A near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy, and its preparation method and application

Abstract

The present invention discloses a near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy and a preparation method thereof. The chemical structural formula of the compound is shown in Formula 4; the preparation method is: first, para-substituted benzaldehyde, 2,4-dimethylpyrrole and boron trifluoride ether are prepared under acid catalysis by the fluoroboron dipyrrole compound; then the obtained fluoroboron dipyrrole compound and the para-substituted benzaldehyde are dehydrated and condensed under the catalysis of acid and base to obtain a target product. The near-infrared difluoroboron pyrrole compound disclosed in the present invention has mild synthesis conditions, short reaction time, high yield, simple post-processing and a wide range of structural applications. The present invention connects thiomethylbenzaldehyde and aniline derivatives to the fluoroboron dipyrrole skeleton, and the prepared compound has absorption and fluorescence emission in the near-infrared region, and has good bioimaging effect; in addition, it can also efficiently generate reactive oxygen species under ultrasound, and can produce excellent sonodynamic effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically 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 most threatening diseases to human health, and the proportion of cancer deaths among all causes of death has gradually increased in recent years. Sonodynamic therapy (SDT) is an emerging non-invasive treatment method that combines the effects of sonosensitizers, ultrasound, and oxygen for the treatment of various diseases, especially deep-seated tumors. The basic principle of SDT is to use ultrasound to activate sonosensitizers, causing them 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 demonstrated significant anti-tumor activity in various 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) Multifunctionality: BODIPY can adjust its photophysical and photochemical properties through structural modification to meet different therapeutic needs. (4) Fluorescence properties: The fluorescence 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, as well as 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, whose chemical structure is shown in Formula 4:

[0009]

[0010] In formula 4, R1 is selected from

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

[0012] (1) Add dichloromethane to a reaction flask, purge with nitrogen, and then add 2,4-dimethylpyrrole, the benzaldehyde of the para-substituted 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, add DDQ and stir at room temperature for 50-70 minutes. Then, add triethylamine and boron trifluoride ether in sequence and stir for 8-12 hours. After extracting the organic layer, rotary evaporate and purify to obtain the fluoroboron dipyrrolidone 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 fluoroboron dipyrrole compound of formula 2 is Wherein, R1 is selected from

[0016] (2) Add the fluoroborane dipyrrole compound of formula 2 to the 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 fluoroborane dipyrrole compound of formula 2 and the para-substituted benzaldehyde 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 the compound of formula 2, a fluoroborane dipyrrole compound.

[0024] Furthermore, in step (2), the specific steps of drying, rotary evaporation and purification after extracting the organic layer are as follows: extracting the organic layer with DCM and water, drying with anhydrous sodium sulfate and rotary evaporation, and purifying by 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-mentioned 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-mentioned 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 difluoroborane pyrrole compounds to carry out a 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-processing is simple, and it is convenient for industrial scale-up production.

[0031] (2) The near-infrared fluoroboron 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 fluoroboron 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 in 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 of the near-infrared fluoroborane dipyrrole compound prepared in Example 1 (10 -5 M) UV-visible absorption spectrum;

[0036] Figure 3 The dichloromethane solution of the near-infrared fluoroborane dipyrrole compound prepared in Example 1 (10 -5 M) fluorescence spectrum;

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

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

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

[0040] Example 1: Synthesis of Near-Infrared Difluoroborane Pyrrole Compound 4A

[0041]

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

[0043] After adding the magnetic bar, the single-necked bottle was sealed and 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 separately using a syringe. One drop of TFA was added, and 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 capping the bottle and continuing stirring for 1 h, 17 mL of TEA (triethylamine) and 15 mL of boron trifluoride etherate were slowly added dropwise and stirred for 8 h. The reaction process was monitored by spot plate (developing solvent PE:DCM = 2:1). The reaction solution was extracted with water (3 × 300 mL), and the fraction containing the compound was collected. The solvent was removed under reduced pressure, and the product was separated and purified by column chromatography using 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 difluoroborane pyrrole compound 4A:

[0046] 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, vented, and 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 sieves (1 g) were added in sequence. The mixture was reacted at 90 ° C. for 3 h, then the heat was turned off and the reaction progress was monitored by thin layer plate TLC (developing solvent PE: DCM = 1: 1); after the reaction solution cooled to room temperature, the 4A molecular sieves were removed by filtration, and the filtrate was extracted with DCM and water. The portion with the compound was collected, dried over 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 is shown in the accompanying drawings. 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 Difluoroborane Pyrrole Compound 4B

