Micro-molecular near-infrared probe with superoxide anion response, preparation method thereof and application of micro-molecular near-infrared probe in imaging of tumor organs

Abstract

The invention provides a superoxide anion-responsive small-molecule near-infrared probe, a preparation method thereof and tumor organ imaging application, and belongs to the field of analysis and detection. According to the invention, trifluoromethanesulfonate is used as a superoxide anion response group, a water-soluble hemicyanine matrix skeleton with excellent biocompatibility is used as a fluorophore, and high selectivity and sensitivity are shown on superoxide anions. The superoxide anion response type hemicyanine fluorescent probe disclosed by the invention has relatively large conjugation and a strong push-pull electron system, so that the superoxide anion response type hemicyanine fluorescent probe is relatively fast in response to superoxide anions, emits long-wavelength near-infrared fluorescence (725nm), and has relatively low background fluorescence and good penetration depth. Especially aiming at a matrigel system widely applied in organoid culture, the long wavelength emission characteristic can effectively avoid interference of matrigel autofluorescence on a detection signal, and the operation of removing matrigel through enzymolysis or chemical treatment in traditional probe detection is avoided.

Description

Technical Field

[0001] The present invention relates to the field of analytical detection, and particularly relates to a small molecule near-infrared probe responsive to superoxide anion, a preparation method thereof, and an application in tumor organoid imaging. Background Art

[0002] Reactive oxygen species (ROS) include superoxide anion (O2 ·- ), hydrogen peroxide (H2O2), hydroxyl radical (·OH), singlet oxygen ( 1 O2), and peroxynitrite (ONOO-), which play an important role in maintaining the cellular redox balance. The generation and elimination of ROS are regulated by a series of antioxidant defense mechanisms. Among them, O2 ·- plays a wide role in cell function regulation and is closely related to physiological and pathway processes such as innate immunity, metabolic homeostasis, and cell signal transduction.

[0003] O2 ·- The abnormal generation of O2 is related to diseases caused by oxidative damage, including oxidative damage to iron-sulfur (Fe-S) proteins, cell death of mammalian cells, degenerative diseases, and ischemia-reperfusion (IR) injury. In addition, the abnormal increase in O2 ·- will increase the levels of other endogenous ROS. Therefore, it is particularly important to provide a detection method for O2 with high sensitivity and good selectivity. ·-

[0004] Currently, the traditional methods for detecting O2 ·- usually include high performance liquid chromatography (HPLC), electron paramagnetic resonance (EPR), and mass spectrometry (MS). However, the traditional methods generally have defects such as complex pretreatment, destruction of cell tissue structure, and high cost. Small molecule fluorescent probes have received extensive attention due to their advantages such as simple operation, high sensitivity, good selectivity, and can be directly used to monitor living cells, tissues, and animals. In organoid culture, matrix gels (such as Matrigel) are widely used due to their biocompatibility and structural support, but their autofluorescence will interfere with fluorescence detection. Traditional methods need to remove the matrix gel by enzymatic digestion or chemical treatment, but these steps may damage the organoids or introduce experimental variables. If a near-infrared fluorescent probe that can directly detect without eliminating the matrix gel is developed, it will simplify the experimental process, maintain the integrity of the organoid sample, and provide a more efficient and non-destructive detection scheme for organoid research. Currently, the reported superoxide anion fluorescent probes generally have characteristics such as short emission wavelength and poor biocompatibility. The short emission wavelength leads to the test of the probe at the cellular level being easily interfered by background fluorescence. Therefore, it is particularly important to design a superoxide anion small molecule fluorescent probe with a long emission wavelength and good biocompatibility. Summary of the Invention

[0005] ​The present invention provides a superoxide anion-responsive small molecule near-infrared probe, a preparation method thereof, and an application in tumor organoid imaging. The superoxide anion-responsive hemicyanine fluorescent probe of the present invention has a long emission wavelength and good biocompatibility.

[0006] The present invention provides a superoxide anion-responsive hemicyanine fluorescent probe having a structural formula shown in Formula I:

[0007]

[0008] The present invention also provides a preparation method of the superoxide anion-responsive hemicyanine fluorescent probe described in the above technical solution, including the following steps:

[0009] Mix 2,3,3-trimethyl-3H-indole, 1,3-propane sultone, and a nitrile organic solvent for a ring-opening addition reaction to obtain Compound 4; Compound 4 has a structural formula shown in Formula 4;

[0010]

[0011] Mix Compound 4, 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde, a liquid organic base, and an alcohol solvent for a condensation reaction to obtain Compound 5; Compound 5 has a structural formula shown in Formula 5:

[0012]

[0013] Mix Compound 5, trifluoromethanesulfonic anhydride, an acid-binding agent, and a first polar organic solvent for a substitution reaction to obtain the superoxide anion-responsive hemicyanine fluorescent probe.

