Si rhodamine derivative for hypoxia imaging as well as preparation method and application of Si rhodamine derivative

Abstract

The invention discloses a Si rhodamine derivative for hypoxia imaging and a preparation method and application of the Si rhodamine derivative. The structural formula of the Si rhodamine derivative is # imgabs0 #. The invention further specifically discloses a preparation method of the Si rhodamine derivative for hypoxia imaging and application of the Si rhodamine derivative in imaging of hypoxia environment distribution in cells and cell hypoxia detection. According to the invention, Si rhodamine is used as a parent to prepare a fluorescent dye with a large conjugate structure and hypoxia marker targeting, namely the Si rhodamine derivative, and the Si rhodamine derivative has a symmetrical conjugate group julolidine, positive charges and a hypoxia targeting benzenesulfonamide group. The benzenesulfonamide structure contained in the dye can enable the dye to target hypoxia high-expression carbonic anhydrase, and detection and imaging of cell hypoxia distribution can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of fluorescent dyes and their biological imaging applications, and specifically relates to a Si rhodamine derivative (Si-Rh-CAIX) for hypoxia imaging and a preparation method and application thereof. Background Art

[0002] At present, cancer is one of the important factors threatening human health. In recent years, the incidence and mortality of cancer have increased year by year, and most cancers present a hypoxic microenvironment compared to normal tissues. By identifying the different degrees of hypoxia, it can be used as a basis for distinguishing normal or tumor cells. Fluorescence imaging has the ability to detect multiple molecular events and high spatiotemporal resolution, making it one of the most promising research methods for studying life activities and physical and chemical properties in complex organisms. Fluorescence microscopy can more accurately distinguish different processes or biological molecules. Combining imaging technology can better help people understand the occurrence and development of diseases, including further diagnosis and treatment of diseases. In this process, fluorescent dyes are needed as tools. In recent years, fluorescent dyes have become an important research direction due to their higher sensitivity, higher resolution, deep tomography, avoidance of fluorescence bleaching, avoidance of autofluorescence interference, and avoidance of tissue scattering. Since the discovery of rhodamine dyes in 1887, they have been widely used in biotechnology as fluorescent markers or small molecule detection due to their good optical properties, and have become a powerful tool for observing biological systems. Combined with microscopic imaging technology, it can illuminate the molecules or life processes that people want to see, achieving the purpose of detection and imaging in complex living organisms. In addition, the effectiveness of fluorescent dyes in disease diagnosis and treatment, especially hypoxia imaging, depends on the basic properties of fluorescent dyes, including wavelength, stability, photobleaching and other factors. Therefore, the development of Si rhodamine derivative fluorescent dyes with excellent performance will help people further understand the hypoxia distribution and development mechanism of diseases. Summary of the invention

[0003] The technical problem solved by the present invention is to provide a Si rhodamine derivative for hypoxia imaging and a preparation method thereof, wherein the method uses Si rhodamine as a matrix to prepare a fluorescent dye having a large conjugated structure and targeting a hypoxia marker, namely, a Si rhodamine derivative. The Si rhodamine derivative has a symmetrical conjugated group, julolidine, a positive charge, and a hypoxia-targeting benzenesulfonamide group, and the benzenesulfonamide structure contained in the derivative can enable the dye to target carbonic anhydrase, which is highly expressed in hypoxia, and can realize the detection and imaging of cellular hypoxia distribution. The Si rhodamine derivative provides an effective tool for imaging and evaluating cellular hypoxia.

[0004] The present invention adopts the following technical solution to solve the above technical problems: a Si rhodamine derivative for hypoxia imaging, characterized in that the Si rhodamine derivative is 9-({2-[4-(aminodioxy-6-thio)phenyl]ethyl}amino)-18,18-dimethyl-2,3,5,6,7,11,12,13,15,16,17,18-dodecahydro-1H-pyrido[3,2,1-ij]pyrido[3",2",1":8',1']quinolino[7',6':2,3]silanol[6,5-f]quinoline-4-cation, and its structure is shown in Formula VI:

[0005]

[0006] The compound represented by formula VI provided by the present invention, namely the Si rhodamine derivative, has good water solubility and pH stability in solution. Meanwhile, the Si rhodamine derivative itself has a longer emission wavelength, a larger Stokes shift, and good fluorescence luminescence.