[0049]

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

[0051] Under nitrogen atmosphere, p-anisaldehyde (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. Ten drops of trifluoroacetic acid (TFA) were added, and the mixture was stirred 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, and stirring was continued for 12 h. The reaction process was monitored by spot plate chromatography (developing solvent PE:DCM = 2:1). The reaction solution was extracted with water (3 × 300 mL), and the fractions containing the compound were collected. The solvent was removed under reduced pressure, and the product was separated and purified by column chromatography using an 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 difluoroborane pyrrole compound 4B:

[0054] 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, vented, and 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 sieves (0.9 g) were added in sequence. The mixture was reacted at 90 ° C. for 3 h, then the heating was turned off and the reaction progress was monitored by thin layer plate TLC (developing solvent PE: DCM = 1: 1); after the reaction solution cooled to room temperature, the 4A molecular sieves were removed by filtration, and the filtrate was extracted with DCM and water. The portion with the compound was collected, dried over 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 difluoroborane pyrrole compound 4A prepared in Example 1 are as follows:

[0057] The spectrum test concentration used in the present invention is 5 μM, and dichloromethane is used as the test solvent. Figure 2 As shown in Figure 2, 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 an excitation wavelength of 720nm. 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 difluoroborane 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 that 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 difluoroborane pyrrole compound 4A solution to it, mix well, and place it on the probe of an ultrasonic therapeutic device for ultrasound. The ultrasound power is 1.5W and the frequency is 1MHz. The ultraviolet absorption spectrum is tested 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 difluoroborane pyrrole compound 4A did not change significantly with the extension of the ultrasonic time; Figure 4 As shown in the figure, after the near-infrared difluoroboryl pyrrole compound 4A was added, 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 generate singlet oxygen under the action of ultrasound.

[0060] The CCK8 cytotoxicity test process and results of the near-infrared difluoroborane 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 4Each well was cultured for 24 hours at 37°C and 5% CO2. After removing the stale culture medium, the cells were cultured with cell culture medium containing different concentrations of near-infrared difluoroboryl pyrrole compound 4A (1-20 μM). After 24 hours, the cells were ultrasonically treated and cultured for 12 hours. 10 μL CCK8 (5 mg / mL) was added to each well and cultured for 4 hours before terminating the culture. The culture medium was removed and 150 μL DMSO was added to each well. After shaking on a shaker for 10 minutes, the OD570 was measured using a microplate reader. The results of the MTT cytotoxicity test are shown in the figure. Figure 5 As shown, under light-shielded conditions and without ultrasound, the near-infrared difluoroboryl pyrrole compound 4A maintained a cell viability exceeding 80% after 24 hours of culture at concentrations ranging from 1 to 50 μM, demonstrating the low dark cytotoxicity of this sonosensitizer. When the sonosensitizer-treated cells were sonicated, their cell viability decreased rapidly with increasing incubation concentration, demonstrating the excellent sonodynamic therapeutic effect of the near-infrared difluoroboryl pyrrole compound 4A as a sonosensitizer.

Claims

1. A near-infrared fluoroboron 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 or .

2. A method for preparing a near-infrared fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy according to claim 1, characterized in that: The specific steps are: (1) Add dichloromethane to the 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 turns red instantly. After stirring for 5-7 hours, add DDQ and stir at room temperature for 50-70 minutes. Then, add triethylamine and boron trifluoride ether in sequence and stir for 8-12 hours. After extracting the organic layer, rotary evaporate and purify to obtain the compound of formula 2, a fluoroboron dipyrrole compound. 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 or ; The structural formula of the fluoroboron dipyrrole compound of formula 2 is , wherein R1 is selected from or ; (2) Add the fluoroborane dipyrrole compound of formula 2 to the 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 2-3h, 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 fluoroborane dipyrrole compound of formula 2 and the para-substituted benzaldehyde 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 fluoroboron 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 fluoroboron 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 fluoroboron 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 fluoroboron 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 fluoroboron dipyrrole compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: In step (2), the mass ratio between the fluoroboron dipyrrole compound of formula 2 and the molecular sieve is 1:

10.

7. The method for preparing a near-infrared fluoroboron 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 as follows: 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 fluoroboron 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 as follows: 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 the near-infrared fluoroborane dipyrrole compound of formula 4.

9. Use of the near-infrared fluoroboron dipyrrole compound according to claim 1 in preparing a sonosensitizer for sonodynamic therapy.

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