A cyanine dye derivative, and a method for synthesizing and using the same

Abstract

The application provides a flower cation dye derivative, a synthesis method and application of the derivative, and the specific Chinese name of the derivative is 2-((E)-2-(E)-2-(4-((3-((1-(13,16-dioxo-16-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)-3,6,9-trioxa-12-azahexadecyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-((5-nitrofuran-2-yl)methyl)benzyl)oxy)carbonyl)piperazin-1-yl)-3-(2-(E)-1,3-trimethylene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl. The application also provides a synthesis method of the derivative and a method for enhancing the uptake of probes by cancer cells based on the flower cation dye derivative, wherein the concentration change of nitroreductase is qualitatively detected by a fluorescence spectrophotometer in a phosphate buffer solution (pH=7.3). The method realizes the detection of hypoxia in cancer cells and the efficient uptake of probes by cancer cells.

Description

Technical Field

[0001] This invention relates to hypoxia-activated near-infrared fluorescent probes, specifically to a cyanine dye derivative, its synthesis method, and its applications. Background Technology

[0002] Oxygen is essential for the cellular activities of aerobic organisms; healthy cells require high levels of oxygen to maintain their vital functions. However, hypoxia occurs in certain tissues under certain conditions, resulting in insufficient oxygen supply within the body. Hypoxia is a key characteristic of tumors; tumor hypoxia refers to solid tumor regions characterized by low oxygen levels. In some solid tumors, oxygen concentrations can be ~4%, and locally even drop to 0%. Furthermore, tumor hypoxia can lead to an aggressive phenotype, promote malignancy, and increase resistance to standard treatments such as cytotoxic chemotherapy and radiotherapy. Therefore, utilizing the characteristics of tumor hypoxia to identify tumor tissue and assess the degree of tumor hypoxia is crucial. Small molecule fluorescent probes, with their excellent biocompatibility, in-situ dynamic labeling, and high spatiotemporal resolution imaging, have been widely used for real-time in-situ fluorescence imaging analysis of biological samples. This demonstrates significant application potential in early disease diagnosis and surgical navigation.

[0003] In recent years, many small-molecule fluorescent probes designed using nitroreductase, which is highly expressed under tumor hypoxia, have been developed for specific activation. However, their application is limited by poor reaction rates and poor targeting ability to cancer cells. This presents higher requirements and challenges for developing small-molecule fluorescent probes that are rapidly activated under hypoxia and efficiently absorbed by cancer cells. Summary of the Invention

[0004] The purpose of this invention is to provide a cyanine dye derivative, Az-Fu-Cy, and its synthesis method, as well as the application of Az-Fu-Cy in cancer cells for effective absorption and hypoxia detection.

[0005] This invention provides a cyanine dye derivative, Az-Fu-Cy, with the Chinese name 2-((E)-2-(E)-2-(4-((3-((1-(13,16-dioxo-16-((5-aminosulfonyl-1,3,4-thiadiazol-2-yl)amino)-3,6,9-trioxa-12-azahexadecyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-((5-nitrofuran-2-yl)methyl)benzyl)oxy)carbonyl)piperazin-1-yl)-3-(2-(E)-1,3-trimethylene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl The base, with the English name 2-((E)-2-((E)-2-(4-(((3-methoxy-4-((5-nitrofuran-2-yl)methoxy)benzyl)oxy)carbonyl)piperazin-1-yl)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium, has the following structural formula:

[0006]

[0007] The synthesis method of Az-Fu-Cy involves the following steps:

[0008] 1) Add 4.94 mL of concentrated HCl to an ethanolic solution of acetazolamide suspension. Reflux the reaction mixture for 4 hours to evaporate the solvent; slowly pour saturated sodium bicarbonate into the reaction mixture. Extract the compound with ethyl acetate. Wash the ethyl acetate layer with brine. Dry the residue with anhydrous sodium sulfate. Filter the reaction mixture and concentrate to give compound 1;

[0009] 2) Compound 1 and succinic anhydride were suspended in acetonitrile at a molar ratio of 1:3. The mixture was stirred at 70°C for 16 h, filtered, and the filter cake was washed with acetonitrile. The mixture was dried under vacuum to obtain compound 2 as a white solid.