[0014] Preferably, the preparation method of the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde includes the following steps:

[0015] Mix phosphorus oxychloride, cyclohexanone, and a second polar organic solvent for a Vilsmeier-Haack formylation reaction to obtain Compound 1; Compound 1 has a structural formula shown in Formula 1:

[0016]

[0017] Mix Compound 1, 4-methoxy-2-hydroxybenzaldehyde, cesium carbonate, and a third polar organic solvent for a substitution reaction-Knoevenagel condensation tandem reaction to obtain Compound 2; Compound 2 has a structural formula shown in Formula 2:

[0018]

[0019] Boron tribromide, Compound 2 and a fourth polar organic solvent are mixed to carry out the demethylation reaction of the methoxy group, and a demethylation reaction product of the methoxy group is obtained;

[0020] The demethylation reaction product of the methoxy group is mixed with water for hydrolysis to obtain the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde.

[0021] Preferably, the dosage ratio of phosphorus oxychloride to cyclohexanone is 7.1-8.0 mL: 50 mmol.

[0022] Preferably, the molar ratio of Compound 1 to 4-methoxy-2-hydroxybenzaldehyde is 3-3.5: 2.0;

[0023] The molar ratio of 4-methoxy-2-hydroxybenzaldehyde to cesium carbonate is 2.0: 5.5-6.0.

[0024] Preferably, the molar ratio of 2,3,3-trimethyl-3H-indole to 1,3-propanesultone is 6.3: 9.0-10.0.

[0025] Preferably, the molar ratio of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde to Compound 4 is 1.0: 1.0-1.5.

[0026] Preferably, the liquid organic base includes one or more of piperidine, triethylamine and pyrrolidine.

[0027] Preferably, the molar ratio of Compound 5 to the acid-binding agent is 1.0: 3.3-3.7;

[0028] The molar ratio of Compound 5 to trifluoromethanesulfonic anhydride is 1.0: 2.8-3.2.

[0029] The present invention also provides the application of the superoxide anion-responsive hemicyanine fluorescent probe described in the above technical solution or the superoxide anion-responsive hemicyanine fluorescent probe prepared by the preparation method described in the above technical solution in the preparation of a superoxide anion detector.

[0030] The present invention uses trifluoromethanesulfonate as a superoxide anion-responsive group and a water-soluble semi-cyanine parent skeleton with excellent biocompatibility as a fluorophore, showing high selectivity and sensitivity to superoxide anions. The superoxide anion-responsive semi-cyanine fluorescent probe (LW-SO3F) of the present invention has a large conjugation and a strong electron-donating and electron-withdrawing system, enabling a fast response to superoxide anions and emitting near-infrared fluorescence with a long wavelength (725 nm), having low background fluorescence and good penetration depth. Particularly for the Matrigel system widely used in organoid culture, its long-wavelength emission characteristics can effectively avoid the interference of the Matrigel's autofluorescence on the detection signal, eliminating the need for the operation of removing Matrigel by enzymatic digestion or chemical treatment in traditional probe detection. Therefore, this probe can be directly used for the detection of organoids containing Matrigel, avoiding possible damage to organoids or introduction of additional experimental variables during the Matrigel removal process. Through this technology, non-invasive, in-situ near-infrared fluorescence imaging of human ovarian cancer cells SKOV-3 and human-derived tumor organoids can be achieved, providing a more efficient detection scheme for organoid research that maintains the integrity of the samples.

[0031] Compared with the prior art, the advantages of the present invention are as follows:

[0032] (1) This probe is a fluorescent probe with a trifluoromethanesulfonic acid group as the recognition unit. Practice has shown that the fluorescent probe molecule of the present invention exhibits high selectivity and sensitivity when detecting superoxide anions;

[0033] (2) This fluorescent probe has a long emission wavelength, reaching 725 nm, and has low background fluorescence in the detection of living cells;

[0034] (3) Due to the structure of benzoxindole salt in the probe molecule, it is easier to interact with superoxide anions. At the same time, the probe molecule has a sulfonate group structure, showing good biocompatibility;

[0035] (4) This probe has good selectivity. After the probe reacts with analytes K2CO3, NaCl, NaClO, TBHP (tert-butyl hydroperoxide), GSH (glutathione), H2O2, and O2 ·- respectively, only when reacting with O2 ·- does it show a significant fluorescence enhancement. Description of the Drawings

[0036] Figure 1 is the synthetic route of the probe LW-SO3F in the example;

[0037] Figure 2 is the 1 1H NMR spectrum of the probe LW-SO3F in the example;

[0038] Figure 3Absorption spectra of the probe LW-SO3F in Example before and after reaction with superoxide anion;

[0039] Figure 4 Fluorescence emission spectra of the probe LW-SO3F in Example before and after reaction with superoxide anion;

[0040] Figure 5 Bar graph of fluorescence intensity at 725 nm after the probe LW-SO3F in Example reacts with other oxides, amino acids, common cations and anions;

[0041] Figure 6 Graph of the results of the cytotoxicity experiment of the probe LW-SO3F in Example;

[0042] Figure 7 Fluorescence effect diagram of the probe LW-SO3F reacting with superoxide anion in human ovarian cancer cell line SKOV-3;

