Hydrogen peroxide-responsive small-molecule near-infrared probe, preparation method thereof and application of hydrogen peroxide-responsive small-molecule near-infrared probe as tumor organ imaging probe

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a hydrogen peroxide-responsive small-molecule near-infrared probe, a preparation method thereof and application of the hydrogen peroxide-responsive small-molecule near-infrared probe as a tumor organ imaging probe. The hydrogen peroxide-responsive small-molecule near-infrared probe provided by the invention takes pentafluorophenyl sulfonyl chloride as a recognition unit, the indole sulfonate structure in the molecule is easier to interact with hydrogen peroxide, efficient and rapid detection of hydrogen peroxide is realized, and the indole salt structure enables the probe to have good cell membrane penetrability, so that the hydrogen peroxide-responsive small-molecule near-infrared probe can be used for detecting hydrogen peroxide. And conditions are provided for in-vivo detection of hydrogen peroxide. The hydrogen peroxide-responsive small-molecule near-infrared probe provided by the invention has long emission wavelength, near-infrared fluorescence emission exceeds 720nm, can penetrate through a matrigel layer to realize imaging of living cells, is not easily interfered by the fluorescence background of matrigel in the aspect of detection of the living cells, can directly detect without eliminating the matrigel, and is high in sensitivity and high in sensitivity. The probe can be directly used as a tumor organ imaging probe.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a small molecule near-infrared probe (Hcy-SO3-5F) responsive to hydrogen peroxide, a preparation method thereof, and an application as a tumor organoid imaging probe. Background Art

[0002] Oxygen is an important substance for life. In cells, reactive oxygen species (ROS) are mainly produced by the mitochondrial electron transport chain. Normal levels of ROS are the main mediators for phagocytes to exert phagocytosis and killing effects. However, the level of ROS will be abnormal under pathological conditions in the human body, thereby damaging the human body. Hydrogen peroxide (H2O2) is one of the important reactive oxygen species. Qualitatively and quantitatively studying the level of ROS in the human body with H2O2 as the entry point has become the focus of attention of medical workers.

[0003] H2O2 is an intermediate product of cellular oxygen metabolism commonly present in aerobic organisms. Current research shows that an increase in the content of H2O2 in cells will directly or indirectly induce malignant transformation of cells. At the same time, reducing the content of H2O2 in cells can also reverse the malignant manifestations of tumor cells. Compared with normal cells, tumor cells are more sensitive to H2O2 and are more likely to die due to excessive or insufficient levels of H2O2. Some clinical anti-tumor drugs achieve therapeutic purposes by regulating the content of H2O2 in cells. Understanding the role of H2O2 in tumor cells helps to deeply understand the treatment of tumors. Therefore, highly sensitive and selective hydrogen peroxide detection methods are particularly important.

[0004] Fluorescent probe detection methods are widely favored by scientific researchers due to their advantages such as high sensitivity and low toxicity. In organoid culture, matrix gels (such as Matrigel) are widely used because of their biocompatibility and structural support, but their autofluorescence will interfere with fluorescence detection. Traditional fluorescent probe detection requires the removal of matrix gel through enzymatic digestion or chemical treatment, but these steps may damage the organoids or introduce experimental variables. Summary of the Invention

[0005] The purpose of the present invention is to provide a small molecule near-infrared probe responsive to hydrogen peroxide, a preparation method thereof, and an application as a tumor organoid imaging probe. The small molecule near-infrared probe responsive to hydrogen peroxide provided by the present invention has a longer emission wavelength and can be directly detected without eliminating the matrix gel.

[0006] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a small molecule near-infrared probe responsive to hydrogen peroxide, and its structure is shown in formula A:

[0008]

[0009] The present invention also provides a preparation method of the hydrogen peroxide-responsive small molecule near-infrared probe described in the above solution, including the following steps:

[0010] Mix indole-hemi-cyanine, pentafluorophenylsulfonyl chloride and an acid-binding agent for a hydroxyl substitution reaction to obtain the hydrogen peroxide-responsive small molecule near-infrared probe; the structure of the indole-hemi-cyanine is shown in Formula B:

[0011]

[0012] Preferably, the mass ratio of the indole-hemi-cyanine to the pentafluorophenylsulfonyl chloride is (0.9-1.1):(1-1.1); the mass ratio of the indole-hemi-cyanine to the acid-binding agent is 1:(0.5-2).

[0013] Preferably, the temperature of the hydroxyl substitution reaction is 23-27 °C, and the heat preservation time is 4.5-5.5 h; the hydroxyl substitution reaction is carried out in a protective atmosphere.

[0014] Preferably, the preparation method of the indole-hemi-cyanine includes the following steps: mixing compound 1, compound 4, an alcohol solvent and piperidine for a reflux reaction; the structure of compound 1 is shown in Formula I:

[0015]

[0016] The structure of the compound 4 is shown in Formula IV:

[0017]

[0018] Preferably, the preparation method of the compound 1 includes the following steps: mixing 2,3,3-trimethyl-3H-indole and 1,3-propanesultone for a nucleophilic addition reaction.