[0010] 3) Compound 2 (1 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1 eq), and N,N-diisopropylethylamine (1 eq) were mixed in DMF and stirred at room temperature for 20 minutes. Then, compound 1 (1 eq) and triethylamine (2 eq) were added at room temperature and stirred overnight. After removing volatiles under reduced pressure, the crude product was purified by chromatography using dichloromethane and methanol as eluents to give white compound 3.

[0011] 4) In a dry, double-necked flask with a nitrogen inlet, sodium hydride (2 equivalents) was added in small portions to anhydrous N,N-dimethylformamide (50 mL). The mixture was stirred and cooled to 0°C. Anhydrous N,N-dimethylformamide (60 mL) containing 3,4-dihydroxybenzaldehyde (1 equivalent) was then added dropwise to the flask. The reaction mixture was stirred at room temperature for 30 minutes, and then bromopropyne (1 equivalent) was added dropwise via syringe, with stirring overnight at room temperature. The mixture was poured onto ice, neutralized with 1 M hydrochloric acid, and the product was extracted with ethyl acetate. The combined organic extract was concentrated to approximately 250 mL, washed with brine, and dried over anhydrous sodium sulfate. The solvent was removed under vacuum after filtration to give a crude product as a brown powder. Chromatography on a silica gel column with an eluent of ethyl acetate / petroleum ether / acetic acid yielded compound 4.

[0012] 5) A mixture of compound 4 (1 equivalent), (5-nitrofuran-2-yl)methanol (1 equivalent), and 1,1'-(azodicarbonyl)bispyridine (1 equivalent) was dissolved in 10 mL of dry tetrahydrofuran in a two-necked round-bottom flask under argon atmosphere. Tributylphosphine (1 equivalent) dissolved in 3 mL of tetrahydrofuran was added, and the mixture was stirred at room temperature for 48 h. At the end of the reaction, a certain amount of distilled water was added to the residue. The product was then extracted from the aqueous layer with ethyl acetate; the organic layer was washed with brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified on silica gel with petroleum ether and dichloromethane to give compound 5, which was pale yellow.

[0013] 6) A solution of compound 5 (1 equivalent) was added to 20 mL of methanol, and sodium borohydride (1.5 equivalent) was added at 0 °C. The mixture was stirred at room temperature for 2 h, and the reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate. After removing volatiles under reduced pressure, an orange solid, compound 6, was obtained, which could be used without further purification.

[0014] 7) 2,3,3-Trimethylbenzoindole (1 equivalent) and iodomethane (1 equivalent) were dissolved in 40 mL of toluene; the reaction was stirred at 100 °C for 20 hours and then cooled to room temperature. The product was filtered, washed with ether, and dried to give a pale pink compound 7.

[0015] 8) N,N-dimethylformamide (10 mL) and dichloromethane (10 mL) were mixed in an ice bath, and then phosphorus oxychloride (10 mL) was added dropwise to the above solution. After stirring for 30 minutes, 2.5 g of cyclohexanone was added and the mixture was refluxed at 80 °C. After 6 hours, the mixture was poured into ice water, and the precipitate was filtered to give compound 8;

[0016] 9) Compound 7 (2 equivalents), compound 8 (1 equivalent), and potassium acetate (2 equivalents) were added to 40 mL of acetic anhydride in a flask. The mixture was heated to 70°C and stirred for 0.5 h to obtain a green solution. The mixture was poured into a large volume of ice water and filtered to obtain compound 9;

[0017] 10) Anhydrous piperazine (10 equivalents) was added to compound 9 (1 equivalent) and dissolved in anhydrous N,N-dimethylformamide (10 mL). The mixture was stirred. The reaction was carried out at 80 °C under a nitrogen atmosphere for 2 hours, and then concentrated. The residue was purified by silica gel chromatography with dichloromethane and methanol to give compound 10;

[0018] 11) Triethylamine (2 equivalents) was added to a solution of 4-nitrobenzene chloroformate (1 equivalent) containing 3 mL of anhydrous tetrahydrofuran at 0 °C. After 20 minutes, compound 6 (0.86 equivalents) was slowly added to a solution in 4 mL of anhydrous dichloromethane, and the mixture was stirred overnight at room temperature. Dichloromethane was added to the mixture, and the resulting solution was washed with brine. The organic solvent was removed under reduced pressure. Compound 11 was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluents.

