Tumor hypoxia specific response BODIPY fluorescent probe as well as preparation and application thereof

Abstract

The invention relates to a tumor hypoxia specific response BODIPY fluorescent probe as well as preparation and application thereof. The invention relates to a BODIPY fluorescent probe which is synthesized by utilizing tumor hypoxia to respond to azobenzene groups. The probe has no signal to normal oxygen cells, shows strong near-infrared fluorescence to hypoxia cancer cells, and can be used for visually detecting deep solid tumors. According to the present invention, the tumor specific response BODIPY fluorescent probe is adopted as the research object, the human liver cancer cells (HepG2) and the human non-small cell lung cancer cells (A549) are adopted as the tested cell strains, the fluorescence detection ability research under the normal oxygen and oxygen deficiency conditions is developed, and the foundation is established for the real-time accurate tumor detection. The fluorescent probe has the advantages of lower preparation cost, simpler synthesis method, easily available raw materials, less side reaction, high yield, easiness in purification, easiness in industrial production and the like.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probe design and synthesis, and specifically relates to a tumor hypoxia-specific response fluoroboron dipyrrole fluorescent probe and a preparation method and application thereof. Background Art

[0002] Accurate detection and early screening of tumor tissue is one of the key strategies to achieve precise cancer treatment and prognosis. It can effectively guide cancer treatment and provide important indicators for cancer recovery.

[0003] Near-infrared fluorescence imaging has many advantages in tumor detection, such as high resolution, low background signal interference, high sensitivity, non-invasiveness, and real-time detection. Fluorescence imaging technology uses molecular probes injected into the human body to perform real-time imaging of the lesion site. This type of probe will produce bright fluorescence signals after being excited by near-infrared light, and generate fluorescence images after being captured by the instrument, which can visually observe the tumor site. However, traditional bamboo red fungus, chlorophyll, and porphyrin fluorescent probes cannot be used as ideal fluorescent probes due to their uncertain composition, weak tissue penetration, and strong background interference. Compared with these traditional photosensitizers, boron dipyrromethene (BODIPY) dyes have advantages such as a certain chemical composition, stable chemical properties, and a maximum emission wavelength in the near-infrared region, and have become a type of fluorescent probe with application prospects. It is important that the structure of BODIPY dyes is easy to modify, and the modified boron dipyrromethene structure can derive a variety of photophysical and photochemical properties, making it a practical fluorescent probe. In addition, BODIPY also has the advantages of high quantum fluorescence yield, high molar extinction coefficient, good photostability, and acid and alkali resistance. It has been widely used in fluorescent probes, drug tracking, photosensitizers, disease detection and diagnosis, and other fields.

[0004] In the molecular structure design of fluorescent probes, how to accurately distinguish tumor tissue from normal tissue is one of the key issues. Hypoxia is considered to be a typical feature of malignant tumors, which is caused by uncontrolled proliferation of cancer cells, insufficient blood oxygen supply and metabolic disorders. A large number of studies have shown that hypoxia is closely related to the invasiveness and metastasis of tumors. In addition, cancer cells with hypoxia will overexpress azobenzene reductase and nitroreductase, and the expression of these enzymes is positively correlated with the degree of tumor hypoxia. The fluoroboron dipyrrole fluorescent probe developed by this patent can specifically respond to the hypoxic microenvironment of the tumor, causing the probe to release near-infrared fluorescence, which has the effect of accurate tumor imaging. This activation imaging method provides vital assistance for the early screening and diagnosis of tumors, and can guide surgical treatment to avoid unnecessary resection of healthy tissue. The fluoroboron dipyrrole fluorescent probe developed by this patent can detect tumor tissue in real time and image the tumor area. Moreover, the probe has high biological safety and provides a sensitive contrast agent for accurate detection of tumors. Summary of the invention

[0005] The object of the present invention is to provide a fluoroboron dipyrrole fluorescent probe that specifically responds to tumor hypoxia and its preparation and application. The present invention uses azobenzene groups that respond to tumor hypoxia to prepare a fluoroboron dipyrrole fluorescent probe, wherein the ultrafast isomer mutual conversion of the azobenzene group will quench the fluorescence of fluoroboron dipyrrole; when the azobenzene group in the probe is reduced and cleaved, the fluorescence of fluoroboron dipyrrole is restored. The fluorescence signal of the fluorescent probe changes from "off" to "on", which can well detect tumor hypoxic cells to sensitively distinguish between normal oxygen cells and hypoxic cancer cells. Studies have shown that the probe will not generate a fluorescent signal after being taken up by normal oxygen cells; when the fluorescent probe is taken up by hypoxic cancer cells, it will be reduced by overexpressed azobenzene reductase, and the generated fluoroboron dipyrrole metabolite will show strong near-infrared fluorescence, which can be used for real-time detection of tumor cells. The synthetic method of the fluorescent probe synthesized by the present invention is simple, the yield is high, the raw materials are easily available, and the cost is low, which is conducive to industrial production.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A tumor hypoxia-specific responsive fluoroboron dipyrrole fluorescent probe, the chemical structure of which is:

[0008]

[0009] The preparation method of the above compound comprises the following steps: As the starting material, the fluoroborane dipyrrole fluorescent probe was synthesized Specifically: Add to a round bottom flask and add 2.1-10 equivalents of In a flask, dissolve it with 60 mL of anhydrous toluene. Add glacial acetic acid (1.0 mL), piperidine (1.2 mL) and a small amount of anhydrous magnesium perchlorate to the above solution and heat to 135 ° C. Reflux for 2 hours under nitrogen protection. The water generated during the reaction is removed by a Dean-stark device. After the reaction is completed, the solution is cooled to room temperature, and the toluene solvent is removed by decompression using a rotary evaporator. The residue is extracted three times with dichloromethane and a saturated sodium chloride solution, and the organic phase is dried with anhydrous sodium sulfate and concentrated. The crude product is purified by silica gel column chromatography using dichloromethane / petroleum ether (volume ratio 1:1) as eluent to separate the azobenzene-fluoroboron dipyrrole probe. The 2.1-10 equivalents Compound The molar measurement.

[0010] The compound The preparation comprises the following steps:

[0011] (1) Using benzyl alcohol, HCl solution, NaNO2, sodium acetate and p-aminobenzyl alcohol as starting materials, the compound was synthesized

[0012] (2) Compounds Dess-Martin oxidant was used as the starting material to synthesize the compound

[0013] (3) Compounds 2,4-Dimethylpyrrole, trifluoroacetic acid, 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), triethylamine and boron trifluoride etherate were used as starting materials to synthesize the compound Specifically:

[0014] (1) Benzyl alcohol was dissolved in 30 mL of a mixed solvent of tetrahydrofuran and water (volume ratio 1:1), 3 M HCl solution was added to the above solution, and the reaction solution was stirred in an ice-water bath for 30 min. NaNO2, p-aminobenzyl alcohol, and sodium acetate were added to the reaction flask in a molar ratio of 1-2:1:1.5-2.5, and the resulting reaction solution was stirred continuously for 18 h at room temperature. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, and the residue was purified by silica gel column chromatography using dichloromethane-methanol as the eluent to obtain a yellow solid.

[0015] (2) Compound and Dess-Martin oxidant in a molar ratio of 1:0.5-1.2 were added to the reaction round-bottom flask and dissolved with anhydrous tetrahydrofuran. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was cooled to room temperature and the THF solvent was removed under reduced pressure using a rotary evaporator. The crude product was purified by silica gel column chromatography using dichloromethane-ethyl acetate as the eluent to obtain an orange solid.

[0016] (3) Compound and 2,4-dimethylpyrrole in a molar ratio of 1:2-3 are added to a round-bottom flask and fully dissolved with anhydrous dichloromethane. Add an appropriate amount of trifluoroacetic acid to the above solution, and the reaction is stirred overnight under nitrogen protection. Weigh 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and add it to the reaction bottle for 4 hours. Under ice-water bath conditions, add 12mL of triethylamine to the reaction bottle and stir for 30 minutes, then measure 12mL of boron trifluoride ether to a constant pressure dropping funnel and slowly add it dropwise to the reaction bottle. The reaction is stirred overnight under nitrogen protection. After the reaction is completed, the reaction solution is filtered with silica gel and transferred to a separatory funnel for extraction. After extraction 3 times with an appropriate amount of saturated sodium bicarbonate aqueous solution and dichloromethane, the organic layer is collected and dried with anhydrous sodium sulfate, and the crude product is spin-dried. The crude product is purified by silica gel column chromatography using petroleum ether-dichloromethane as eluent to obtain a red solid.

[0017] The azobenzene-modified fluoroboron dipyrrole fluorescent probe is used for detecting hypoxic cancer cells.