[0019] Preferably, the preparation method of the compound 4 includes the following steps: mixing boron tribromide, compound 3 and a substituted hydrocarbon solvent for a hydroxylation reaction; the structure of the compound 3 is shown in Formula III:

[0020]

[0021] Preferably, the temperature of the hydroxylation reaction is 23-27 °C, and the heat preservation time is 33-37 h; the hydroxylation reaction is carried out under stirring conditions.

[0022] Preferably, the preparation method of the compound 3 includes the following steps: mixing 2-chlorocyclohexene-1-carbaldehyde, 4-methoxy-2-hydroxybenzaldehyde, cesium carbonate and an amide solvent for a Knoevenagel condensation-substitution cyclization tandem reaction; the structure of the 2-chlorocyclohexene-1-carbaldehyde is shown in Formula II:

[0023]

[0024] The present invention also provides the use of the small molecule near-infrared probe responsive to hydrogen peroxide described in the above scheme or the small molecule near-infrared probe responsive to hydrogen peroxide obtained by the preparation method described in the above scheme as a tumor organoid imaging probe.

[0025] The present invention provides a small molecule near-infrared probe that responds to hydrogen peroxide. The small molecule near-infrared probe that responds to hydrogen peroxide provided by the present invention uses pentafluorophenylsulfonyl chloride as a recognition unit, and the indolesulfonate structure in the molecule is more likely to interact with hydrogen peroxide, thereby achieving efficient and rapid detection of hydrogen peroxide. In addition, the indole salt structure enables it to have good cell membrane permeability, providing conditions for the in vivo detection of hydrogen peroxide. The small molecule near-infrared probe that responds to hydrogen peroxide provided by the present invention has a long emission wavelength, and the near-infrared fluorescence emission exceeds 720nm. It can penetrate the matrix gel layer to achieve imaging of living cells, and is not easily interfered by the fluorescence background of the matrix gel itself in the detection of living cells, and can be directly detected without eliminating the matrix gel. Practice has shown that the small molecule near-infrared probe that responds to hydrogen peroxide provided by the present invention exhibits high selectivity and sensitivity in hydrogen peroxide detection, has strong specificity and selectivity, a detection limit of 0.88μM, good biocompatibility, and good water solubility. In addition, the small molecule near-infrared probe that responds to hydrogen peroxide provided by the present invention can resist the interference of various ions, such as O2 - 、Na + , K + , NaClO, TBHP (tert-butyl peroxide) and GSH (glutathione).

[0026] The present invention also provides a method for preparing the hydrogen peroxide-responsive small molecule near-infrared probe described in the above scheme. In the present invention, pentafluorophenylsulfonyl chloride is connected to the skeleton of indole-hemicyanine through a hydroxyl substitution reaction to construct a fluorine-containing hydrogen peroxide-responsive small molecule near-infrared probe. The preparation method provided by the present invention has simple steps, good stability, and is suitable for industrial production.

[0027] The present invention also provides the use of the hydrogen peroxide-responsive small molecule near-infrared probe described in the above scheme or the hydrogen peroxide-responsive small molecule near-infrared probe obtained by the preparation method described in the above scheme as a tumor organoid imaging probe. The hydrogen peroxide-responsive small molecule near-infrared probe provided by the present invention can be used for hydrogen peroxide detection, especially the detection of hydrogen peroxide content in cells or tumor organoids, and can realize near-infrared fluorescence imaging of human ovarian cancer cells SKOV-3 and their organoids, with excellent indicators, and can also be directly used for the detection of organoids containing matrix glue, realizing non-destructive detection, avoiding organoid damage that may be caused by degumming treatment, and avoiding the introduction of additional experimental variables, and is simple to operate, and has important application significance in the fields of materials science and biomedicine. Brief Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0029] Figure 1 1H NMR spectrum of Hcy-SO3-5F prepared for Example 1 1 ;

[0030] Figure 2 19F NMR spectrum of Hcy-SO3-5F prepared for Example 1 19 ;

[0031] Figure 3 Absorption spectra of Hcy-SO3-5F before and after reaction with hydrogen peroxide at different concentrations; where the concentrations of hydrogen peroxide are 0, 50, 100, and 200 μM;

[0032] Figure 4 Fluorescence emission spectra of Hcy-SO3-5F before and after reaction with hydrogen peroxide at different concentrations; where the concentrations of hydrogen peroxide are 0, 20, 50, 100, 150, and 200 μM;

[0033] Figure 5 Time-dependent fluorescence intensity spectra of Hcy-SO3-5F at 720 nm before and after adding hydrogen peroxide;

[0034] Figure 6 Fluorescence change spectra of Hcy-SO3-5F at 720 nm before and after adding hydrogen peroxide; where the concentrations of hydrogen peroxide are 0, 20, 50, 100, 150, and 200 μmol / L;

[0035] Figure 7 Time-dependent fluorescence intensity spectra of Hcy-SO3-5F at 720 nm before and after adding hydrogen peroxide;

[0036] Figure 8 Bar charts of fluorescence intensity of Hcy-SO3-5F at 720 nm after reaction with other amino acids, common cations, and anions; where the samples are Na + , NaClO, K + , TBHP, GSH, and O2 - ;

[0037] Figure 9Cytotoxicity diagram of Hcy-SO3-5F in ovarian cancer cell line SKOV-3; SKOV-3 cells were incubated with 100 μM H2O2 for 1 h and then washed, followed by incubation with 10 μM Hcy-SO3-5F for 1 h.

