Near-infrared BODIPY compound for biological imaging and sonodynamic therapy as well as preparation method and application of near-infrared BODIPY compound

Abstract

The invention discloses a near-infrared BODIPY compound for biological imaging and sonodynamic therapy and a preparation method of the near-infrared BODIPY compound. The chemical structural formula of the compound is as shown in formula 4. The preparation method comprises the following steps: firstly, preparing a BODIPY compound from para-substituted benzaldehyde, 2, 4-dimethyl pyrrole and boron trifluoride diethyl etherate under the catalysis of acid; and then carrying out dehydration condensation on the obtained BODIPY compound and para-substituted benzaldehyde under the catalysis of acid and alkali to obtain a target product. The near-infrared difluoro boron pyrrole compound disclosed by the invention is mild in synthesis condition, short in reaction time, high in yield, simple in post-treatment and wide in structural application range. According to the present invention, the sulfur methyl benzaldehyde and the aniline derivative are connected to the BODIPY skeleton, such that the prepared compound has absorption and fluorescence emission in the near-infrared region, and has good biological imaging effect; in addition, active oxygen can be efficiently generated under the ultrasonic action, and an excellent sonodynamic effect can be generated. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy, and a preparation method and application thereof. Background Art

[0002] Cancer has become one of the diseases that pose the greatest threat to human life and health, and the proportion of cancer deaths in all causes of death in humans has gradually shown an upward trend in recent years. Sonodynamic therapy (SDT) is an emerging non-invasive treatment method that combines the effects of sonosensitizers, ultrasound and oxygen to treat a variety of diseases, especially deep tumors. The basic principle of SDT is to use ultrasound to activate sonosensitizers to produce highly toxic reactive oxygen species (ROS) such as singlet oxygen and free radicals in cells. These ROS can oxidize biomolecules, causing damage to cell membranes, proteins and DNA, and ultimately inducing cell apoptosis or necrosis.

[0003] Compared with traditional photodynamic therapy (PDT), SDT has higher tissue penetration ability and can effectively treat tumors in deep tissues. In addition, SDT has high spatiotemporal controllability, which can accurately locate and treat the lesion area and reduce damage to surrounding healthy tissues. In recent years, SDT has shown good therapeutic effects in preclinical studies and has shown significant anti-tumor activity in a variety of cancer models. Therefore, sonodynamic therapy shows great potential and good application prospects in the efficient treatment of deep tumors.

[0004] BODIPY (Boron-Dipyrromethene) is a class of fluorescent dyes with unique photophysical and photochemical properties. Due to its high fluorescence quantum yield, excellent photostability and tunable absorption and emission spectra, BODIPY has been widely used in bioimaging, sensing and drug delivery. In recent years, BODIPY has also been studied as a sonosensitizer for sonodynamic therapy.

[0005] The main advantages of BODIPY as a sonosensitizer include: (1) High ROS generation ability: BODIPY can efficiently generate ROS, especially singlet oxygen, under the action of ultrasound, thereby effectively killing cancer cells. (2) Good biocompatibility: BODIPY has low systemic toxicity and less damage to normal cells, making it suitable for in vivo applications. (3) Versatility: BODIPY can adjust its photophysical and photochemical properties through structural modification to meet different therapeutic needs. (4) Fluorescence properties: The fluorescent properties of BODIPY enable it to be used for imaging during treatment, achieving dual functions of treatment and diagnosis. Although BODIPY shows great potential as a sonosensitizer, there are still some challenges: (1) Stability issues: The stability of existing BODIPY compounds in vivo needs to be improved, especially when exposed to physiological conditions for a long time. (2) ROS generation efficiency: Although BODIPY can generate ROS, its generation efficiency still has room for improvement to enhance its therapeutic effect. Summary of the invention

[0006] The purpose of the present invention is to provide a near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy, and a preparation method and application thereof. The method has mild reaction conditions, short reaction time, high yield, and a wide range of substrate applications. The prepared near-infrared difluoroboron pyrrole compound has broad application prospects in near-infrared bioimaging and sonodynamic therapy.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy, the chemical structure of which is shown in Formula 4:

[0009]

[0010] In formula 4, R1 is selected from

[0011] To achieve the above object, the present invention also provides a method for preparing the near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy, the specific steps of which are:

[0012] (1) Add dichloromethane to a reaction flask, purge with nitrogen, and then add 2,4-dimethylpyrrole, benzaldehyde substituted at the para position of the compound of formula 1, and trifluoroacetic acid to the reaction flask. The color of the solution instantly turns red. After stirring for 5-7 hours, DDQ is added and stirred at room temperature for 50-70 minutes. Then, triethylamine and boron trifluoride ether are added in sequence and stirred for 8-12 hours. After extracting the organic layer, rotary evaporation and purification are performed to obtain a fluoroboron dipyrrole compound of formula 2;

[0013] The molar ratio between the para-substituted benzaldehyde and 2,4-dimethylpyrrole in the compound of formula 1 is (1-2.2):1;

[0014] The structural formula of the para-substituted benzaldehyde of the compound of formula 1 is Wherein R1 is selected from

[0015] The structural formula of the fluoroborane dipyrrole compound of formula 2 is Wherein R1 is selected from

[0016] (2) Add the fluoroborane dipyrrole compound of Formula 2 to a reaction flask, purge with nitrogen, add toluene and stir to dissolve, then add the para-substituted benzaldehyde, piperidine, acetic acid and molecular sieve of Formula 3, react at 90° C. for 2-3 hours, filter to remove the molecular sieve, extract the organic layer, dry, rotary evaporate and purify to obtain the near-infrared fluoroborane dipyrrole compound of Formula 4;

[0017] The molar ratio between the fluoroboron dipyrrole compound of formula 2 and the para-substituted benzaldehyde of the compound of formula 3 is 1:2.5;

[0018] The structural formula of the para-substituted benzaldehyde of the compound of formula 3 is

[0019] Preferably, in step (1), the molar ratio of the para-substituted benzaldehyde of the compound of formula 1 to DDQ is 2.2:1; the molar volume ratio of the para-substituted benzaldehyde of the compound of formula 1 to triethylamine and boron trifluoride ether is 24 mmol:(10-17) mL:(10-15) mL.

[0020] Preferably, in step (1), the molar volume ratio between the para-substituted benzaldehyde of the compound of formula 1 and dichloromethane is 1 mmol:(4-21) mL.

[0021] Preferably, in step (2), the molar volume ratio of the fluoroboron dipyrrole compound of formula 2 to piperidine and acetic acid is 1 mmol:2.8 mL:2.4 mL.

[0022] Preferably, in step (2), the mass ratio between the fluoroboron dipyrrole compound of formula 2 and the molecular sieve is 1:10.

[0023] Furthermore, in step (1), the specific steps of extracting the organic layer and then rotary evaporating and purifying are: extracting the organic layer with water and then rotary evaporating, and purifying by silica gel column chromatography to obtain a fluoroboron dipyrrole compound of formula 2.

[0024] Furthermore, in step (2), the specific steps of extracting the organic layer, drying, rotary evaporation and purification are: extracting the organic layer with DCM and water, drying with anhydrous sodium sulfate and rotary evaporation, and purifying with silica gel column chromatography to obtain a near-infrared fluoroborane dipyrrole compound of formula 4.

[0025] To achieve the above objectives, the present invention also provides the use of the above near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy as a fluorescent dye in bioimaging.

[0026] To achieve the above objectives, the present invention also provides the use of the above near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy as a sonosensitizer in sonodynamic therapy.

[0027] The reaction scheme of the present invention is as follows:

[0028]

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention uses para-substituted benzaldehyde and difluoroboryl pyrrole compounds to carry out condensation reaction in the presence of molecular sieves to generate the target product. The reaction conditions are mild, the yield is high, the reaction time is short, the post-treatment is simple, and it is convenient for industrial scale-up production.

[0031] (2) The near-infrared fluoroborane dipyrrole compound prepared by the present invention has the characteristics of near-infrared absorption and emission, has a strong tissue penetration depth, can effectively reduce the interference of background fluorescence signals, and can be used for near-infrared biological imaging.

[0032] (3) The near-infrared fluoroborane dipyrrole compound prepared by the present invention has good stability in vivo and a long excited state lifetime. It can efficiently generate singlet oxygen under the action of ultrasound and can be used as an efficient, safe and highly targeted sonosensitizer for sonodynamic therapy to promote the development of sonodynamic therapy in clinical applications.