[0043] Figure 8 Fluorescence effect diagram of the probe LW-SO3F reacting with superoxide anion in organoids. Detailed implementation mode

[0044] The present invention provides a superoxide anion-responsive hemicyanine fluorescent probe with the structural formula shown in Formula I:

[0045]

[0046] The present invention also provides a preparation method of the superoxide anion-responsive hemicyanine fluorescent probe described in the above technical solution, including the following steps:

[0047] Mix 2,3,3-trimethyl-3H-indole, 1,3-propane sultone and a nitrile organic solvent for a ring-opening addition reaction to obtain Compound 4; Compound 4 has the structural formula shown in Formula 4;

[0048]

[0049] Mix Compound 4, 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde, a liquid organic base and an alcohol solvent for a condensation reaction to obtain Compound 5; Compound 5 has the structural formula shown in Formula 5:

[0050]

[0051] Mix Compound 5, trifluoromethanesulfonic anhydride, an acid-binding agent and a first polar organic solvent for a substitution reaction to obtain the superoxide anion-responsive hemicyanine fluorescent probe.

[0052] The present invention mixes 2,3,3-trimethyl-3H-indole, 1,3-propane sultone and a nitrile organic solvent to carry out a ring-opening addition reaction to obtain Compound 4.

[0053] In the present invention, the molar ratio of 2,3,3-trimethyl-3H-indole to 1,3-propane sultone is preferably 6.3:9.1 to 9.7. In specific embodiments of the present invention, the molar ratio of 2,3,3-trimethyl-3H-indole to 1,3-propane sultone can be 6.3:9.1, 6.3:9.4, 6.3:9.5, 6.3:9.6 or 6.3:9.7.

[0054] In the present invention, the dosage ratio of 2,3,3-trimethyl-3H-indole to the nitrile organic solvent is preferably 6.3 mmol:4.0 to 5.0 mL. In specific embodiments of the present invention, the dosage ratio of 2,3,3-trimethyl-3H-indole to the nitrile organic solvent can be 6.3 mmol:4.0 mL, 6.3 mmol:4.2 mL, 6.3 mmol:4.5 mL, 6.3 mmol:4.6 mL, 6.3 mmol:4.8 mL or 6.3 mmol:5.0 mL; the nitrile organic solvent preferably includes acetonitrile.

[0055] In the present invention, the ring-opening addition reaction is preferably carried out under reflux conditions; the time of the ring-opening addition reaction is preferably 18 to 20 h.

[0056] After the ring-opening addition reaction, the present invention preferably cools the obtained product to room temperature and then filters it, and then washes and dries the obtained solid to obtain Compound 4.

[0057] After obtaining Compound 4, the present invention mixes Compound 4, 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde, a liquid organic base and an alcohol solvent to carry out a condensation reaction to obtain Compound 5.

[0058] In the present invention, the preparation method of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde preferably includes the following steps:

[0059] Mix phosphorus oxychloride, cyclohexanone and a second polar organic solvent to carry out a Vilsmeier-Haack formylation reaction to obtain Compound 1; Compound 1 has the structural formula shown in Formula 1:

[0060]

[0061] Mix Compound 1, 4-methoxy-2-hydroxybenzaldehyde, cesium carbonate and a third polar organic solvent to carry out a substitution reaction-Knoevenagel condensation tandem reaction to obtain Compound 2; Compound 2 has the structural formula shown in Formula 2:

[0062]

[0063] Boron tribromide, Compound 2 and a fourth polar organic solvent are mixed to carry out the demethylation reaction of the methoxy group, and a demethylation reaction product of the methoxy group is obtained;

[0064] The demethylation reaction product of the methoxy group is mixed with water for hydrolysis to obtain the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde.

[0065] In the present invention, phosphorus oxychloride, cyclohexanone and a second polar organic solvent are mixed to carry out the Vilsmeier-Haack formylation reaction to obtain Compound 1.

[0066] In the present invention, the mixing is preferably carried out under a protective atmosphere and an ice bath.

[0067] In the present invention, the mixing preferably includes dropping phosphorus oxychloride into the second polar organic solvent and then stirring, and then adding cyclohexanone to the obtained mixture in several portions.

[0068] In the present invention, the dosage ratio of phosphorus oxychloride to cyclohexanone is preferably 7-8.0 mL: 50 mmol. In specific examples of the present invention, the dosage ratio of phosphorus oxychloride to cyclohexanone can be 7.1 mL: 50 mmol, 7.3 mL: 50 mmol, 7.5 mL: 50 mmol, 7.7 mL: 50 mmol or 8 mL: 50 mmol.

[0069] In the present invention, the volume ratio of the second polar organic solvent to phosphorus oxychloride is preferably 8.0: 7.1-8.0. In specific examples of the present invention, the volume ratio of the second polar organic solvent to phosphorus oxychloride can be 8.0: 7.1, 8.0: 7.3, 8.0: 7.5, 8.0: 7.7 or 8.0: 8.0; the second polar organic solvent preferably includes N,N-dimethylformamide.