[0007] The method for preparing the Si rhodamine derivative for hypoxia imaging of the present invention comprises the following specific steps:

[0008] Step S1, 3-bromoaniline and 1-bromo-3-chloropropane are mixed evenly, a catalyst is added, and then the mixture is refluxed at 120-150° C. to obtain a compound of formula I, a DMF solution is directly added without purification, and then the mixture is refluxed at 150-160° C. to obtain a compound of formula II, wherein the catalyst is one or more of potassium carbonate, sodium acetate, sodium carbonate or sodium bicarbonate, and the reaction process and the structural formulas of the compounds of formula I and formula II are as follows:

[0009]

[0010] Reaction process and structural formula I and II;

[0011] Step S2, dissolving the compound represented by formula II in acetic acid, adding formaldehyde solution, and reacting at 60-90° C. to obtain the compound represented by formula III. The reaction flow and the structural formula of the compound represented by formula III are:

[0012]

[0013] Reaction process and structural formula III;

[0014] Step S3, adding the compound represented by formula III to anhydrous THF, treating with sec-BuLi under -78°C argon environment, further stirring the obtained mixture at -78°C, treating with dichlorodimethylsilane, and then stirring and reacting at room temperature. After the reaction is completed, quenching with HCl solution, neutralizing with saturated NaHCO3 solution, extracting to obtain the compound represented by formula IV, directly adding acetone and KMnO4 without purification, and then stirring and reacting at -15°C to obtain the compound represented by formula V. The reaction process and the structural formulas of the compounds represented by formula IV and formula V are:

[0015]

[0016] Reaction process and structural formula IV and V;

[0017] Step S4, adding trifluoromethanesulfonic anhydride dropwise to an anhydrous CH2Cl2 solution of the compound represented by Formula V at 0°C, stirring the resulting green reaction mixture at room temperature for reaction, then adding 4-(2-aminoethyl)benzenesulfonamide, stirring the reaction mixture at room temperature for reaction, and after the reaction is completed, purifying the crude product on a silica gel column to obtain the compound represented by Formula VI, the reaction process and the structural formula of the compound represented by Formula VI are:

[0018]

[0019] Reaction scheme and structure of Formula VI.

[0020] Preferably, the molar ratio of 3-bromoaniline to 1-bromo-3-chloropropane in step S1 is 1:(6-9).

[0021] Preferably, the formaldehyde content in the formaldehyde solution in step S2 is 35wt%-45wt%.

[0022] Preferably, the molar ratio of the compound represented by formula IV to potassium permanganate in step S3 is 1:(1-3).

[0023] Preferably, in step S4, the molar ratio of the compound represented by formula V to 4-(2-aminoethyl)benzenesulfonamide is 1:(2-5).

[0024] The Si rhodamine derivative for hypoxia imaging of the present invention is used for cell imaging of the distribution of hypoxia environment in cells and cell hypoxia detection.

[0025] The Si rhodamine derivative for hypoxia imaging of the present invention exhibits different fluorescence intensities in Hela cells under different hypoxic environments.

[0026] The principle that the Si rhodamine derivatives for hypoxia imaging described in the present invention exhibit different fluorescence intensities in Hela cells for different hypoxic environments is achieved based on the different carbonic anhydrase contents, because carbonic anhydrase is a biological enzyme expressed by hypoxia, and the more severe the hypoxia, the higher the content and the stronger the fluorescence.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects: the Si rhodamine derivative prepared by the present invention for hypoxia imaging has good water-oil amphiphilicity and can enter cells; because it has a symmetrical conjugated group julolidine and a positive charge and a hypoxia-targeted benzenesulfonamide group, it can emit long wavelengths, and the distribution of the imaging cell hypoxia microenvironment and cell hypoxia detection can be achieved through the action of benzenesulfonamide and the hypoxia marker carbonic anhydrase. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the H NMR spectrum of the compound of formula II in Example 1.

[0029] Figure 2 This is the H NMR spectrum of the compound represented by Formula III in Example 1.

[0030] Figure 3 This is the H NMR spectrum of the compound represented by Formula V in Example 1.

[0031] Figure 4 This is the H NMR spectrum of the compound represented by Formula VI in Example 1.

[0032] Figure 5 It is the water-soluble Si-Rh-CAIX, a rhodamine derivative.

[0033] Figure 6 is the emission spectrum of the Si rhodamine derivative Si-Rh-CAIX.

[0034] Figure 7 is the pH stability of the Si-rhodamine derivative Si-Rh-CAIX.

[0035] Figure 8 is the efficiency of Si-Rh-CAIX, a Si-rhodamine derivative, entering cells.