[0033] In summary, the near-infrared fluoroboron dipyrrole compound prepared by the present invention has broad application prospects in bioimaging and sonodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the near-infrared fluoroborane dipyrrole compound prepared in Example 1;

[0035] Figure 2 The dichloromethane solution (10 -5 M) UV-visible absorption spectrum;

[0036] Figure 3 The dichloromethane solution (10 -5 M) fluorescence spectrum;

[0037] Figure 4 The absorbance change curve of the near-infrared fluoroborane dipyrrole compound and the singlet oxygen indicator DPBF solution in Example 1 under ultrasound at different time periods;

[0038] Figure 5 This is a statistical chart of the CCK8 cytotoxicity test results of the near-infrared fluoroboron dipyrrole compound prepared in Example 1 under light-proof conditions and ultrasonic conditions. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following examples.

[0040] Example 1: Synthesis of near-infrared difluoroboryl pyrrole compound 4A

[0041]

[0042] (1) Synthesis of the fluoroborane dipyrrole compound of formula 2:

[0043] After adding the magnetic bar, the single-mouth bottle was sealed, filled with nitrogen, 500 mL of redistilled DCM was added, and then 4-(methylmercaptobenzaldehyde) (24 mmol) (1.16 mL) and 2,4-dimethylpyrrole (24 mmol, ρ = 0.924 g / mL, 2.47 mL) were added with a syringe, and 1 drop of TFA was added. After stirring at room temperature for 6 h, 2.5 g (11 mmol) of DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) was quickly added after removing the stopper. After covering the stopper and continuing to stir for 1 h, 17 mL of TEA (triethylamine) and 15 ml of boron trifluoride ether were slowly added dropwise and stirred for 8 h. The reaction process was monitored by a spot plate (developing agent PE: DCM = 2: 1), the reaction solution was extracted with water (3×300 mL), the part with the compound was collected, the solvent was removed under reduced pressure, and the product was separated and purified by column chromatography with an eluent (PE: DCM = 5: 1) to obtain the product.

[0044] Its structural characterization data are: 1 H NMR (400MHz, CDCl3) 7.32 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.0 (s, 2H), 2.6 (s, 6H), 2.53 (s, 3H), 1.42 (s, 6H).

[0045] (2) Synthesis of near-infrared difluoroboryl pyrrole compound 4A:

[0046] The compound of formula 2, fluoroborane dipyrrole compound (0.6 mmol), was added to a 25 mL round-bottom flask with a branch tube equipped with a stirrer, and the flask was vented. 10 mL of anhydrous toluene was added under a nitrogen atmosphere. After the fluoroborane dipyrrole compound of formula 2 was dissolved in toluene, p-methylthiobenzaldehyde (0.75 mmol, ρ = 1.144 g / mL, 1 mL), piperidine (17.17 mmol, ρ = 0.86 g / mL, 1.7 mL), acetic acid (1.42 mL) and 4A molecular sieve (1 g) were added in sequence. The mixture was reacted at 90 ° C for 3 h and then the heating was turned off. The reaction progress was monitored by thin layer plate TLC (developing agent PE: DCM = 1: 1); after the reaction solution was cooled to room temperature, the 4A molecular sieve was filtered out, and the filtrate was extracted with DCM and water. The part with the compound was collected, dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product (68.1 mg, 85%) was separated and purified by column chromatography.

[0047] The structural representation of the attached figure is as follows Figure 1 As shown, the characterization data are: 1 H NMR (400MHz, CDCl3)7.72(d,J=6.4Hz,2H),7.61(d,J=6.0Hz,4H),7.32(d,J=2.4 Hz,2H),7.19-7.25(m,8H),6.66(s,2H),2.63(s,3H),2.55(s,6H),1.52(s,6H).

[0048] Example 2: Synthesis of near-infrared difluoroboryl pyrrole compound 4B

[0049]

[0050] (1) Synthesis of the fluoroborane dipyrrole compound of formula 2:

[0051] Under nitrogen atmosphere, p-methoxybenzaldehyde (24 mmol, 3.2676 g) and 2,4-dimethylpyrrole (11 mmol, ρ=0.924 g / mL, 1.13 mL) were dissolved in 100 mL of degassed anhydrous CH2Cl2, and ten drops of trifluoroacetic acid (TFA) were added. After stirring at room temperature for 6 h, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ, 11 mmol, 2.4971 g) was added, and stirring was continued for 1 h. Then, 10 mL of triethylamine (TEA) and 10 mL of boron trifluoride etherate (BF3·Et2O) were added dropwise in sequence, and stirring was continued for 12 h. The reaction process was monitored by spot plate (developing agent PE:DCM=2:1), the reaction solution was extracted with water (3×300 mL), the part with compound was collected, the solvent was removed under reduced pressure, and the product was separated and purified by column chromatography with eluent (PE:DCM=5:1) to obtain the product.

