A near-infrared emitting fluorescent small molecule probe and preparation method thereof

Abstract

This case involves a near-infrared emitting fluorescent small molecule probe and a preparation method thereof, which is prepared by condensation reaction with BODIPY as the main luminescent unit and azo group as the specific recognition group, and has the following structural formula: The present invention constructs a class of BODIPY-azobenzene derivatives with novel structures. This series of molecules are all based on anaerobic enzymes or azo-specific recognition objects as targets. The absorption and emission wavelengths of the prepared materials are both in the near-infrared region 1, which exceeds the spectral recognition range of conventional anaerobic enzyme probes, and pushes the application of the material forward a step. The material designed in this case is relatively simple to synthesize, and the molecules reserve reaction groups for further functional recognition, which prepares the conditions for the subsequent application of the material.

Description

Technical Field

[0001] The present invention relates to the technical field of organic luminescent materials, and in particular to a near-infrared emitting fluorescent small molecule probe and a preparation method thereof. Background Art

[0002] Fluorescence imaging technology has the advantages of visualization, in situ, non-destructive, deep imaging depth, real-time, and economical, and is widely used in immunodiagnosis and treatment, medical imaging, cancer diagnosis and treatment, etc. BODIPY fluorescent molecules have high fluorescence quantum yield, high molar absorption coefficient, relatively good light and thermochemical stability, good solubility, high sensitivity, fluorescence signal is insensitive to the pH value and polarity of the solvent, easy to modify the structure, and emission wavelength to the near-infrared region. It is a material with great potential in the field of biological imaging and medical diagnosis and treatment research.

[0003] Organic fluorescent small molecules have outstanding advantages in cancer diagnosis. For example, hypoxia in cancer cells is a common phenomenon in most solid tumor cells. Hypoxia can induce accelerated biological reduction reactions and lead to the expression of intracellular reductases. Among them, azoreductase is one of the most representative reductases. The enzyme-catalyzed reduction of azo to amino groups has been proven to be an effective positioning and hypoxic imaging principle. Currently, most of the cancer diagnosis and treatment fluorescent probes based on azo recognition are based on rhodamine and cyanine dye molecules. However, cyanine dyes are not very stable and their own luminescence properties are unstable due to environmental influences such as solvents. This has limited the application of organic small molecule fluorescent probes in hypoxic enzyme cancer diagnosis to a certain extent, and therefore has also stimulated the interest of our research group in developing such specific recognition fluorescent dyes with good application prospects. In the previous work, the applicant constructed a fluorescent small molecule probe based on perylene imide as the main luminescent unit, azo derivatives as the modifying groups, and BODIPY as the intramolecular energy donor. The present invention is essentially different from the previous work in that a near-infrared absorbing molecule with an intramolecular large conjugated system is constructed based on BODIPY and azobenzene. The azo group serves as a potential hypoxia recognition functional group, and its other function is to quench the emission of the main BODIPY based on the intramolecular (electron transfer) PET mechanism. At the same time, the addition of the azo group expands the molecular conjugated plane to a certain extent, extending the overall absorption of the molecule to the near-infrared region. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention is based on molecular groups such as BODIPY and azobenzene, and achieves molecular conjugation enlargement through conjugation linkage, greatly extending the molecular absorption wavelength, and overcoming the shortcomings of conventional small molecule fluorescent hypoxia enzyme probes such as short emission wavelength and little change in wavelength before and after identification.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A near-infrared emitting fluorescent small molecule probe, which is prepared by condensation of a witting-horner reaction with BODIPY as the main luminescent unit and an azo group as the specific recognition group, and has the following structural formula:

[0007]

[0008] Wherein, R1 is -CH2CH2CH3, -CH2CH=CH2, -CH2C≡CH; R2 is -N(CH2CH3)2, -OCH2CH2CH3, -SCH2CH2CH3. .