[0019] 12) Compound 10 (1 equivalent) and Compound 11 (1 equivalent), triethylamine (2 equivalents), and a catalytic amount of dimethylaminopyridine were added to 4 mL of anhydrous dichloromethane. The mixture was stirred at room temperature for 8 hours, and then the solvent was removed under reduced pressure. The crude product was purified by column chromatography using dichloromethane and methanol as eluents to obtain Compound 12.

[0020] 13) Compound 12 (1 equivalent) and Compound 3 (1 equivalent) were mixed with sodium ascorbate (0.1 equivalent) and stirred at room temperature for 15 minutes in a tetrahydrofuran:N,N-dimethylformamide (0.5:0.5 mL). 0.05 equivalents of CuSO4 dissolved in water were added to the above solution, and the mixture was stirred overnight. After the reaction was complete, an appropriate amount of water was added to the residue, and the product was extracted with dichloromethane. The organic layer was then washed with brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by column chromatography using dichloromethane and methanol as eluents to obtain Az-Fu-Cy.

[0021] The synthesized cyanine dye derivative Az-Fu-Cy can be used for rapid and specific qualitative fluorescence detection of nitroreductase in vitro, as well as for its efficient uptake and hypoxia detection in cancer cells.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The cyanine dye derivative Az-Fu-Cy of this invention offers a simple detection method, enabling in vitro qualitative and quantitative detection of nitroreductase using fluorescence spectroscopy. The effective integration of rapid hypoxia activation, near-infrared emission, and efficient uptake by cancer cells was verified by spectroscopy and confocal microscopy.

[0024] 2. The probe Az-Fu-Cy was used to achieve a rapid response to hypoxia and effective absorption of the probe by cancer cells. Attached Figure Description

[0025] Figure 1 Az-Fu-Cy 1H NMR spectrum

[0026] Figure 2 Az-Fu-Cy carbon NMR spectrum

[0027] Figure 3 Az-Fu-Cy mass spectrum

[0028] Figure 4 Fluorescence spectral changes of the reaction between Az-Fu-Cy and nitroreductase

[0029] Figure 5 Figure 1. Fluorescence intensity change over time in the reaction of Az-Fu-Cy with nitroreductase

[0030] Figure 6 Linear relationship between fluorescence intensity and nitroreductase concentration after the reaction of nitroreductase with Az-Fu-Cy.

[0031] Figure 7 Fluorescence imaging after Az-Fu-Cy and compound 12 were incubated in cells for 15 minutes. Detailed Implementation

[0032] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0033] Example 1

[0034] Preparation and characterization of Az-Fu-Cy:

[0035]

[0036] 1) Add 4.94 mL of concentrated HCl to an ethanolic solution of acetazolamide suspension (5 g, 22.00 mmol). Reflux the reaction mixture for 4 hours and evaporate the solvent. Slowly pour saturated NaHCO3 into the reaction mixture. Extract the compound with ethyl acetate. Wash the ethyl acetate layer with brine. Dry the residue with anhydrous Na2SO4. Filter the reaction mixture and concentrate to give compound 1;

[0037] 2) Compound 1 (12.5 g, 13.9 mmol) and succinic anhydride (4.17 g, 41.7 mmol) were suspended in MeCN. The mixture was stirred at 70 °C for 16 h, filtered, and the filter cake was washed with MeCN. The mixture was dried under vacuum to give compound 2 as a white solid (60% yield).

