solution in each well, monitor the fluorescence intensity changes at different time points using an enzyme marker. The test results are as follows Figure 6 As shown in the figure, after adding XO, the fluorescence intensity of the probe rises rapidly, reaches the maximum value in less than 1 minute, and then enters a stable period. This shows that the probe has a rapid response to XO and good stability, and can provide a reliable analytical method for the rapid detection of XO.

[0064] Example 7

[0065] The fluorescent probe SQ-T1 prepared in Example 1 is used to compare the imaging of normal human liver cells (LO2) producing endogenous XO and human skin fibroblasts (HSF) producing XO. The specific steps include:

[0066] LO2 and HSF cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum at 37°C and 5% CO2 in a humidified environment. When the cells were in good condition, they were inoculated into different confocal culture dishes and cultured for 24 hours. Then, the probe SQ-T1 DMSO solution with a final concentration of 10 μM was added and incubated for another hour. After washing the cells three times with PBS solution, the fluorescence intensity of the intracellular probe was recorded using a Nikon C2 fluorescence confocal microscope, (a1-a3) HSF cells; (b1-b3) LO2 cells. Scale bar = 100 μm, λ ex =561nm. The results are as follows Figure 7 As shown, SQ-T1 was able to enter LO2 cells and emit significant fluorescence signals in the cytoplasm, while no obvious fluorescence signals were observed in the control HSF cells. This indicates that the probe SQ-T1 can distinguish hepatocytes from other cells based on the difference in XO expression levels through fluorescence imaging.

Claims

1. A fluorescent probe of a squaryl cyanine amide structure, characterized in that: The structural formula of the probe is as follows:

2. A method for preparing a fluorescent probe having a squaryl cyanine amide structure as claimed in claim 1, characterized in that: The preparation method steps are as follows: (1) Using glacial acetic acid as a solvent, 4-hydrazinobenzoic acid and 3-methyl-2-butanone were dissolved in a single-necked bottle. After the reflux reaction was completed, the solvent was removed by distillation under reduced pressure, and compound 1 was obtained after purification by column chromatography; (2) Using acetonitrile as solvent, compound 1 and iodoethane were dissolved in a single-mouth bottle, refluxed and then cooled to room temperature, solid precipitated, and then cooled to 0°C. After suction filtration, the filter cake was rinsed with cyclohexane and dried to obtain compound 2; (3) Using acetonitrile as solvent, 2,3,3-trimethyl-3H-indole and iodoethane were dissolved in a single-mouth bottle, refluxed and then cooled to room temperature, solid precipitated, and then cooled to 0°C. After suction filtration, the filter cake was rinsed with cyclohexane and dried to obtain compound 3; (4) Using ethanol as solvent, compound 3 and diethyl thiourea were dissolved in a single-necked bottle, triethylamine was added as alkaline conditions, and after the reflux reaction was completed, the solvent was distilled off under reduced pressure, and compound 4 was purified by column chromatography; (5) Using acetonitrile as solvent, compound 4 was dissolved in a single-mouth bottle, glacial acetic acid and hydrochloric acid were added, and after the reflux reaction was completed, the pH was adjusted to 5-7 with sodium bicarbonate aqueous solution, and then extracted with saturated brine and ethyl acetate, and finally purified by column chromatography to obtain compound 5; (6) Mix toluene and n-butanol as a solvent, and then dissolve compound 5 and compound 2 in a single-necked bottle. After heating and refluxing, remove the solvent by reduced pressure distillation, and purify by column chromatography to obtain compound SQ; (7) Using N,N-dimethylformamide as solvent, compound SQ was dissolved in a single-necked bottle, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU and N,N-diisopropylethylamine DIPEA were added under ice bath conditions. After stirring for 0.5-2 hours, 4-(aminomethyl)pyrimidine was added and stirred at room temperature for 6-8 hours. After the reaction was completed, the mixture was extracted with saturated brine and dichloromethane, the solvent was distilled off under reduced pressure, and the final product SQ-T1 was obtained after purification by column chromatography.

3. The method for preparing the fluorescent probe of the squaraine amide structure as claimed in claim 2, characterized in that: In step (1), the molar ratio of 4-hydrazinobenzoic acid to 3-methyl-2-butanone is 13:18, and the reflux reaction is carried out at 118° C. for 12 hours.

4. The method for preparing the fluorescent probe of the squaraine amide structure as claimed in claim 2, characterized in that: In step (2), the molar ratio of compound 1 to ethyl iodide is 2.46:4.93, and the reflux reaction is carried out at 90° C. for 4 hours.

5. The method for preparing the fluorescent probe of the squaraine amide structure as claimed in claim 2, characterized in that: In step (3), the molar ratio of 2,3,3-trimethyl-3H-indole to iodoethane is 13:19, and the reflux reaction is carried out at 90° C. for 12 hours.

6. The method for preparing the fluorescent probe of the squaraine amide structure as claimed in claim 2, characterized in that: In step (4), the molar ratio of compound 3 to diethyl thiourea is 1:1, and the reflux reaction is carried out at 78° C. for 6 hours.

7. The method for preparing the fluorescent probe of the squaraine amide structure as claimed in claim 2, characterized in that: In step (5), the mass volume ratio of compound 4, glacial acetic acid and hydrochloric acid is: 200 mg: 0.9 mL: 0.3 mL, and the reflux reaction is: reflux reaction at 60° C. for 8 hours.

8. The method for preparing the fluorescent probe of the squaraine amide structure as claimed in claim 2, characterized in that: In step (6), the volume ratio of toluene to n-butanol is 1:1; the molar ratio of compound 5 to compound 2 is 0.88:0.94; and the reflux reaction is carried out at 120° C. for 2 hours.

9. The method for preparing the fluorescent probe of the squaraine amide structure according to claim 2, characterized in that: In step (7), the molar ratio of compound SQ, HATU and DIPEA is 1:1:3 to 1:2:5, and the molar ratio of the added 4-(aminomethyl)pyrimidine to compound SQ is 1:1 to 1.2:

1.

10. The use of the fluorescent probe of the squaraine amide structure as claimed in claim 9, characterized in that: The fluorescent probe recognizes xanthine oxidase XO in a 10 mM HEPES buffer solution by fluorescence enhancement.

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