[0009] The present invention further provides a method for preparing the near-infrared emitting fluorescent small molecule probe as described above, comprising the following steps:

[0010] S1: Preparation of azobenzene derivatives: Compound 4-aminobenzaldehyde was dissolved in DMF under ice bath conditions, and then hydrochloric acid was added dropwise to the reaction solution, followed by slow dropwise addition of NaNO2. After the addition was complete, stirring was continued for 30 minutes. The reaction solution was transferred and added dropwise to a sodium hydroxide solution containing phenol at 0°C. After the reaction was completed, the solid was extracted under reduced pressure, dried, and recrystallized from ethanol to obtain a light yellow solid.

[0011] S2: Preparation of azobenzene derivatives containing terminal hydrocarbon groups: Under nitrogen protection, the azobenzene derivative of S1, halogenated hydrocarbon, and anhydrous potassium carbonate are mixed and dissolved in N,N-dimethylformamide and reacted for 24 hours; after the reaction, cool to room temperature and pour into ice water, adjust the pH of the reaction solution to acidic until a yellow solid is generated, and filter and purify by column chromatography to obtain the solid;

[0012] S3: Preparation of BODIPY-azobenzene derivatives: under nitrogen protection, add the azobenzene derivative containing terminal hydrocarbon group of S2 and 2,4-dimethylpyrrole to a two-necked bottle containing dichloromethane, and use trifluoroacetic acid to catalyze the reaction at room temperature; after the reaction, add tetrachlorobenzoquinone to the reaction solution for oxidation, stir at room temperature for 30 minutes, and then continue to add triethylamine dropwise, continue to stir at room temperature for 30 minutes, continue to add boron trifluoride ether complexing reagent dropwise, and continue to stir. After the reaction is completed, reduce the pressure and filter, and obtain the product by column chromatography;

[0013] S4: Preparation of near-infrared absorbing BODIPY-azobenzene derivatives: Under nitrogen protection, add the BODIPY-azobenzene derivative of S3, benzaldehyde derivative, piperazine and p-toluenesulfonic acid into a 250 mL two-necked bottle containing toluene, and react in an oil bath for 48 hours. After the reaction is completed, cool to room temperature, remove the solvent under reduced pressure, and purify by column chromatography.

[0014] Furthermore, in step S2, the halogenated hydrocarbon is propyne bromide, allyl bromide or propane bromide.

[0015] Furthermore, in step S4, the benzaldehyde derivative is 4-N,N-diethylaminobenzaldehyde, 4-propoxybenzaldehyde or 4-propylmercaptobenzaldehyde.

[0016] In this case, hypoxic enzyme molecules with near-infrared absorption and fluorescence emission were prepared based on BODIPY and azobenzene derivatives. First, azobenzene derivatives containing aldehyde groups and terminal alkynyl groups or olefins were prepared, and a series of azo BODIPY derivatives were prepared through a typical BODIPY synthesis method. Then, the active methyl group on BODIPY was used to condense a series of molecules containing aldehyde groups with it under the catalysis of piperidine and benzenesulfonic acid to prepare a large π molecule with a larger conjugated plane. At the same time, an olefin or alkyne reaction site with certain reaction activity was reserved at the end of the molecule. After preliminary ultraviolet and fluorescence tests and analysis of its spectral data, the material designed and constructed by the present invention fully achieved the expected conception and will provide important reference value for the development of subsequent materials.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention constructs a class of BODIPY-azobenzene derivatives with novel structures. This series of molecules are all based on anaerobic enzymes or azo-specific recognition objects as targets. The absorption and emission wavelengths of the prepared materials are both in the near-infrared region, which exceeds the spectral recognition range of conventional anaerobic enzyme probes, and pushes the application of the materials forward by one step. The synthesis of the materials designed in this case is relatively simple, and the molecules reserve reaction groups for further functional identification, which prepares the conditions for the subsequent application of the materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the UV absorption spectrum of the probe molecule BODIPY-2.

[0020] Figure 2 This is the fluorescence emission spectrum of the probe molecule BODIPY-2.