[0070] In the present invention, the temperature of the Vilsmeier-Haack formylation reaction is preferably 20-25 °C, and the time is preferably 4-6 h. The time of the Vilsmeier-Haack formylation reaction is counted from the addition of cyclohexanone.

[0071] In the present invention, after the Vilsmeier-Haack formylation reaction, the product obtained from the Vilsmeier-Haack formylation reaction is preferably poured into ice water and then neutralized with a saturated Na2CO3 solution. The neutralized product is extracted with ethyl acetate to obtain an organic phase. The organic phase is washed and then dried by adding a solid desiccant. After drying is completed, the solvent of the organic phase after removing the solid desiccant is rotary evaporated to obtain the compound 1.

[0072] The present invention has no special limitation on the reduced pressure distillation, and the solvent can be removed.

[0073] After obtaining the compound 1, the present invention mixes the compound 1, 4-methoxy-2-hydroxybenzaldehyde, cesium carbonate and a third polar organic solvent to carry out a substitution reaction-Knoevenagel condensation tandem reaction to obtain the compound 2.

[0074] In the present invention, the molar ratio of the compound 1 to 4-methoxy-2-hydroxybenzaldehyde is preferably 3-3.5:2.0. In a specific embodiment of the present invention, the molar ratio of the compound 1 to 4-methoxy-2-hydroxybenzaldehyde is preferably 3:2.0, 3.1:2.0, 3.2:2.0, 3.3:2.0 or 3.5:2.0.

[0075] In the present invention, the molar ratio of 4-methoxy-2-hydroxybenzaldehyde to cesium carbonate is preferably 2.0:5.5-6.0. In a specific embodiment of the present invention, the molar ratio of 4-methoxy-2-hydroxybenzaldehyde to cesium carbonate can be 2.0:5.5, 2.0:5.6, 2.0:5.7, 2.0:5.8, 2.0:5.9 or 2.0:6.0.

[0076] In the present invention, the dosage ratio of the compound 1 to the third polar organic solvent is preferably 3 mmol:4 mL; the third polar organic solvent preferably includes N,N-dimethylformamide.

[0077] In the present invention, the temperature of the substitution reaction-Knoevenagel condensation tandem reaction is preferably room temperature, and the time is preferably 16-18 h; the substitution reaction-Knoevenagel condensation tandem reaction is preferably carried out under stirring conditions.

[0078] After the substitution reaction-Knoevenagel condensation tandem reaction, the present invention filters the product obtained from the substitution reaction-Knoevenagel condensation tandem reaction, and then mixes the obtained filtrate with water for washing to remove the second polar organic solvent, obtaining a water-containing mixture; the water-containing mixture is extracted to obtain an organic phase; a solid desiccant is added to the organic phase for drying, and after drying is completed, the solvent of the organic phase after removing the solid desiccant is rotary-evaporated and separated by column chromatography to obtain the compound 2.

[0079] In the present invention, the extraction preferably includes extracting alternately with dichloromethane and ethyl acetate.

[0080] In the present invention, the eluent for the column chromatography separation is preferably petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is preferably 2:1.

[0081] After obtaining the compound 2, the present invention mixes boron tribromide, the compound 2 and a fourth polar organic solvent to carry out the demethylation reaction of the methoxy group, obtaining a demethylation reaction product of the methoxy group;

[0082] The demethylation reaction product of the methoxy group is mixed with water for hydrolysis to obtain the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde.

[0083] In the present invention, the mixing is preferably carried out under ice bath conditions.

[0084] In the present invention, the dosage ratio of the boron tribromide to the compound 2 is preferably 2.2-2.3 mL: 15 mmol.

[0085] In the present invention, the volume ratio of the boron tribromide to the fourth polar organic solvent is preferably 2.2-2.3: 30; the fourth polar organic solvent preferably includes dichloromethane.

[0086] In the present invention, the temperature of the demethylation reaction of the methoxy group is preferably 20-25 °C, and the time is preferably 36-40 h; the demethylation reaction of the methoxy group is preferably carried out under stirring conditions.

[0087] After obtaining the demethylation reaction product of the methoxy group, the present invention mixes the demethylation reaction product of the methoxy group with water for hydrolysis to obtain the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde.

[0088] In the present invention, the water is preferably ice water. The water in the ice water directly participates in the hydrolysis of the intermediate, provides hydroxy hydrogen, and generates 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde.

[0089] After the hydrolysis, the present invention preferably filters the hydrolysis product to obtain a crude product; the crude product is filtered and separated by column chromatography to obtain compound 3.

[0090] In the present invention, the eluents for column chromatography separation are preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 20:1.

[0091] In the present invention, the molar ratio of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde to compound 4 is preferably 1.0:1.0-1.2.

[0092] In the present invention, the dosage ratio of the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde and the liquid organic base is preferably 1.0 mmol: 0.2-0.3 mL. The liquid organic base preferably includes one or more of piperidine, pyrrolidine and triethylamine. The base can capture the H of the indole 2-methyl group to generate an indole-2-methyl carbon anion (nucleophile), and then the nucleophile attacks the aldehyde group to cause a condensation reaction.