[0038] 3) Compound 2 (2.48 g, 8.86 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.37 g, 8.86 mmol), and N,N-diisopropylethylamine (1.54 mL, 8.86 mmol) were mixed in 7 mL of DMF and stirred at room temperature for 20 min. Then, compound 1 (2.13 g, 9.74 mmol) and triethylamine (2.47 mL, 17.72 mmol) were added at room temperature and stirred overnight. After removing volatiles under reduced pressure, the crude product was purified by column chromatography using dichloromethane and methanol (v / v, 20:1) as eluent to give white compound 3 (yield 35%).

[0039] 4) In a dry double-necked flask with a nitrogen inlet, NaH (2.4 g, 100 mmol) was added in small portions to anhydrous DMF (50 mL). The mixture was stirred and cooled to 0 °C. Anhydrous DMF (60 mL) containing 3,4-dihydroxybenzaldehyde (6.9 g, 50 mmol) was added dropwise to the flask. The reaction mixture was stirred at room temperature for 30 minutes, then bromopropyne (5 mL, 50 mmol) was added dropwise via syringe, and the mixture was stirred overnight at room temperature. The mixture was poured onto ice, neutralized with 1 M hydrochloric acid, and the product was extracted with ethyl acetate. The combined organic extract was concentrated to approximately 150 mL, washed with brine, and dried over anhydrous sodium sulfate. The solvent was removed under vacuum after filtration to give a crude product as a brown powder. Chromatography on a silica gel column with an ethyl acetate / petroleum ether (v / v, 1:4) eluent yielded compound 4 (50% yield).

[0040] 5) A mixture of 4 (848 mg, 4.8 mmol), (5-nitrofuran-2-yl)methanol (688 mg, 4.8 mmol), and 1,1'-(azodicarbonyl)bispyridine (1.21 g, 4.8 mmol) was dissolved in 10 mL of dry tetrahydrofuran in a double-necked round-bottom flask under argon atmosphere. Tributylphosphine (971 mg, 4.8 mmol) dissolved in 3 mL of THF was added, and the mixture was stirred at room temperature for 48 h. At the end of the reaction, a certain amount of distilled water was added to the residue. The product was then extracted from the aqueous layer with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified on silica gel with petroleum ether and dichloromethane (v / v, 2:1) to give compound 5 (40% yield).

[0041] 6) Add a solution of 5 (300 mg, 1 mmol) to 20 mL of methanol, and add sodium borohydride (56 mg, 1.5 mmol) at 0 °C. Stir at room temperature for 2 h, extract the reaction mixture with ethyl acetate and water, and dry the organic layer with NaSO4. After removing volatiles under reduced pressure, give an orange solid compound 6, which can be used without further purification (yield 80%).

[0042] 7) 2,3,3-Trimethylbenzoindole (5.25 g, 25.0 mmol) and iodomethane (5.25 g, 25.0 mmol) were dissolved in 40 mL of toluene. The reaction was stirred at 100 °C for 20 hours and then cooled to room temperature. The product was filtered, washed with diethyl ether, and dried to give a pale pink compound 7 (yield 80%).

[0043] 8) N,N-dimethylformamide (10 mL) and dichloromethane (10 mL) were mixed in an ice bath, and then POCl3 (10 mL) was added dropwise to the above solution. After stirring for 30 min, 2.5 g of cyclohexanone was added and the mixture was refluxed at 80 °C. After 6 hours, the mixture was poured into ice water, and the precipitate was filtered to give compound 8 (yield 80%).

[0044] 9) Compounds 7 (7.3 g, 20 mmol), 8 (1.7 g, 10 mmol), and potassium acetate (2.0 g, 20 mmol) were added to 40 mL of acetic anhydride in a flask. The mixture was heated to 70°C and stirred for 0.5 h to give a green solution. The mixture was poured into a large volume of ice water and filtered to give compound 9 (75% yield).

[0045] 10) Anhydrous piperazine (2.0 mmol) was added to compound 9 (0.2 mmol) and dissolved in anhydrous DMF (10 mL). The mixture was stirred. The reaction was carried out at 80 °C under a nitrogen atmosphere for 2 hours, and then concentrated. The residue was purified by silica gel chromatography with dichloromethane and methanol (v / v, 40:1) to give compound 10 (60% yield).