[0018] BODIPY is a type of fluorescent dye. Its molecular structure is mainly composed of two pyrrole rings connected by a methine bridge bond and a fluorine-boron bridge bond, forming a planar conjugated structure with 12 π electrons. It is often used in biomedical imaging, fluorescent probes, photodynamic therapy and other fields. BODIPY has the following characteristics:

[0019] (1) High molar absorption coefficient: BODIPY dye has a high molar absorption coefficient and strong light capture ability, which enables it to produce strong fluorescence signals even at low concentrations.

[0020] (2) High fluorescence quantum yield: The fluorescence quantum yield of BODIPY dyes is usually high and can effectively convert the absorbed light into fluorescent signals.

[0021] (3) High sensitivity and resolution: The fluorescence emission peak of BODIPY dye is narrow, and the emitted fluorescence has little interference from biological background.

[0022] (4) Good photostability: Porphin and porphyrin fluorescent dyes are easily photobleached under light conditions, while BODIPY dyes exhibit strong photostability and can perform long-term fluorescence imaging.

[0023] (5) Adjustable spectral properties: By modifying the molecular structure of BODIPY, its absorption and emission spectra can be adjusted to meet different experimental needs.

[0024] Based on this, the present invention utilizes the tumor hypoxia response azobenzene group to covalently modify the near-infrared fluorophore BODOIPY, and the ultrafast conformational isomerization of azobenzene will quench the fluorescence signal of BODIPY. The fluorescence of the fluorescent probe in normal oxygen cells is very weak. When the probe is enriched in hypoxic tumor cells, the overexpressed azobenzene reductase will reduce the azobenzene group and release the fluorescence signal of BODIPY. Therefore, the fluorescent probe can selectively detect the location and size of the tumor, and can be used to guide cancer treatment methods such as surgical resection, radiotherapy and chemotherapy in the later stage, laying the foundation for accurately locating the site of tumor occurrence.

[0025] The significant advantages of the present invention are:

[0026] (1) The fluorescence of fluoroborane dipyrrole modified with azobenzene is in a quenched state. In hypoxic cancer cells, the fluorescence of this fluorescent probe is restored, which can accurately detect tumor cells;

[0027] (2) After activation, the fluoroborane dipyrrole fluorescent probe emits fluorescence at around 700 nm, which is in the near-infrared region. The fluorescence biological background interference in this band is small, and the detection sensitivity is high;

[0028] (3) The target compound has low synthesis cost, simple synthesis method, readily available raw materials, fewer side reactions, higher yield, easy purification, and easy industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In the drawings of the specification:

[0030] Figure 1 Fluorescence detection imaging of HepG2 and A549 cells under normal oxygen or hypoxia by azobenzene-modified fluoroboron dipyrrole derivatives (a); quantification of probe channel fluorescence intensity (b). (*P<0.05, **P<0.01, ***P<0.001). Cell scale: 20μm;

[0031] Figure 2 For compound In DMSO-d6 1 H NMR spectrum;

[0032] Figure 3 For compound In CDCl3 1 H NMR spectrum;

[0033] Figure 4 For compound In CDCl3 1 HNMR spectrum;

[0034] Figure 5 For compound In CDCl3 1 H NMR spectrum; DETAILED DESCRIPTION

[0035] In order to explain in detail the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0036] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in the embodiments can be combined in any way to form a corresponding implementable technical solution.

[0037] Unless otherwise defined, the technical terms used in this document have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms in this document is only for describing specific embodiments and is not intended to limit this application.

[0038] Hereinafter, the embodiments of the present application are specifically disclosed in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there may be cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0039] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0040] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0041] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0042] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0043] The terms "above" and "below" used in this application include the number, for example, "one or more" means one or more, and "one or more of A and B" means "A", "B" or "A and B".

[0044] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0045] like Figures 1 to 5 As shown, the specific preparation process of the azobenzene-modified fluoroboron dipyrrole derivative with the function of fluorescent tumor detection is as follows:

[0046] (1) Benzyl alcohol was dissolved in 30 mL of a mixed solvent of tetrahydrofuran and water (volume ratio 1:1), 3 M HCl solution was added to the above solution, and the reaction solution was stirred in an ice-water bath for 30 min. NaNO2, p-aminobenzyl alcohol, and sodium acetate were added to the reaction flask in a molar ratio of 1-2:1:1.5-2.5, and the resulting reaction solution was stirred continuously for 18 h at room temperature. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, and the residue was purified by silica gel column chromatography using dichloromethane-methanol as the eluent to obtain a yellow solid. Yield 31-40%;