[0093] In the present invention, the volume ratio of the alcohol solvent to the liquid organic base is preferably 1.5 mL:0.3 mL; and the alcohol solvent preferably includes ethanol.

[0094] In the present invention, the Knoevenagel-type condensation reaction is preferably carried out under reflux.

[0095] After the Knoevenagel-type condensation reaction, the present invention preferably cools the product obtained by the Knoevenagel-type condensation reaction to room temperature and then distills it under reduced pressure, and then separates the obtained crude product by column chromatography to obtain compound 5.

[0096] In the present invention, the eluents for column chromatography separation are preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 30:1.

[0097] After obtaining compound 5, the present invention mixes compound 5, trifluoromethanesulfonic anhydride, an acid-binding agent and a first polar organic solvent to carry out a substitution reaction to obtain the superoxide anion responsive hemicyanine fluorescent probe.

[0098] In the present invention, the mixing preferably comprises: first mixing compound 5 with a first polar organic solvent, then adding an acid binding agent to the obtained first mixture to obtain a second mixture; and dropping trifluoromethanesulfonic anhydride into the second mixture.

[0099] In the present invention, the dropping is preferably performed under ice bath conditions.

[0100] In the present invention, the molar ratio of the compound 5 to the acid binding agent is preferably 1.0:3.5-3.7;

[0101] The molar ratio of the compound 5 to trifluoromethanesulfonic anhydride is preferably 1.0:3.0 - 3.2; the acid-binding agent preferably includes triethylamine.

[0102] In the present invention, the dosage ratio of the compound 5 to the first polar organic solvent is preferably 1.0 mmol:5 - 6 mL; the polar organic solvent preferably includes ethanol.

[0103] In the present invention, the substitution reaction preferably includes a first substitution reaction and a second substitution reaction. The first substitution reaction is preferably carried out under ice bath conditions, and the time of the first substitution reaction is preferably 20 - 30 min. The reaction of trifluoromethanesulfonic anhydride with hydroxyl groups is a strongly exothermic process. Direct mixing at room temperature may cause local overheating and the formation of by-products. The reaction rate slows down under ice bath conditions, and the heat is released slowly, avoiding the out-of-control of the reaction. At the same time, the generated trifluoromethanesulfonate is more stable at low temperature, avoiding its decomposition or further reaction.

[0104] In the present invention, the second substitution reaction is preferably carried out at room temperature, and the time of the second substitution reaction is preferably 30 - 35 min. The second substitution reaction can drive the reaction to completion. Under ice bath conditions, it may not be completely converted. Heating to room temperature can accelerate the sulfonylation of the remaining -OH, ensuring a high yield.

[0105] After the substitution reaction, in the present invention, the product obtained from the substitution reaction is preferably diluted with dichloromethane and water, and then the obtained mixture is extracted to obtain an organic phase; the obtained organic phase is dried and the solvent is rotary evaporated to obtain a crude product, and the crude product is subjected to column chromatography separation to obtain the superoxide anion-responsive hemicyanine fluorescent probe.

[0106] In the present invention, the eluent for column chromatography separation is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 20:1.

[0107] The present invention also provides the application of the superoxide anion-responsive hemicyanine fluorescent probe described in the above technical solution or the superoxide anion-responsive hemicyanine fluorescent probe prepared by the preparation method described in the above technical solution in the preparation of a superoxide anion detector.

[0108] The following is a detailed description of the superoxide anion-responsive small molecule near-infrared probe provided by the present invention, its preparation method and its application in tumor organoid imaging in combination with examples, but they should not be construed as limiting the protection scope of the present invention.

[0109] In the examples, the dosage ratios of the reagents used for column chromatography separation are all volume ratios.

[0110] Example 1

[0111] Figure 1 Synthesis route of the probe LW-SO3F for the example:

[0112] Synthesis of Compound 1:

[0113]

[0114] Under nitrogen protection and at 0 °C, phosphorus oxychloride (7.7 mL) was slowly dropped into dry N,N-dimethylformamide (8 mL) and stirred for 30 min. Subsequently, cyclohexanone (4.9 g, 50 mmol) was added to the system in small portions and the reaction was continued at 25 °C for 4 h. After the reaction was completed, the reaction solution was poured into ice water and neutralized to pH = 7 with saturated Na2CO3 solution. Subsequently, the aqueous solution was poured into ethyl acetate, washed three times with saturated NaCl solution, the organic phase was collected, dried over anhydrous Na2SO4, the Na2SO4 was removed, and the solvent was rotary evaporated to obtain an orange-yellow liquid.