[0046] 11) Triethylamine (0.14 mL, 1 mmol) was added to a solution of 4-nitrobenzene chloroformate (110 mg, 0.5 mmol) containing 3 mL of anhydrous tetrahydrofuran at 0 °C. After 20 min, the solution of compound 6 (130 mg, 0.43 mmol) in anhydrous DCM was slowly added, and the mixture was stirred overnight at room temperature. DCM was added to the mixture, and the resulting solution was washed with brine. The organic solvent was removed under reduced pressure. Compound 11 was purified by silica gel column chromatography using petroleum ether ethyl acetate (v / v, 4:1) as eluent to obtain a 35% yield.

[0047] 12) Compounds 10 (24 mg, 0.05 mmol) and 11 (35 mg, 0.05 mmol), triethylamine (14 μL, 0.1 mmol), and a catalytic amount of dimethylaminopyridine were added to 4 mL of anhydrous dichloromethane. The mixture was stirred at room temperature for 8 hours, and then the solvent was removed under reduced pressure. The crude product was purified by column chromatography using dichloromethane and methanol (v / v, 50:1) as eluent to give compound 12 (60% yield).

[0048] 13) Compound 12 (49 mg, 0.05 mmol) and compound 3 (24 mg, 0.05 mmol) were mixed with sodium ascorbate (10 mol%) and stirred at room temperature for 15 min in a tetrahydrofuran:N,N-dimethylformamide (0.5:0.5 mL) solution. 0.05 equivalents of CuSO4 dissolved in water were added to the above solution, and the mixture was stirred overnight. After the reaction was complete, an appropriate amount of water was added to the residue, and the product was extracted with dichloromethane. The organic layer was then washed with brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by column chromatography using dichloromethane and methanol (v / v, 10:1) as eluent to obtain Az-Fu-Cy (yield 20%). 1 H NMR (600MHz, DMSO-d6) δ8.18 (s, 1H), 7.98 (t, J = 5.4Hz, 1H), 7.69 (s, 1H), 7. 68–7.64(m,2H),7.49(d,J=7.3Hz,2H),7.37–7.32(m,3H),7.27(d,J=8.0Hz ,2H),7.13(t,J=6.9Hz,2H),6.99(d,J=8.2Hz,1H),6.93(d,J=3.7Hz,1H),5 .95(d,J=13.6Hz,2H),5.19(d,J=15.9Hz,3H),5.12(s,2H),4.50(t,J=5.2H z,2H),3.79(t,J=5.2Hz,2H),3.67(d,J=55.6Hz,8H),3.54(s,6H),3.49–3. 46(m,2H),3.43(s,6H),3.15(dd,J=11.4,5.7Hz,2H),2.68(t,J=6.8Hz,2H) ,2.60(d,J=18.0Hz,1H),2.52(s,3H),2.45(t,J=7.0Hz,2H),2.04–1.96(m, 1H),1.78–1.73(m,2H),1.58(s,12H),1.23(s,5H),0.85(t,J=6.9Hz,1H).( Figure 1 ) 13CNMR(151MHz,DMSO-d6)δ171.46,171.35,170.06,154.91,154.65,152.10,148.43,147.34, 143.66,142.83,141.13,140.68,131.02,130.12,128.73,125.60,124.74,123.92,122.59,1 21.39,115.35,114.63,114.50,114.06,110.44,97.44,70.11,70.02,69.97,69.93,69.50,69.09,66.98,62.88,62.20,54.07,49.87,48.16,31.22,28.50,27.01,24.88,22.57,21.81. Figure 2 ESI-MS: [M] + Calcd.For1342.5384,Found 1342.5383( Figure 3 ).

[0049] Example 2

[0050] Take 5 μL of the probe stock solution and 2.0 mL of the test system into a cuvette, add 20 μL of 500 μg / mL nitroreductase aqueous solution, and monitor the change in fluorescence intensity at 783 nm over time using a fluorescence spectrophotometer with 710 nm UV-Vis absorption as the excitation source. The fluorescence intensity at the fluorescence peak gradually increases over time. Analysis of the change in fluorescence intensity at 783 nm over time shows that the fluorescence intensity tends to stabilize after approximately 5 minutes of reaction between the probe Az-Fu-Cy and nitroreductase. Figure 4 , Figure 5 ).