[0021] Figure 3 This is a photograph of the probe molecule BODIPY-2 imaging cancer cells and normal cells. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Embodiment 1:

[0025] Step 1

[0026]

[0027] Under ice bath conditions, 5 g (41 mmol) of compound 4-aminobenzaldehyde was dissolved in 100 ml of DMF, and then 10 ml of hydrochloric acid was added dropwise to the reaction solution, and then 2.8 g (42 mmol) of NaNO2 was slowly added dropwise. After the addition was completed, stirring was continued for 30 minutes. After the reaction was completed by starch test paper, the reaction solution was transferred and added dropwise to a sodium hydroxide solution containing 3.8 g (42 mmol) of phenol at 0°C. A large amount of yellow solid was generated. After the reaction was completed, it was vacuum-evacuated, dried, and recrystallized from ethanol to obtain a light yellow solid ONB-1, 7.6 g, with a yield of 82%. 1 H NMR (300MHz, CDCl3): δ9.88(s,1H),9.48(s,1H),8.10-8.04(m,4H),7.73-6.92(m,4H).

[0028] Step 2:

[0029]

[0030] Under nitrogen protection, compound ONB-1 (5.0 g, 22 mmol), propylene bromide (4.4 g, 30 mmol), anhydrous potassium carbonate (4.2 g, 30 mmol) were mixed in N, N-dimethylformamide (50 mL) and reacted at 80 ° C for 24 h. After the reaction, the reaction mixture was cooled to room temperature and poured into ice water (100 mL). The pH of the reaction solution was adjusted to acidic, and a large amount of yellow solid was generated. Filtered and dried, column chromatography was performed to obtain yellow solid ONB-2 4.5, yield: 85%. 1 H NMR (300MHz, CDCl3): δ9.92(s,1H),8.12-8.04(m,4H),7.75-6.94(m,4H),6.68-5.84(m,3H),4.63(d,2H).

[0031] Step 3:

[0032]

[0033] Under nitrogen protection, add 1.5g (5.7mmol) of compound ONB-2, 1.2g (12mmol) of compound 2,4-dimethylpyrrole, and 1-2 drops of trifluoroacetic acid to a 250mL two-necked bottle containing 100ml of dichloromethane, and react at room temperature for 8-12 hours. After the reaction, add 2.8g of tetrachlorobenzoquinone to the reaction solution, stir at room temperature for 30min, and then drop 2.2g (24mmol) of triethylamine, continue to stir at room temperature for 30min, and then drop 1.8g (24mmol) of boron trifluoride ether, and continue to stir for 2h. After the reaction is completed, filter under reduced pressure, and column chromatography (dichloromethane / petroleum ether) is performed to obtain 0.25g of compound BODIPY-1, with a yield of 22%. 1 H NMR (300MHz, CDCl3): 8.12-8.04(m,4H),7.75-6.92(m,4H),6.68-5.84(m,C=C,3H),6.16(s,2H),4.64(d,2H),2.65(s,6H),1.45(s,6H).

[0034] Step 4:

[0035]

[0036] Under nitrogen protection, add 1.2g (4.2mmol) of compound BODIPY-1, 2.0g (8.4mmol) of compound 4-N,N-diethylaminobenzaldehyde, 1.0g (10mmol) of piperazine, and 1.8g (10mmol) of p-toluenesulfonic acid to a 250mL two-necked bottle containing 100ml of toluene, and react in an oil bath at 110-120℃ for 48 hours. After the reaction is completed, cool to room temperature, remove the solvent under reduced pressure, and obtain 3g of compound BODIPY-2 by column chromatography (dichloromethane / petroleum ether), with a yield of 70%. 1 H NMR(300MHz, CDCl3):8.08-8.01(m,4H),7.71-6.89(m,4H),7.54-6.84(m,8H),6.65- 5.81(m,C=C,7H),6.12(s,2H),4.62(d,2H),3.24(m,8H),1.45(s,6H),1.12(t,12H).