[0047] (2) Compound and Dess-Martin oxidant in a molar ratio of 1:0.5-1.2 were added to the reaction round-bottom flask and dissolved with anhydrous tetrahydrofuran. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was cooled to room temperature and the THF solvent was removed under reduced pressure using a rotary evaporator. The crude product was purified by silica gel column chromatography using dichloromethane-ethyl acetate as the eluent to obtain an orange solid. Yield 80-94%;

[0048] (3) Compound and 2,4-dimethylpyrrole in a molar ratio of 1:2-3 are added to a round-bottom flask and fully dissolved with anhydrous dichloromethane. Add an appropriate amount of trifluoroacetic acid to the above solution, and the reaction is stirred overnight under nitrogen protection. Weigh 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and add it to the reaction bottle for 4 hours. Under ice-water bath conditions, add 12mL of triethylamine to the reaction bottle and stir for 30 minutes, then measure 12mL of boron trifluoride ether to a constant pressure dropping funnel and slowly add it dropwise to the reaction bottle. The reaction is stirred overnight under nitrogen protection. After the reaction is completed, the reaction solution is filtered with silica gel and transferred to a separatory funnel for extraction. After extraction 3 times with an appropriate amount of saturated sodium bicarbonate aqueous solution and dichloromethane, the organic layer is collected and dried with anhydrous sodium sulfate, and the crude product is spin-dried. The crude product is purified by silica gel column chromatography using petroleum ether-dichloromethane as eluent to obtain a red solid. Yield 25-36%;

[0049] (3) Add to a round bottom flask and add 2.1-10 equivalents of In a flask, dissolve it with 60 mL of anhydrous toluene. Add glacial acetic acid (1.0 mL), piperidine (1.2 mL) and a small amount of anhydrous magnesium perchlorate to the above solution and heat to 135 ° C. Reflux for 2 hours under nitrogen protection. The water generated during the reaction is removed by a Dean-stark device. After the reaction is completed, the solution is cooled to room temperature, and the toluene solvent is removed by decompression using a rotary evaporator. The residue is extracted three times with dichloromethane and a saturated sodium chloride solution, and the organic phase is dried with anhydrous sodium sulfate and concentrated. The crude product is purified by silica gel column chromatography using dichloromethane / petroleum ether (volume ratio 1:1) as eluent to separate the azobenzene-fluoroboron dipyrrole probe. The 2.1-10 equivalents Compound The yield is 22-31%.

[0050] The present invention is further described in the following examples, but the present invention is not limited thereto.

[0051] Example 1

[0052] The preparation of azobenzene-modified fluoroboron dipyrrole derivatives comprises the following specific steps:

[0053] (1) Dissolve benzyl alcohol (1.00 g, 7.99 mmol) in 30 mL of a mixed solvent (THF:H2O=1:1), add 3 M HCl solution (5 mL) to the above solution, and stir the reaction solution in an ice-water bath for 30 min. Continue to add NaNO2 (0.66 g, 9.59 mmol), p-aminobenzyl alcohol (1.10 g, 7.99 mmol), and sodium acetate (1.31 g, 15.98 mmol) to the reaction flask, and stir the resulting reaction solution at room temperature for 18 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, and purify the residue by silica gel column chromatography using dichloromethane / methanol (50:1, v / v) as the eluent to obtain a yellow solid. (0.29g, 31%).

[0054] 1 H NMR (400MHz, DMSO-d6): δ7.87(d,J=8.0Hz,4H,ArH), 7.54(d,J=8.0Hz,4H,ArH), 5.40(t,J=5.6Hz,2H,OH), 4,61(d,J=6.0Hz,4H,CH2).

[0055] (2) The yellow solid (0.50 g, 0.65 mmol) and Dess-Martin periodinane (0.23 g, 0.65 mmol) were dissolved in a 100 mL round-bottom flask with 50 mL of anhydrous tetrahydrofuran. The reaction solution was stirred at room temperature for 1 h. After the reaction was completed, the reaction solution was cooled to room temperature and the THF solvent was removed under reduced pressure using a rotary evaporator. The crude product was purified by silica gel column chromatography using dichloromethane / ethyl acetate (3:1, v / v) as the eluent to obtain an orange-yellow solid (0.19g, 94%).

[0056] 1 H NMR (400MHz, CDCl3): δ10.11(s,1H,CHO),8.05(s,4H,ArH),7.98(d,J=7.6Hz,2H,ArH),7.56(d,J=8.0Hz,2H,ArH),4.83(s,2H,CH2).