[0052] Its structural characterization data are: 1 H NMR (400MHz, CDCl3) 7.35 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 6.12 (s, 2H), 3.78 (s, 3H), 2.39 (s, 6H), 1.41 (s, 6H).

[0053] (2) Synthesis of near-infrared difluoroboryl pyrrole compound 4B:

[0054] The compound of formula 2, fluoroboron dipyrrole compound (0.6 mmol), was added to a 25 mL round-bottom flask with a branch tube equipped with a stirrer, and the flask was vented. 10 mL of anhydrous toluene was added under a nitrogen atmosphere. After the fluoroboron dipyrrole compound of formula 2 was dissolved in toluene, p-methylthiobenzaldehyde (0.75 mmol, ρ = 1.144 g / mL, 1 mL), piperidine (17.17 mmol, ρ = 0.86 g / mL, 1.7 mL), acetic acid (1.42 mL) and 4A molecular sieve (0.9 g) were added in sequence. The mixture was reacted at 90 ° C for 3 h and then the heating was turned off. The reaction progress was monitored by thin layer plate TLC (developing agent PE: DCM = 1: 1); after the reaction solution was cooled to room temperature, the 4A molecular sieve was filtered out, and the filtrate was extracted with DCM and water. The part with the compound was collected, dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product (72.5 mg, 86%) was separated and purified by column chromatography.

[0055] Its structural characterization data are: 1H NMR (400MHz, CDCl3)7.81(d,J=6.4Hz,2H),7.66(d,J=6.0Hz,4H),7.37(d,J=2.4 Hz,2H),7.18-7.23(m,8H),6.72(s,2H),3.76(s,3H),2.42(s,6H),1.46(s,6H).

[0056] The UV absorption and fluorescence emission spectroscopy characterization process and results of the near-infrared difluoroboryl pyrrole compound 4A prepared in Example 1 are as follows:

[0057] The spectral test concentration used in the present invention is 5 μM, and dichloromethane is used as the test solvent. Figure 2 As shown, the near-infrared difluoroboryl pyrrole compound 4A exhibits strong absorption in the near-infrared range of 600-750nm. The emission spectrum of the near-infrared difluoroboryl pyrrole compound 4A was measured with 720nm as the excitation wavelength, as shown in FIG. Figure 3 As shown, its emission peak is relatively broad, the emission peak is located at 805 nm, and it has near-infrared light emission, which is more conducive to biological imaging.

[0058] The experimental process and results of the near-infrared difluoroboryl pyrrole compound 4A prepared in Example 1 generating singlet oxygen in a dichloromethane solution under ultrasound are as follows:

[0059] DPBF is a commonly used singlet oxygen indicator, which can react with singlet oxygen to cause a decrease in the absorption peak. Prepare a DPBF solution of appropriate concentration, add 1 μL of near-infrared difluoroboryl pyrrole compound 4A solution to it, mix well, place it on the probe of an ultrasonic therapy device for ultrasound, with an ultrasound power of 1.5W and a frequency of 1MHz. Test the ultraviolet absorption spectrum every 30 seconds. Continuously record the changes in the absorption peak of DPBF at 410nm. Figure 4 It can be seen that the absorbance of DPBF in the DPBF solution without the addition of the near-infrared difluoroborylpyrrole compound 4A did not change significantly with the extension of the ultrasonic time; Figure 4 As shown, after the addition of the near-infrared difluoroboryl pyrrole compound 4A, the absorbance of DPBF decreased rapidly with the extension of the ultrasonic time, which means that the near-infrared difluoroboryl pyrrole compound 4A can effectively produce singlet oxygen under the action of ultrasound.