[0036] Fig. 9 It is the toxicity of Si-Rh-CAIX, a Si-rhodamine derivative, in cells.

[0037] Fig.10 The Si-rhodamine derivative Si-Rh-CAIX was validated for hypoxia imaging in cells. DETAILED DESCRIPTION

[0038] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0039] The synthesis steps of Si-Rh-CAIX, a Si-rhodamine derivative for hypoxia imaging, are as follows:

[0040] Example 1

[0041] (1) Synthesis of the compound represented by formula II

[0042]

[0043] Reaction process and structural formula I and II

[0044] In a round-bottom flask, 3-bromoaniline (171 mg, 1 mmol), 1-bromo-3-chloropropane (1.25 g, 8 mmol) and Na2CO3 (336 mg, 4 mmol) were injected respectively, and the reaction was stirred at 140°C for 48 h. After the reaction solution was cooled to room temperature, it was transferred to a separatory funnel and extracted with CH2Cl2 (3×100 mL). The organic component was dried with Na2SO4, filtered, and concentrated under reduced pressure. The residual 1-bromo-3-chloropropane was removed under reduced pressure at 60°C. The crude dialkylated intermediate was dissolved in DMF (15 mL) and stirred at 160°C for 24 h. After cooling to room temperature, it was concentrated under reduced pressure, rinsed with water (200 mL), and extracted with CH2Cl2 (3×100 mL). The synthesized organic component was dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography on a silica gel column to obtain a light yellow oily compound, namely the compound shown in Formula II, with a yield of 65.1%. H NMR characterization: 1 H NMR (600MHz, CDCl3) δ6.89 (d, J = 8.0Hz, 1H), 6.75 (d, J = 8.0Hz, 1H), 3.26–3.19 (dt, 4H), 2.91 (t, J = 6.7Hz, 2H), 2.82 (t, J = 6.5Hz, 2H), 2.11–2.06 (m, 4H). The H NMR spectrum of the obtained product is shown in Figure 1 shown.

[0045] (2) Synthesis of the compound represented by formula III

[0046]

[0047] Reaction process and structural formula III

[0048] The compound of formula II (2.3 g, 9.2 mmol) was dissolved in acetic acid (35 mL), and formaldehyde solution (37 wt%, 10 mmol, 2.8 g) was added, and heated at 60°C for 30 min. The white solid was filtered and precipitated with acetic acid and methanol to obtain the compound of formula III, with a yield of 72%. Characterization by hydrogen nuclear magnetic resonance spectrum: 1 H NMR (600MHz,CDCl3)δ6.48(s,2H),3.96(s,2H),3.13–3.05(m,8H),2.84(t,J=6.7Hz,4H),2.65(t,J=6.6Hz,4H),2.07–1.90(m,8H). The H NMR spectrum of the obtained product is shown in Figure 2 shown.

[0049] (3) Synthesis of the compound represented by formula V

[0050]

[0051] Reaction process and structural formula IV and formula V

[0052] The compound represented by formula III (200 mg, 0.39 mmol) was dissolved in anhydrous THF (15 mL), sec-BuLi (1.4 M, 0.84 mL, 1.17 mmol) was added at -78 ° C, argon was introduced, and the resulting mixture was further stirred at -78 ° C for 2 h. The reaction mixture was treated with dichlorodimethylsilane (85 μL, 0.7 mmol) at -78 ° C, then stirred at room temperature for 3 h, quenched with 1N HCl, then neutralized with saturated NaHCO3 solution and extracted with dichloromethane (2×10 mL), the organic layer was washed with brine, and then dried over anhydrous Na2SO4. The crude product obtained by evaporation of the organic solvent was used in the next step without further purification. Subsequently, KMnO4 (185 mg, 1.17 mmol) and the compound obtained above were dissolved in acetone (15 mL) at -15 ° C, and the resulting mixture was stirred at -15 ° C for 2.5 h. The reaction mixture was diluted with dichloromethane (15 mL), then filtered, the filtrate was concentrated, and the residue was purified by column chromatography to obtain the compound represented by Formula IV with a yield of 20%. Characterization by hydrogen nuclear magnetic resonance spectrum: 1 H NMR (600MHz, CDCl3) δ8.06 (s, 2H), 3.36–3.22 (m, 8H), 2.95–2.77 (m, 8H), 2.03–1.99 (m, 8H), 0.59 (s, 6H). The H NMR spectrum of the obtained product is shown in Figure 3 shown.