[0037] Embodiment 2:

[0038] Step 1

[0039]

[0040] The same method as step 2 of Example 1 was used, except that allyl bromide was replaced by propyne bromide to obtain 5 g of yellow solid ONB-3 with a yield of 85%. 1 H NMR (300MHz, CDCl3): δ9.92(s,1H),8.12-8.04(m,4H),7.75-6.94(m,4H),4.63(d,2H),3.37(s,1H).

[0041] Step 2

[0042]

[0043] The same as step 3 of Example 1, except that ONB-2 was replaced by ONB-3, to obtain 0.3 g of compound BODIPY-A, with a yield of 25%. 1 H NMR (300MHz, CDCl3): 8.12-8.04(m,4H),7.75-6.92(m,4H),6.16(s,2H),4.64(d,2H),3.37(s,1H),2.65(s,6H),1.45(s,6H).

[0044] Step 3

[0045]

[0046] Under nitrogen protection, add 1.2g (4.2mmol) of compound BODIPY-A, 2.0g (8.4mmol) of compound 4-N,N-diethylaminobenzaldehyde, 1.0g (10mmol) of piperazine, and 1.8g (10mmol) of p-toluenesulfonic acid to a 250mL two-necked bottle containing 100ml of toluene, and react in an oil bath at 110-120℃ for 48 hours. After the reaction is completed, cool to room temperature, remove the solvent under reduced pressure, and obtain 3.2g of compound BODIPY-B by column chromatography (dichloromethane / petroleum ether), with a yield of 72%. 1 H NMR(300MHz, CDCl3):8.08-8.02(m,4H),7.72-6.90(m,4H),7.54-6.86(m,8H),6.64-5.82( m,C=C,4H),6.12(s,2H),4.62(d,2H),3.37(s,1H),3.24(m,8H),1.45(s,6H),1.12(t,12H).

[0047] Embodiment 3:

[0048]

[0049] The preparation steps are the same as those in Example 2, except that in step 4, 4-N,N-diethylaminobenzaldehyde is replaced by 4-propoxybenzaldehyde to obtain 2.9 g of compound BODIPY-D with a yield of 72%. 1 H NMR(300MHz, CDCl3):8.08-8.02(m,4H),7.72-6.90(m,4H),7.54-6.86(m,8H),6.64-5.82(m,C=C ,4H),6.12(s,2H),4.62(d,2H),4.01(m,4H),3.37(s,1H),3.24(m,8H),1.76(t,6H),1.45(s,6H).

[0050] Embodiment 4:

[0051]

[0052] The preparation steps are the same as those in Example 1, except that in step 4, 4-N,N-diethylaminobenzaldehyde is replaced by 4-propylmercaptobenzaldehyde to obtain 3.1 g of compound BODIPY-E with a yield of 74%. 1 H NMR (300MHz, CDCl3):8.06-8.01(m,4H),7.70-6.88(m,4H),7.54-6.82(m,8H),6.62-5.80(m,C=C ,4H),6.10(s,2H),4.56(d,2H),4.01(m,4H),3.34(s,1H),3.22(m,8H),1.74(t,6H),1.42(s,6H).

[0053] Taking the material BODIPY-2 prepared in Example 1 as an example, fluorescence and ultraviolet spectrum tests were performed: the material was configured into 10 -5 ~10 -6 mol / L concentration of dichloromethane solution, using ultraviolet absorption spectrometer and fluorescence spectrometer to measure the absorption spectrum and emission spectrum of the material respectively ( Figure 1-2 ). From the measured ultraviolet absorption spectrum, it can be seen that the compound BODIPY-2 has the strongest absorption at 720nm, which meets the absorption range of the near-infrared region. From its fluorescence emission spectrum, it can be seen that the compound BODIPY-2 has a weaker emission above 800nm, which is in line with the original intention of molecular design. Based on the intramolecular electron transfer mechanism, because the azo group in the molecule quenches the emission of the main molecule, when the azo group reacts with the specific recognition molecule or other catalysts, the azo group is reduced, the fluorescence of the main molecule will be turned on, and the emission spectrum of the molecule will be blue-shifted.