[0060] The CCK8 cytotoxicity test process and results of the near-infrared difluoroboryl pyrrole compound 4A prepared in Example 1 are as follows:

[0061] The digested cells were seeded in a 96-well plate at a seeding density of 10 4 / well, and continue to culture for 24 hours at 37°C and 5% CO2. After removing the stale culture medium, continue to culture the cells with cell culture medium containing different concentrations of near-infrared difluoroboryl pyrrole compound 4A (1-20μM). After 24 hours, the cells are ultrasonically treated and cultured for 12 hours after ultrasound. Add 10μL CCK8 (5mg / mL) to each well and continue to culture for 4 hours. After terminating the culture, remove the culture medium, add 150μL DMSO to each well, shake on a shaker for 10 minutes, and then use a microplate reader to test OD570. The results of the MTT cytotoxicity experiment are as follows Figure 5 As shown, under the condition of light protection and no ultrasound, when the concentration of near-infrared difluoroboryl pyrrole compound 4A was 1-50 μM, the cell survival rate after 24 hours of culture was greater than 80%, proving that the sonosensitizer has low dark cytotoxicity. When the cells incubated with the sonosensitizer were treated with ultrasound, the cell survival rate decreased rapidly with the increase of the incubation concentration, indicating that the near-infrared difluoroboryl pyrrole compound 4A has excellent sonodynamic therapeutic effect as a sonosensitizer.

Claims

1. A near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy, characterized in that: Its chemical structure is shown in Formula 4: In formula 4, R1 is selected from 2. A method for preparing a near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy as claimed in claim 1, characterized in that: The specific steps are: (1) Add dichloromethane to a reaction flask, purge with nitrogen, and then add 2,4-dimethylpyrrole, benzaldehyde substituted at the para position of the compound of formula 1, and trifluoroacetic acid to the reaction flask. The color of the solution instantly turns red. After stirring for 5-7 hours, DDQ is added and stirred at room temperature for 50-70 minutes. Then, triethylamine and boron trifluoride ether are added in sequence and stirred for 8-12 hours. After extracting the organic layer, rotary evaporation and purification are performed to obtain a fluoroboron dipyrrole compound of formula 2; The molar ratio between the para-substituted benzaldehyde and 2,4-dimethylpyrrole in the compound of formula 1 is (1-2.2):1; The structural formula of the para-substituted benzaldehyde of the compound of formula 1 is Wherein R1 is selected from The structural formula of the fluoroborane dipyrrole compound of formula 2 is Wherein R1 is selected from (2) Add the fluoroborane dipyrrole compound of Formula 2 to a reaction flask, purge with nitrogen, add toluene and stir to dissolve, then add the para-substituted benzaldehyde, piperidine, acetic acid and molecular sieve of Formula 3, react at 90° C. for 2-3 hours, filter to remove the molecular sieve, extract the organic layer, dry, rotary evaporate and purify to obtain the near-infrared fluoroborane dipyrrole compound of Formula 4; The molar ratio between the fluoroboron dipyrrole compound of formula 2 and the para-substituted benzaldehyde of the compound of formula 3 is 1:2.5; The structural formula of the para-substituted benzaldehyde of the compound of formula 3 is 3. The method for preparing a near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy according to claim 2, characterized in that: In step (1), the molar ratio of the para-substituted benzaldehyde of the compound of formula 1 to DDQ is 2.2:1; the molar volume ratio of the para-substituted benzaldehyde of the compound of formula 1 to triethylamine and boron trifluoride ether is 24 mmol:(10-17) mL:(10-15) mL.

4. The method for preparing a near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: In step (1), the molar volume ratio between the para-substituted benzaldehyde of the compound of formula 1 and dichloromethane is 1 mmol:(4-21) mL.

5. A method for preparing a near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: In step (2), the molar volume ratio of the fluoroborane dipyrrole compound of formula 2 to piperidine and acetic acid is 1 mmol:2.8 mL:2.4 mL.

6. The method for preparing a near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: In step (2), the mass ratio between the fluoroborane dipyrrole compound of formula 2 and the molecular sieve is 1:

10.

7. The method for preparing a near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: In step (1), the specific steps of extracting the organic layer and then performing dry rotary evaporation and purification are: extracting the organic layer with water and then performing dry rotary evaporation, and purifying by silica gel column chromatography to obtain a fluoroboron dipyrrole compound of formula 2.

8. The method for preparing a near-infrared fluoroborane dipyrrole compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: In step (2), the specific steps of extracting the organic layer, drying, rotary evaporation and purification are: extracting the organic layer with DCM and water, drying with anhydrous sodium sulfate and rotary evaporation, and purifying with silica gel column chromatography to obtain a near-infrared fluoroborane dipyrrole compound of formula 4.

9. Use of the near-infrared fluoroboron dipyrrole compound of claim 1 as a fluorescent dye in biological imaging.

10. Use of the near-infrared fluoroboron dipyrrole compound of claim 1 as a sonosensitizer in sonodynamic therapy.

Images