[0057] (3) Weigh a certain amount of orange solid (0.84g, 5.26mmol) was added to a 250mL round-bottom flask, and a certain amount of freshly distilled dichloromethane was added to fully dissolve it, and then 2,4-dimethylpyrrole (1.03g, 10mmol) was added. A drop of trifluoroacetic acid was added, and the reaction was stirred overnight under nitrogen protection. Weigh 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) (1.19g, 5.26mmol) and dissolved in dichloromethane. The solid components were dissolved with an ultrasonic cleaner and then added to the reaction bottle and reacted for 4h. Under ice bath conditions, triethylamine (12mL) was added to the reaction bottle and stirred for 30min. Boron trifluoride·ether (12mL) was measured and transferred to a constant pressure dropping funnel and slowly added dropwise to the round-bottom flask. The reaction was stirred overnight under nitrogen protection. After the reaction is completed, the reaction solution is filtered with silica gel and transferred to a separatory funnel for extraction. The organic layer is extracted three times with an appropriate amount of saturated sodium bicarbonate aqueous solution and dichloromethane (50 mL × 3) and then collected and dried with anhydrous sodium sulfate, filtered, and spin-dried to obtain a crude product. The crude product is purified by silica gel column chromatography using petroleum ether: dichloromethane (1:1, v / v) as the eluent to obtain a red solid. (0.72g, 36%).

[0058] 1 H NMR (400MHz, CDCl3): δ8.06 (d, J=7.2Hz, 2H, ArH), 7.97 (d, J=6.8Hz, 2H, ArH), 7.55 (d, J=7.6Hz, 2H, ArH) ,7.47(d,J=6.8Hz,ArH),6.00(s,2H,pyrrole-H),4.82(s,2H,CH2),2.57(s,6H,CH3),1.46(s,6H,CH3).

[0059] (4) The red solid (0.20g, 0.33mmol), (0.89mg, 3.3mmol), glacial acetic acid (1.0mL), piperidine (1.2mL) and a small amount of anhydrous magnesium perchlorate were dissolved in 60mL of anhydrous toluene in a 100mL round-bottom flask and heated to 135°C. The mixture was refluxed for 2h under nitrogen protection. The water generated during the reaction was removed by a Dean-stark device. After the reaction, the solution was cooled to room temperature and the toluene solvent was removed by decompression using a rotary evaporator. The residue was extracted three times with dichloromethane and a saturated sodium chloride solution, and the organic phase was dried with anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography using dichloromethane / petroleum ether (1:1, v / v) as the eluent to obtain a blue solid, i.e., a fluoroboron dipyrrole derivative modified with azobenzene: (60mg, 31%).

[0060] 1H NMR (400MHz, CDCl3): δ8.08(d,J=7.6Hz,2H,ArH),7.98(d,J=7.2Hz,2H,ArH),7.62(d,J=16.0Hz,2H,CH=CH),7.5 8(d,J=8.0Hz,4H),7.56(d,J=8.4Hz,2H,ArH),7.52(d,J=8.4Hz,2H,ArH),7.22(d,J=16.0Hz,2H,CH=CH),6.95(d ,J=7.6Hz,4H,ArH),6.63(s,2H,pyrrole-H),4.83(s,2H,CH2),4.19(t,J=6.4Hz,4H,CH2),3.90(t,J=4.8Hz,4H, CH2),3.80-3.75(m.4H,CH2),3.73-3.65(m,8H,CH2),3.59-3.55(m,4H,CH2),3.39(s,6H,CH3),1.61(s,6H,CH3).

[0061] Application Example 1

[0062] (1) Cell culture: HepG2 cells and A549 cells with good growth status were selected and incubated in a cell culture flask with DMEM medium (containing 1% penicillin, streptomycin, and 10% fetal bovine serum). The cell culture flask was placed in a carbon dioxide cell culture incubator and cultured for 12 h until the cells were fully grown and well attached to the wall of the cell culture flask;

[0063] (2) Laying laser confocal dish: remove the old culture medium in the culture bottle, wash twice with PBS solution, add 1 mL of trypsin to digest the adherent cells, and place them in a 1.5 mL centrifuge tube. 5 The cells were plated at a density of 100 / mL in a laser confocal dish and cultured in a cell culture incubator for 12 h.

[0064] (3) Adding drugs: Prepare a cell culture medium solution of azobenzene-modified fluoroboron dipyrrole derivatives (concentration of 10 μM). Remove the old culture medium in the laser confocal dish, add the prepared nano drug solution to the laser confocal dish, and then place the laser confocal dish in normal oxygen (21% O2) or hypoxic (1% O2) conditions for further culture for 12 hours;

[0065] (4) Photography: The laser confocal microscopy dish was washed three times with PBS, 1 mL of serum-free and phenol red-free cell culture medium was added, and the laser confocal microscopy was used to scan and take pictures (probe: excitation wavelength is 633 nm, detection wavelength is 650-750 nm).