[0053] (4) Synthesis of the compound represented by formula VI

[0054]

[0055] Reaction process and structure of formula VI

[0056] Trifluoromethanesulfonic anhydride (12 μL, 0.075 mmol) was added dropwise to a solution of the compound of formula V (20 mg, 0.047 mmol) in anhydrous CH2Cl2 (5 mL) at 0°C, and the resulting green reaction mixture was stirred at room temperature for 20 min, and then 4-(2-aminoethyl)benzenesulfonamide (36.74 mg, 0.141 mmol) was added, and the reaction mixture was further stirred at room temperature for 4 h. After the reaction, the crude product was purified on a silica gel column to obtain a dark red solid fluorescent dye Si rhodamine derivative Si-Rh-CAIX, i.e., the compound of formula VI, with a yield of 35%. Characterization by hydrogen nuclear magnetic resonance spectrum 1 H NMR (600MHz, MeOD) δ7.73 (s, 2H), 7.22 (s, 4H), 4.14 (s, 2H), 3.38 (s, 8H), 3.11 (s, 2H), 2.82 (d, J = 22.7 Hz, 8H), 1.98 (s, 8H), 0.49 (s, 6H). The H NMR spectrum of the obtained product is shown in Figure 4 shown.

[0057] Example 2

[0058] (1) Synthesis of the compound represented by formula II

[0059]

[0060] Reaction process and structural formula I and II

[0061] In a round-bottom flask, 3-bromoaniline (171 mg, 1 mmol), 1-bromo-3-chloropropane (0.94 g, 6 mmol) and Na2CO3 (336 mg, 4 mmol) were injected respectively. The reaction was stirred at 140°C for 48 h. After the reaction solution was cooled to room temperature, it was transferred to a separatory funnel and extracted with CH2Cl2 (3×100 mL). The organic component was dried with Na2SO4, filtered, and concentrated under reduced pressure. The residual 1-bromo-3-chloropropane was removed under reduced pressure at 60°C. The crude dialkylated intermediate was dissolved in DMF (15 mL) and stirred at 160°C for 24 h. After cooling to room temperature, it was concentrated under reduced pressure, rinsed with water (200 mL), and extracted with CH2Cl2 (3×100 mL). The synthesized organic component was dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography on a silica gel column to obtain a light yellow oily compound, namely the compound of formula II, with a yield of 57.4%.

[0062] (2) Synthesis of the compound represented by formula III

[0063]

[0064] Reaction process and structural formula III

[0065] The compound of formula II (2.3 g, 9.2 mmol) was dissolved in acetic acid (35 mL), and formaldehyde solution (45 wt%, 10 mmol, 2.8 g) was added, and heated at 60°C for 30 min. The precipitate was filtered with acetic acid and methanol to obtain a white solid, namely the compound of formula III, with a yield of 72%.

[0066] (3) Synthesis of the compound represented by formula V

[0067]

[0068] Reaction process and structural formula IV and formula V

[0069] The compound of formula III (200 mg, 0.39 mmol) was dissolved in anhydrous THF (15 mL), sec-BuLi (1.4 M, 0.84 mL, 1.17 mmol) was added at -78 ° C, argon was introduced, and the resulting mixture was further stirred at -78 ° C for 2 h. The reaction mixture was treated with dichlorodimethylsilane (85 μL, 0.7 mmol) at -78 ° C, then stirred at room temperature for 3 h, quenched with 1N HCl, then neutralized with saturated NaHCO3 solution and extracted with dichloromethane (2×10 mL), the organic layer was washed with brine, and then dried over anhydrous Na2SO4. The crude product obtained by evaporation of the organic solvent was used in the next step without further purification. Subsequently, KMnO4 (101 mg, 0.64 mmol) and the compound obtained above were dissolved in acetone (15 mL) at -15 ° C, and the resulting mixture was stirred at -15 ° C for 2.5 h. The reaction mixture was diluted with dichloromethane (15 mL), then filtered, the filtrate was concentrated, and the residue was purified by column chromatography to obtain the compound represented by Formula IV with a yield of 14%.

[0070] (4) Synthesis of the compound represented by formula VI

[0071]

[0072] Reaction process and structure of formula VI

[0073] Trifluoromethanesulfonic anhydride (12 μL, 0.075 mmol) was added dropwise to a solution of the compound of formula V (20 mg, 0.047 mmol) in anhydrous CH2Cl2 (5 mL) at 0°C, and the resulting green reaction mixture was stirred at room temperature for 20 min. Then 4-(2-aminoethyl)benzenesulfonamide (13.0 mg, 0.05 mmol) was added, and the reaction mixture was further stirred at room temperature for 4 h. After the reaction, the crude product was purified on a silica gel column to obtain a deep red solid fluorescent dye Si rhodamine derivative Si-Rh-CAIX, i.e., the compound of formula III, with a yield of 26%.