[0115] Synthesis of Compound 2:

[0116]

[0117] Compound 1 (2.2 g, 15 mmol), 4-methoxy-2-hydroxybenzaldehyde (1.5 g, 10 mmol), and cesium carbonate (5.8 g, 30 mmol) were added to dry N,N-dimethylformamide (20 mL), and the reaction was stirred at room temperature for 16 h. After the reaction was completed, the filtrate was collected by filtration. After washing the filtrate with water to remove N,N-dimethylformamide, it was alternately extracted with an appropriate amount of dichloromethane and ethyl acetate. The combined organic phase was dried over anhydrous Na2SO4, the Na2SO4 was removed after drying, and then the solvent was rotary evaporated. The crude product was separated by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain a yellow solid.

[0118] Synthesis of Compound 3:

[0119]

[0120] At 0 °C, boron tribromide (2.25 mL) was dropped into dichloromethane (30 mL) of Compound 2 (2.8 g, 15 mmol), and the reaction was stirred at 25 °C for 36 h. After the reaction was completed, the reaction solution was poured into ice water, stirred for 30 min, and then filtered to obtain the crude product. After the crude product was filtered, it was separated by column chromatography (dichloromethane:methanol = 20:1) to obtain an orange-yellow solid.

[0121] Synthesis of Compound 4:

[0122]

[0123] 2,3,3-Trimethyl-3H-indole (1.00 mg, 6.29 mmol) and 1,3-propanesultone (1.16 g, 9.43 mmol) were added to acetonitrile (5 mL), and the mixture was heated under reflux for 20 h. After the reaction, it was cooled to room temperature. The reaction system was filtered to obtain a purple solid, which was washed with acetone (2 mL) and dried to obtain a lavender solid.

[0124] Synthesis of compound 5:

[0125]

[0126] Compound 3 (228 mg, 1.0 mmol) and compound 4 (281 mg, 1.0 mmol) were dissolved in ethanol (10 mL), and then piperidine (0.3 mL) was added. The mixture was refluxed and stirred for 16 h. After the reaction, the reaction system was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was separated by column chromatography (dichloromethane:methanol = 30:1) to obtain a blue solid.

[0127] Synthesis of probe LW-SO3F:

[0128]

[0129] Triethylamine (100 μL, 0.73 mmol) was added dropwise to a solution of compound 5 (98 mg, 0.20 mmol) in dichloromethane (6.0 mL). Then, trifluoromethanesulfonic anhydride (100 μL, 0.6 mmol) was added dropwise to the reaction system at 0 °C and stirred for 30 min. The reaction was continued at room temperature for 35 min. After the reaction, the reaction solution was diluted with dichloromethane (30 mL) and water (20 mL), and then the aqueous layer was extracted twice with dichloromethane (30 mL). The organic phase was collected and dried over anhydrous Na2SO4. After distillation under reduced pressure, the crude product was purified by column chromatography (dichloromethane:methanol = 20:1) to obtain a dark blue solid.

[0130] Figure 2 For the 1 1H NMR spectrum of probe LW-SO3F in the examples:

[0131] 11H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 15.2 Hz, 1H), 7.62 - 7.45 (m, 5H), 7.48 - 7.35 (m, 2H), 7.31 (d, J = 15.2 Hz, 1H), 7.18 - 7.07 (m, 3H), 6.95 (s, 2H), 4.85 (t, J = 7.8 Hz, 3H), 3.70 - 3.61 (m, 1H), 3.13 - 3.03 (m, 3H), 2.94 (s, 1H), 2.92 (d, J = 6.0 Hz, 2H), 2.85 (q, J = 6.1 Hz, 0H), 2.69 (dt, J = 18.6, 6.2 Hz, 3H), 2.48 - 2.32 (m, 3H), 2.23 - 2.08 (m, 2H), 2.05 (s, 1H), 1.91 (h, J = 7.1 Hz, 3H), 1.82 (s, 2H), 1.80 (s, 7H), 1.43 (t, J = 7.0 Hz, 1H), 1.33 - 1.26 (m, 1H), 1.25 (s, 3H), 0.91 - 0.78 (m, 1H).

[0132] Changes in the absorption spectra of probe LW - SO3F before and after reaction with superoxide anion

[0133] The prepared probe LW - SO3F was dissolved in dimethyl sulfoxide solution to make a 0.5 mM probe stock solution, and O2 ·- stock solution was prepared by KO2, crown ether and dimethyl sulfoxide and diluted to the required concentration of 1.0 mM.

[0134] Take two 4.0 mL centrifuge tubes. One tube was added with 1.98 mL of phosphate buffer solution (PBS, concentration 100 mM, pH = 7.4), and then 20 μL of the 0.5 mM probe stock solution; the other tube was added with 1.78 mL of phosphate buffer solution, then 20 μL of the 0.5 mM probe stock solution, and finally 200 μL of the 1.0 mM O2 ·- stock solution. After incubation at room temperature for 3 min, the absorption spectra were measured respectively, and the results are as Figure 3 shown. Figure 3 Absorption spectra of probe LW - SO3F before and after reaction with superoxide anion for the example.

[0135] As Figure 3 can be seen, without O2 ·- the absorption peak is in the visible light region. After adding O2 ·- the original absorption peak broadens, and at the same time a new set of absorption peaks in the near - ultraviolet region appears.