[0066] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A tumor hypoxia-specific responsive fluoroborate dipyrrole fluorescent probe, characterized in that: The fluorescent probe is an azobenzene-fluoroboron dipyrrole probe, and its chemical structure is:

2. A method for preparing the azobenzene-fluoroboron dipyrrole probe as claimed in claim 1, characterized in that: The steps include: Azobenzene-fluoroborane dipyrrole probe was synthesized as the starting material 3. The method according to claim 2, characterized in that The preparation method comprises the following steps: Add to a round bottom flask and add 2.1-10 equivalents of in a flask and dissolved with 60 mL of anhydrous toluene; 1.0 mL of glacial acetic acid, 1.2 mL of piperidine and a small amount of anhydrous magnesium perchlorate were added to the above solution and the temperature was raised to 135°C, and refluxed for 2 hours under nitrogen protection. The water generated during the reaction was removed by a Dean-stark device; after the reaction was completed, the solution was cooled to room temperature, and the toluene solvent was removed by a rotary evaporator under reduced pressure; the residue was extracted three times with dichloromethane and a saturated sodium chloride solution, and the organic phase was dried with anhydrous sodium sulfate and concentrated; the crude product was purified by silica gel column chromatography using dichloromethane / petroleum ether as eluent to obtain an azobenzene-fluoroboron dipyrrole probe The 2.1-10 equivalents Compound The molar measurement.

4. The method according to claim 2, characterized in that: The compound The preparation method comprises the following steps: S1, using benzyl alcohol, HCl solution, NaNO2, sodium acetate and p-aminobenzyl alcohol as starting materials, synthesize compound S2. Compound Dess-Martin oxidant was used as the starting material to synthesize the compound S3, compound 2,4-Dimethylpyrrole, trifluoroacetic acid, 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ, triethylamine and boron trifluoride etherate were used as starting materials to synthesize the compound 5. The method according to claim 4, characterized in that , the compound The preparation method comprises the following steps: S1. Dissolve benzyl alcohol in 30 mL of a mixed solvent of tetrahydrofuran and water in a volume ratio of 1:1, add 3M HCl solution to the above solution, and stir the reaction solution in an ice-water bath for 30 min; continue to add NaNO2, p-aminobenzyl alcohol, and sodium acetate in a molar ratio of 1-2:1:1.5-2.5 to the reaction flask, and stir the resulting reaction solution at room temperature for 18 h; after the reaction is completed, remove the solvent by vacuum rotary evaporation, and purify the residue by silica gel column chromatography using dichloromethane-methanol as eluent to obtain a yellow solid S2. Compound and Dess-Martin oxidant in a molar ratio of 1:0.5-1.2 were added to a reaction round-bottom flask and dissolved with anhydrous tetrahydrofuran. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was cooled to room temperature and the THF solvent was removed under reduced pressure using a rotary evaporator. The crude product was purified by silica gel column chromatography using dichloromethane-ethyl acetate as the eluent to obtain an orange-yellow solid. S3. Compound and 2,4-dimethylpyrrole in a molar ratio of 1:2-3 are added to a round-bottom flask, and fully dissolved with anhydrous dichloromethane; an appropriate amount of trifluoroacetic acid is added to the above solution, and the reaction is stirred overnight under nitrogen protection; 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ is weighed and added to the reaction bottle for 4 hours; under ice-water bath conditions, 12mL of triethylamine is added to the reaction bottle and stirred for 30min, then 12mL of boron trifluoride ether is measured and added to a constant pressure dropping funnel and slowly added dropwise to the reaction bottle; the reaction is stirred overnight under nitrogen protection; after the reaction is completed, the reaction solution is filtered with silica gel, transferred to a separating funnel for extraction, extracted 3 times with an appropriate amount of saturated sodium bicarbonate aqueous solution and dichloromethane, and then the organic layer is collected and dried with anhydrous sodium sulfate, and the crude product is spin-dried; the crude product is purified by silica gel column chromatography using petroleum ether-dichloromethane as eluent to obtain a red solid 6. Use of the tumor hypoxia-specific responsive fluoroborate dipyrrole fluorescent probe as claimed in claim 1 in cancer cell fluorescence detection.

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