[0074] Example 3

[0075] Test on the water solubility of Si-Rh-CAIX, a derivative of Si-rhodamine

[0076] Take the Si-Rh-CAIX synthesized in Example 1 and add it gradually to 3 mL of H2O (high purity water) according to a certain concentration gradient, and ensure that the final volume of DMSO added is less than one thousandth of the total volume. Test its absorption spectrum, the result is as follows Figure 5 The results show that the Si-Rh-CAIX derivative has good water solubility, which enables it to enter cells.

[0077] Example 4

[0078] Emission Spectrum Detection Experiment of Si-Rh-CAIX, a Si-rhodamine Derivative

[0079] Add 3 mL of different solvents (dichloromethane, methanol, DMSO, ethyl acetate, water, anhydrous ethanol, acetonitrile, DMF) to the cuvette, add Si-Rh-CAIX mother solution of Si rhodamine derivative to the above solution, and keep the final concentration at 6.0 μM. Use fluorescence spectrometer to obtain fluorescence emission spectrum. The results are as follows: Figure 6 The results show that the Si-Rh-CAIX derivative has good long-wavelength emission and strong fluorescence signals in different solvents.

[0080] Example 5

[0081] pH Stability Test of Si-Rh-CAIX, a Si-rhodamine Derivative

[0082] In this experiment, HCl solution and NaOH solution were used as acid-base regulators to adjust the pH of the solution to obtain a solution of the corresponding pH. The Si-Rh-CAIX derivative was added to H2O (high-purity water) to prepare a 6.0 μM solution. Then, the fluorescence emission spectrum of the Si-Rh-CAIX derivative was tested in the pH range of 2 to 12 under the calibration of the pH meter. The results are as follows: Figure 7 The results show that the Si-Rh-CAIX derivative has good stability in the physiological pH range of 4 to 9, which is beneficial to the accuracy of the imaging results.

[0083] Example 6

[0084] Detection of the efficiency of Si-Rh-CAIX entering cells

[0085] Hela cells were used as the research object. After the cells were cultured, Si-Rh-CAIX, a derivative of Si rhodamine, was added to maintain a final concentration of 2.0 μM. Then, a confocal microscope was used for imaging, with a laser wavelength of 488 nm and a fluorescence wavelength range of 620 to 660 nm. Fluorescence images were collected at 2 min, 7 min, and 10 min after the addition of Si rhodamine derivative Si-Rh-CAIX. The imaging results are shown in Figure 2. Figure 8 The results show that the Si-Rh-CAIX derivative begins to enter the cell at 2 minutes and basically maintains the same level at 10 minutes. It enters the cell very quickly, which is beneficial for imaging and biological applications.

[0086] Example 7

[0087] Cell viability test of Si-Rh-CAIX, a Si-rhodamine derivative

[0088] Hela cells and 3A cells were used as the research objects. The cell survival rate was used to characterize the cytotoxicity of Si-Rh-CAIX derivative. 4 100 μL of cells were inoculated in a 96-well plate and cultured at 37°C and 5.0 wt% / vol CO2 for 24 hours. Then, Si-Rh-CAIX solution was added to some wells on one side of the 96-well plate according to a certain concentration gradient (0.6 μM, 0.8 μM, 1 μM, 1.5 μM, 2 μM), which were the damaged wells; the rest were blank wells without any addition. After incubation for 24 hours, 20 μL of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and cultured at 37°C and 5% CO2 for 4 hours. Then the original culture medium was removed and DMSO (150 μL / well) was added, and the cell survival rate was detected. The results are as follows. Fig. 9 The results showed that the Si-Rh-CAIX derivative had little toxicity to Hela and 3A cells and could be used in vivo.