[0136] Changes in the fluorescence spectra of probe LW - SO3F before and after reaction with superoxide anion

[0137] Take out 20 μL from the above probe mother liquor. Take two 1.5 mL centrifuge tubes. Add 990 μL of PBS buffer solution to one tube first, and then add 10 μL of the probe mother liquor; add 890 μL of PBS buffer solution to the other tube, then add 10 μL of the probe mother liquor, and finally add 100 μL of O2 with a concentration of 1.0 mM ·- mother liquor. After incubating at room temperature for 3 min, test the fluorescence spectra respectively. The results are as Figure 4 shown Figure 4 Fluorescence emission spectra of probe LW-SO3F before and after reacting with superoxide anion in the example

[0138] As can be seen from Figure 4 it, without O2 ·- in the system, there is almost no fluorescence. After adding O2 ·- the fluorescence intensity increases significantly, and the fluorescence is the strongest at 725 nm

[0139] Selectivity study of probe LW-SO3F for different analytes

[0140] Select the above probe mother liquor with a concentration of 0.5 mM: Take several 1.5 mL centrifuge tubes, add 890 μL of PBS buffer solution to each tube respectively, then add 10 μL of the probe mother liquor to each tube respectively, and finally add 100 μL of the following different analytes with a concentration of 1 mM: K2CO3, NaCl, NaClO, TBHP, GSH, H2O2 and O2 ·- , after incubating at room temperature for 3 min, perform fluorescence tests respectively. The results are as Figure 5 shown Figure 5 Bar chart of fluorescence intensity of probe LW-SO3F at 725 nm after reacting with other oxides, amino acids and common cations and anions in the example

[0141] As can be seen from Figure 5 it, the measured fluorescence intensity does not change significantly after adding various ions, amino acids and oxides; while the fluorescence intensity at 725 nm after adding O2 ·- increases significantly, indicating that the probe has good selectivity for O2 ·-

[0142] Cytotoxicity assessment of probe LW-SO3F

[0143] ​SKOV-3 cells were seeded in 96-well plates and incubated at 37 °C for 24 h until the cells were completely adherent to the wells. Meanwhile, dimethyl sulfoxide solutions of LW-SO3F with different concentrations (0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, and 2.0 mM) were prepared. 10 μL of the probe solutions with different concentrations were respectively added to 1.0 mL of the culture medium to prepare the probe solutions with the required concentrations. After 24 h, the culture medium in the wells was aspirated, and the probe culture media with different concentrations were respectively added to the 96-well plates, 100 μL for each well, and then incubated for another 24 h. Finally, the cell toxicity was tested by MTT, and the results are as Figure 6 shown. Figure 6 Figure of the cell toxicity experiment results of the probe LW-SO3F in the example

[0144] As Figure 6 can be seen, within the tested concentration range (0 - 20 μM), the probe LW-SO3F basically has no cell toxicity.

[0145] Fluorescence imaging of the probe LW-SO3F in SKOV-3 cells

[0146] Select the probe mother solution with a concentration of 0.5 mM mentioned above.

[0147] Control group: 10 μL of DMSO was added to the culture dish containing SKOV-3 cells and incubated for 1 h (the total volume of the culture medium was 2 mL). After 1 h, the original culture medium was removed, 1990 μL of fresh culture medium was added, and then 10 μL of the probe mother solution was added and incubated for 15 min;

[0148] Experimental group: 10 μL of 4MAPAP was added to the culture dish containing SKOV-3 cells and incubated for 1 h (the total volume of the culture medium was 2 mL). After 1 h, the original culture medium was removed, 1990 μL of fresh culture medium was added, and then 10 μL of the probe mother solution was added and incubated for 15 min. Subsequently, fluorescence imaging was respectively performed with a confocal microscope, and the results are as Figure 7 shown. Figure 7 Figure of the fluorescence effect of the probe LW-SO3F and superoxide anion in human ovarian cancer cell SKOV-3 in the example

[0149] Figure 7 In (B), it is the fluorescence imaging map after SKOV-3 cells were incubated with 10 μL of DMSO for 1 h and then 2.5 μM of the probe LW-SO3F was added and incubated for another 15 min; Figure 7 In (D), it is the fluorescence imaging map after uSKOV-3 cells were incubated with 20 mM APAP (paracetamol) for 1 h and then 2.5 μM of the probe LW-SO3F was added and incubated for another 15 min; Figure 7In (A) and (C), they are the bright-field images of (B) and (D) respectively.

[0150] As can be seen from Figure 7 the experimental group could observe more obvious fluorescence compared with the control group, indicating that the probe could detect superoxide anions in the cellular environment through a confocal microscope.

[0151] Fluorescence imaging of probe LW-SO3F in organoids

[0152] Select the probe mother solution with a concentration of 0.5 mM.