[0054] Compound BODIPY-2 cell imaging experiment:

[0055] In a carbon dioxide atmosphere, the probe molecules were configured to a concentration of 10 -5 mol / L, drop it into the culture medium of HeLa living cells, continue to culture for 5-10 minutes, then wash away the molecules that are not eaten by the cells with PBS solution; configure the probe molecule to a concentration of 10 -5 mol / L, add it dropwise to normal cell culture medium, continue culturing for 5-10 minutes, then wash away the molecules not eaten by cells with PBS solution; transfer the cells to a glass slide and image them under 556nm and UV light sources, respectively, such as Figure 3 As shown, after the compound BODIPY-2 interacts with cancer cells, the probe molecule shows a bright red color, while the probe molecule shows a faint red color after staining with normal cells. This result is consistent with the original intention of the molecular design.

[0056] The reaction conditions designed by the invention are mild, the reaction is efficient, the operation is simple, and the applicability is wide, and a new molecular design idea is provided for enriching and developing detection reagents with selective recognition effects on azo.

[0057] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A near-infrared emitting fluorescent small molecule probe, characterized in that: It is prepared by condensation of BODIPY as the main luminescent unit and azo group as the specific recognition group through a witting-horner reaction and has the following structural formula: Among them, R1 is -CH2CH2CH3, -CH2CH=CH2, -CH2C≡CH; R2 is -N(CH2CH3)2, -OCH2CH2CH3, -SCH2CH2CH3.

2. The method for preparing a near-infrared emitting fluorescent small molecule probe according to claim 1, characterized in that: The steps include: S1: Preparation of azobenzene derivatives: Compound 4-aminobenzaldehyde was dissolved in DMF under ice bath conditions, and then hydrochloric acid was added dropwise to the reaction solution, followed by slow dropwise addition of NaNO2. After the addition was complete, stirring was continued for 30 minutes. The reaction solution was transferred and added dropwise to a sodium hydroxide solution containing phenol at 0°C. After the reaction was completed, the solid was extracted under reduced pressure, dried, and recrystallized from ethanol to obtain a light yellow solid. S2: Preparation of azobenzene derivatives containing terminal hydrocarbon groups: Under nitrogen protection, the azobenzene derivative of S1, halogenated hydrocarbon, and anhydrous potassium carbonate are mixed and dissolved in N,N-dimethylformamide and reacted for 24 hours; after the reaction, cool to room temperature and pour into ice water, adjust the pH of the reaction solution to acidic until a yellow solid is generated, and filter and purify by column chromatography to obtain the solid; S3: Preparation of BODIPY-azobenzene derivatives: under nitrogen protection, add the azobenzene derivative containing terminal hydrocarbon group of S2 and 2,4-dimethylpyrrole to a two-necked bottle containing dichloromethane, and use trifluoroacetic acid to catalyze the reaction at room temperature; after the reaction, add tetrachlorobenzoquinone to the reaction solution for oxidation, stir at room temperature for 30 minutes, and then continue to add triethylamine dropwise, continue to stir at room temperature for 30 minutes, continue to add boron trifluoride ether complexing reagent dropwise, and continue to stir. After the reaction is completed, reduce the pressure and filter, and obtain the product by column chromatography; S4: Preparation of near-infrared absorbing BODIPY-azobenzene derivatives: Under nitrogen protection, add the BODIPY-azobenzene derivative of S3, benzaldehyde derivative, piperazine and p-toluenesulfonic acid into a 250 mL two-necked bottle containing toluene, and react in an oil bath for 48 hours. After the reaction is completed, cool to room temperature, remove the solvent under reduced pressure, and purify by column chromatography.

3. The method for preparing a near-infrared emitting fluorescent small molecule probe according to claim 2, characterized in that: In the step S2, the halogenated hydrocarbon is propyne bromide, allyl bromide or propane bromide.

4. The method for preparing a near-infrared emitting fluorescent small molecule probe according to claim 2, characterized in that: In the step S4, the benzaldehyde derivative is 4-N,N-diethylaminobenzaldehyde, 4-propoxybenzaldehyde or 4-propylmercaptobenzaldehyde.

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