[0089] Example 8

[0090] Imaging test of cell hypoxia using Si-Rh-CAIX, a Si-rhodamine derivative

[0091] Hela cells were used as the research object. The nutrient solution used for cell culture in this experiment was obtained by adding 10wt% fetal bovine serum and 1wt% trypsin to the DMEM basic nutrient solution, and cultured at 37°C in an incubator containing 5.0wt% / vol CO2. After the cell culture is completed, digestion and subculture are performed, and some cells are placed in a confocal culture dish for culture (divided into three groups). After the cells adhere to the wall, one group is cultured normally and the other group is incubated in hypoxia. Then, the Si rhodamine derivative Si-Rh-CAIX is added, and the final concentration is maintained at 2μM. After incubation in the incubator, fluorescence imaging is performed using a confocal imaging microscope. The results are shown as follows. Fig.10 The results show that Si-Rh-CAIX, a derivative of Si-rhodamine, exhibits a strong fluorescence intensity in cells under hypoxia, and a weak fluorescence intensity in normoxia, which can be well used for cell hypoxia degree detection and hypoxia distribution imaging.

[0092] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A Si-rhodamine derivative for hypoxia imaging, characterized in that: The Si rhodamine derivative is 9-({2-[4-(aminodioxy-6-thio)phenyl]ethyl}amino)-18,18-dimethyl-2,3,5,6,7,11,12,13,15,16,17,18-dodecahydro-1H-pyrido[3,2,1-ij]pyrido[3",2",1":8',1']quinolino[7',6':2,3]silanol[6,5-f]quinoline-4-cation, and its structure is shown in Formula VI:

2. A method for preparing a Si-rhodamine derivative for hypoxia imaging according to claim 1, characterized in that The specific steps are: Step S1, 3-bromoaniline and 1-bromo-3-chloropropane are mixed evenly, a catalyst is added, and then the mixture is refluxed at 120-150° C. to obtain a compound of formula I, a DMF solution is directly added without purification, and then the mixture is refluxed at 150-160° C. to obtain a compound of formula II, wherein the catalyst is one or more of potassium carbonate, sodium acetate, sodium carbonate or sodium bicarbonate, and the reaction process and the structural formulas of the compounds of formula I and formula II are as follows: Step S2, dissolving the compound represented by formula II in acetic acid, adding formaldehyde solution, and reacting at 60-90° C. to obtain the compound represented by formula III. The reaction flow and the structural formula of the compound represented by formula III are: Step S3, adding the compound represented by formula III to anhydrous THF, treating with sec-BuLi under -78°C argon environment, further stirring the obtained mixture at -78°C, treating with dichlorodimethylsilane, and then stirring and reacting at room temperature. After the reaction is completed, quenching with HCl solution, neutralizing with saturated NaHCO3 solution, extracting to obtain the compound represented by formula IV, directly adding acetone and KMnO4 without purification, and then stirring and reacting at -15°C to obtain the compound represented by formula V. The reaction process and the structural formulas of the compounds represented by formula IV and formula V are: Step S4, adding trifluoromethanesulfonic anhydride dropwise to an anhydrous CH2Cl2 solution of the compound represented by Formula V at 0°C, stirring the resulting green reaction mixture at room temperature for reaction, then adding 4-(2-aminoethyl)benzenesulfonamide, stirring the reaction mixture at room temperature for reaction, and after the reaction is completed, purifying the crude product on a silica gel column to obtain the compound represented by Formula VI, the reaction process and the structural formula of the compound represented by Formula VI are:

3. The method for preparing Si rhodamine derivatives for hypoxia imaging according to claim 2, characterized in that: The molar ratio of 3-bromoaniline to 1-bromo-3-chloropropane in step S1 is 1:(6-9).

4. The method for preparing Si rhodamine derivatives for hypoxia imaging according to claim 2, characterized in that: The formaldehyde content in the formaldehyde solution in step S2 is 35wt%-45wt%.

5. The method for preparing Si rhodamine derivatives for hypoxia imaging according to claim 2, characterized in that: In step S3, the molar ratio of the compound represented by formula IV to potassium permanganate is 1:(1-3).

6. The method for preparing Si rhodamine derivatives for hypoxia imaging according to claim 2, characterized in that: In step S4, the molar ratio of the compound represented by formula V to 4-(2-aminoethyl)benzenesulfonamide is 1:(2-5).

7. Use of the Si rhodamine derivative for hypoxia imaging according to claim 1 in preparing a cell imaging of the distribution of hypoxia environment in cells and a cell hypoxia detection preparation.

8. The use according to claim 7, characterized in that: The Si rhodamine derivative can display different fluorescence intensity imaging in different hypoxic environments of cells, thereby realizing the evaluation of the degree of hypoxia. The more severe the hypoxia, the higher the content, and the stronger the fluorescence.

9. The use according to claim 8, characterized in that: The cells are Hela cells.

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