[0153] Control group: Take 5 μL of DMSO and add it to the culture dish containing organoids for incubation for 1 h (the total volume of the culture medium is 1 mL). After 1 h, remove the original culture medium, add 995 μL of fresh culture medium, and then add 5 μL of the probe mother solution and incubate for 20 min;

[0154] Experimental group: Take 5 μL of 4MAPAP and add it to the culture dish containing organoids for incubation for 1 h (the total volume of the culture medium is 1 mL). After 1 h, remove the original culture medium, add 995 μL of fresh culture medium, and then add 5 μL of the probe mother solution and incubate for 20 min.

[0155] Subsequently, fluorescence imaging was performed using a confocal microscope respectively, and the results are as Figure 8 shown, Figure 8 which is the fluorescence effect diagram of probe LW-SO3F and superoxide anions in organoids in the example: Figure 8 In (B), it is the fluorescence imaging diagram after incubating the organoids with 5 μL of DMSO for 1 h and then adding 2.5 μM of probe LW-SO3F and incubating for another 20 min; Figure 8 In (E), it is the fluorescence imaging diagram after incubating the organoids with 20 mM APAP (paracetamol) for 1 h and then adding 2.5 μM of probe LW-SO3F and incubating for another 20 min; Figure 8 In (H), it is the fluorescence imaging diagram after incubating the organoids with 20 mM APAP (paracetamol) for 1 h and then adding 2.5 μM of probe LW-SO3F and incubating for another 20 min under the condition of retaining Matrigel; Figure 8 In (A), it is the bright-field image of (B), Figure 8 In (C), it is the superimposed image of (A) and (B); Figure 8 In (D), it is the bright-field image of (E), Figure 8 In (F), it is the superimposed image of (D) and (E); Figure 8 In (G), it is the bright-field image of (H), Figure 8 In (I), it is the superimposed image of (G) and (H).

[0156] As can be seen from Figure 8It can be seen that more obvious fluorescence can be observed in the experimental group compared to the control group, and the experimental group retaining Matrigel has a stronger fluorescence signal compared to the experimental group without Matrigel, indicating that the probe can detect superoxide anions in the organoid environment through a confocal microscope.

[0157] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A superoxide anion-responsive hemicyanine-based fluorescent probe, characterized in that, It has the structural formula shown in Formula I:

2. The preparation method of the superoxide anion-responsive hemicyanine fluorescent probe according to claim 1, characterized in that, It includes the following steps: Mix 2,3,3-trimethyl-3H-indole, 1,3-propane sultone and a nitrile organic solvent for a ring-opening addition reaction to obtain Compound 4; Compound 4 has the structural formula shown in Formula 4; Mix Compound 4, 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde, a liquid organic base and an alcohol solvent for a condensation reaction to obtain Compound 5; Compound 5 has the structural formula shown in Formula 5: Mix Compound 5, trifluoromethanesulfonic anhydride, a base scavenger and a first polar organic solvent for a substitution reaction to obtain the superoxide anion-responsive hemicyanine fluorescent probe.

3. The preparation method according to claim 2, characterized in that, The preparation method of the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde includes the following steps: Mix phosphorus oxychloride, cyclohexanone and a second polar organic solvent for a Vilsmeier-Haack formylation reaction to obtain Compound 1; Compound 1 has the structural formula shown in Formula 1: Mix Compound 1, 4-methoxy-2-hydroxybenzaldehyde, cesium carbonate and a third polar organic solvent for a substitution reaction-Knoevenagel condensation tandem reaction to obtain Compound 2; Compound 2 has the structural formula shown in Formula 2: Mix boron tribromide, Compound 2 and a fourth polar organic solvent for a demethylation reaction of the methoxy group to obtain a demethylation reaction product of the methoxy group; Mix the demethylation reaction product of the methoxy group with water for hydrolysis to obtain the 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde.

4. The preparation method according to claim 3, characterized in that, The dosage ratio of the phosphorus oxychloride to the cyclohexanone is 7.1-8.0 mL: 50 mmol.

5. The preparation method according to claim 3, characterized in that, The molar ratio of Compound 1 to 4-methoxy-2-hydroxybenzaldehyde is 3-3.5: 2.0; The molar ratio of 4-methoxy-2-hydroxybenzaldehyde to cesium carbonate is 2.0: 5.5-6.

0.

6. The preparation method according to claim 2, wherein The molar ratio of 2,3,3-trimethyl-3H-indole to 1,3-propane sultone is 6.3: 9.0-10.

0.

7. The preparation method according to claim 2, characterized in that, The molar ratio of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde to Compound 4 is 1.0: 1.0-1.

5.

8. The preparation method according to claim 2, characterized in that, The liquid organic base includes one or more of piperidine, triethylamine and pyrrolidine.

9. The preparation method according to the preparation method described in claim 2, characterized in that, The molar ratio of Compound 5 to the base scavenger is 1.0: 3.3-3.7; The molar ratio of Compound 5 to trifluoromethanesulfonic anhydride is 1.0: 2.8-3.

2.

10. Use of the superoxide anion-responsive hemicyanine fluorescent probe according to claim 1 or the superoxide anion-responsive hemicyanine fluorescent probe prepared by the preparation method according to any one of claims 2-9 in the preparation of a superoxide anion detector.

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