[0051] Example 3

[0052] Different final concentrations of nitroreductase (0.25 μg / mL, 0.5 μg / mL, 0.75 μg / mL, 1.0 μg / mL, 1.25 μg / mL, 1.5 μg / mL) aqueous solutions were reacted with Az-Fu-Cy at a final concentration of 5 μmol / L in 2.0 mL of test solution for 5 minutes, and the fluorescence intensity at 783 nm was recorded. A linear correlation curve between fluorescence intensity and nitroreductase concentration was plotted. According to the International Union of Pure and Applied Chemistry (IUPAC) rules for calculating the limit of detection, the detection limit of the probe Az-Fu-Cy for nitroreductase was calculated to be 0.98 ng / mL. Figure 6 ).

[0053] Example 4

[0054] Human breast cancer cell line MDA-MB-231 was treated at 37°C for 6 hours under normoxic (21% O2) and hypoxic (0.1% O2) conditions. 10 μL of probe stock solution and compound 12 stock solution were added to a confocal dish and incubated for 15 minutes. The cells were then washed twice with PBS and placed under a fluorescence confocal microscope. The fluorescence intensity of each cell in the red channel within the 739-754 nm wavelength range was monitored using a 633 nm excitation source. The images showed that cells incubated with Az-Fu-Cy exhibited stronger fluorescence intensity compared to those incubated with compound 12. Figure 7 ).

[0055] The above experimental results indicate that Az-Fu-Cy is a good tool for detecting nitroreductases in vitro and intracellularly.

Claims

1. A cyanine dye derivative, Az-Fu-Cy, characterized in that, The structural formula is: 。 2. The method for synthesizing the cyanine dye derivative Az-Fu-Cy as described in claim 1, characterized in that, Includes the following steps: ； 1) Add 4.94 mL of concentrated hydrochloric acid to the ethanol solution of acetazolamide suspension, reflux the reaction mixture for 4 hours, and evaporate the solvent; slowly pour saturated sodium bicarbonate into the reaction mixture, extract with ethyl acetate, wash the ethyl acetate layer with brine, dry the residue with anhydrous sodium sulfate, filter the reaction mixture, and concentrate to obtain compound 1; 2) Compound 1 and succinic anhydride were suspended in acetonitrile at a molar ratio of 1:

3. The mixture was stirred at 70°C for 16 h. After filtration, the filter cake was washed with acetonitrile and dried in vacuum to obtain compound 2 as a white solid. 3) One equivalent of azido-PEG3-amine and 1.3 equivalents of compound 2 were dissolved in DMF. 2.25 equivalents of Et3N and 1.68 equivalents of DCC were added to the mixture. The mixture was stirred for 16 h, quenched in water, and extracted with ethyl acetate. The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography using DCM / MeOH to obtain compound 3. The structural formula of the azido-PEG3-amine is: ; 4) In a dry, double-necked flask with a nitrogen inlet, 2 equivalents of sodium hydride were added in portions to 50 mL of anhydrous N,N-dimethylformamide. The mixture was stirred and cooled to 0°C. 60 mL of anhydrous N,N-dimethylformamide containing 1 equivalent of 3,4-dihydroxybenzaldehyde was added dropwise to the flask. The reaction mixture was stirred at room temperature for 30 minutes, and then 1 equivalent of bromopropyne was added dropwise via syringe. The mixture was stirred overnight at room temperature. The mixture was poured onto ice, neutralized with 1 M hydrochloric acid solution, and the product was extracted with ethyl acetate. The extract was concentrated to approximately 250 mL, washed with brine, and dried over anhydrous sodium sulfate. The solvent was removed under vacuum after filtration to give a crude product as a brown powder. The product was separated on a silica gel column using an ethyl acetate / petroleum ether / acetic acid eluent to give compound 4. 5) Dissolve 1 equivalent of compound 4, 1 equivalent of (5-nitrofuran-2-yl)methanol and 1 equivalent of 1,1'-(azodicarbonyl)bispyridine in 10 mL of dry tetrahydrofuran in a double-necked round-bottom flask under argon atmosphere; Add 1 equivalent of tributylphosphine dissolved in 3 mL of THF and stir at room temperature for 48 h; at the end of the reaction, add a certain amount of distilled water to the residue, then extract the product from the aqueous layer with ethyl acetate, wash the organic layer with brine, dry with anhydrous sodium sulfate, and evaporate under reduced pressure; the crude product is purified on silica gel with petroleum ether and dichloromethane to give a pale yellow compound 5. 6) Add 1 equivalent of a solution of compound 5 to 20 mL of methanol, and add 1.5 equivalents of sodium borohydride at 0 °C; stir at room temperature for 2 h, extract the reaction mixture with ethyl acetate and water, and dry the organic layer with anhydrous sodium sulfate; after removing volatiles under reduced pressure, obtain an orange solid compound 6; 7) Dissolve 1 equivalent of 2,3,3-trimethyl-3H-indole and 1 equivalent of iodomethane in 40 mL of toluene; stir at 100 °C for 20 hours and cool to room temperature. The product is filtered, washed with ether, and dried to obtain a light pink compound 7. 8) Mix 10 mL of N,N-dimethylformamide and 10 mL of dichloromethane in an ice bath, then add 10 mL of phosphorus oxychloride dropwise to the above solution; after stirring for 30 min, add 2.5 g of cyclohexanone and reflux at 80°C; after 6 hours, pour the mixture into ice water and filter to precipitate compound 8; 9) Two equivalents of compound 7, one equivalent of compound 8, and two equivalents of potassium acetate were placed in a flask, and 40 mL of acetic anhydride was added; the mixture was heated to 70 °C and stirred for 0.5 h to obtain a green solution; the mixture was poured into ice water and filtered to obtain compound 9; 10) Add 10 equivalents of anhydrous piperazine to 1 equivalent of compound 9 and dissolve it in 10 mL of anhydrous N,N-dimethylformamide; stir the mixture and react it at 80 °C under a nitrogen atmosphere for 2 hours, then concentrate it; the residue is purified by silica gel chromatography with dichloromethane and methanol to give compound 10. 11) Two equivalents of triethylamine were added to a solution containing 3 mL of anhydrous tetrahydrofuran and 1 equivalent of 4-nitrobenzene chloroformate at 0 °C. After 20 min, 0.86 equivalents of compound 6 dissolved in 4 mL of anhydrous dichloromethane were slowly added, and the mixture was stirred overnight at room temperature. Dichloromethane was added to the mixture, and the resulting solution was washed with brine. The organic solvent was removed under reduced pressure. Compound 11 was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluent. 12) Add 1 equivalent of compound 10 and 1 equivalent of compound 11, 2 equivalents of triethylamine and catalytic amount of 4-dimethylaminopyridine to 4 mL of anhydrous dichloromethane; stir the mixture at room temperature for 8 hours, and then remove the solvent under reduced pressure; purify the crude product by column chromatography using dichloromethane and methanol as eluent to obtain compound 12; 13) Mix 1 equivalent of compound 12 and 1 equivalent of compound 3 with 0.1 equivalent of sodium ascorbate and stir at room temperature for 15 min in 0.5 mL of tetrahydrofuran and 0.5 mL of N,N-dimethylformamide. Add 0.05 equivalent of CuSO4 dissolved in water to the above solution and stir overnight. After the reaction is complete, add an appropriate amount of water to the residue and extract the product with dichloromethane. Then wash the organic layer with brine, dry with anhydrous sodium sulfate, and evaporate under reduced pressure. The crude product is purified by column chromatography using dichloromethane and methanol as eluents to obtain Az-Fu-Cy.

3. The application of the Az-Fu-Cy as described in claim 1 in the preparation of a reagent that can be efficiently taken up by cancer cells.

4. The application of Az-Fu-Cy as described in claim 1 in the preparation of a reagent for detecting hypoxia in cancer cells.

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