[0038] Figure 10 Fluorescence effect diagram of Hcy-SO3-5F and hydrogen peroxide in human ovarian cancer cell line SKOV-3; among them, (B) is the fluorescence imaging diagram of SKOV-3 cells incubated with 100 μL H2O2 for 1 h, then washed, and then incubated with 10 μM Hcy-SO3-5F for 1 h. (E) is the fluorescence imaging diagram of SKOV-3 cells incubated with a mixture of 100 μL H2O2 and 10 μM Hcy-SO3-5F for 1 h. (H) is the fluorescence imaging diagram of SKOV-3 cells incubated with 10 μM Hcy-SO3-5F for 1 h. (A), (D), (G) are the bright-field imaging diagrams of (B), (E), (H) respectively, and (C), (F), (I) are the superimposed diagrams of the fluorescence imaging and bright-field imaging of (B), (E), (H) and (A), (D), (G) respectively.

[0039] Figure 11 Fluorescence effect diagram of Hcy-SO3-5F and hydrogen peroxide in organoids; among them, (B) is the fluorescence imaging diagram of organoids incubated with 10 μM Hcy-SO3-5F for 1 h under the condition of retaining Matrigel. (D) is the fluorescence imaging diagram of organoids incubated with 10 μL H2O2 for 1 h under the condition of retaining Matrigel, then washed, and then incubated with 10 μM Hcy-SO3-5F for 1 h. (A) is the bright-field imaging diagram of (B), and (C) is the bright-field imaging diagram of (D).

[0040] Figure 12 Synthetic route of the hydrogen peroxide-responsive small molecule near-infrared probe provided by the present invention. Detailed implementation mode

[0041] The present invention provides a hydrogen peroxide-responsive small molecule near-infrared probe, the structure of which is shown in Formula A:

[0042]

[0043] In the present invention, the molecular formula of the hydrogen peroxide-responsive small molecule near-infrared probe is C 34 H 28 F5NO7S2.

[0044] The present invention also provides a preparation method of the hydrogen peroxide-responsive small molecule near-infrared probe described in the above scheme, including the following steps:

[0045] Mix indole - hemicyanine, pentafluorophenylsulfonyl chloride and an acid - binding agent to carry out a hydroxyl - substitution reaction to obtain the small - molecule near - infrared probe responsive to hydrogen peroxide.

[0046] The preparation method of the small - molecule near - infrared probe responsive to hydrogen peroxide provided by the present invention has a synthesis route as Figure 12 shown. In the present invention, indole - hemicyanine, pentafluorophenylsulfonyl chloride and an acid - binding agent are mixed to obtain a reaction solution. In the present invention, the structure of the indole - hemicyanine is as shown in Formula B:

[0047]

[0048] In the present invention, the preparation method of the indole - hemicyanine may include the following steps: Mix compound 1, compound 4, an alcohol solvent and piperidine and carry out a reflux reaction.

[0049] In the present invention, the structure of the compound 1 is as shown in Formula I:

[0050]

[0051] In the present invention, the preparation method of the compound 1 may include the following steps: Mix 2,3,3 - trimethyl - 3H - indole and 1,3 - propane sultone to carry out a nucleophilic addition reaction.

[0052] In the present invention, the molar ratio of 2,3,3 - trimethyl - 3H - indole to 1,3 - propane sultone may be (0.9 - 1.1):(0.9 - 1.1), specifically 1:1.

[0053] In the present invention, the temperature of the nucleophilic addition reaction may be 110 - 125 °C, specifically 120 °C, and the heat - preservation time may be 20 - 25 h, specifically 24 h; the nucleophilic addition reaction may be carried out in a protective atmosphere; the protective atmosphere may be nitrogen; the nucleophilic addition reaction may be carried out under solvent - free conditions; the equipment for the nucleophilic addition reaction may be a pressure - resistant bottle.

[0054] In the present invention, after the nucleophilic addition reaction, it may further include successively cooling, removing the solvent and precipitating the obtained reaction system; the final temperature of the cooling may be 20 - 35 °C; the removal of the solvent may be rotary evaporation; the temperature of the rotary evaporation may be 40 - 45 °C, and the vacuum degree may be - 0.07 - - 0.09 MPa; the reagent for precipitation may be an ether; the ether may be petroleum ether.

[0055] In the present invention, the structure of the compound 4 is as shown in Formula IV:

[0056]

[0057] In the present invention, the preparation method of the compound 4 may include the following steps: boron tribromide, the compound 3 and a substituted hydrocarbon solvent are mixed (denoted as the first mixing) for a hydroxylation reaction.

[0058] In the present invention, the structure of the compound 3 is shown in Formula III:

[0059]

[0060] In the present invention, the preparation method of the compound 3 may include the following steps: 2-chlorocyclohexene-1-carbaldehyde (denoted as the compound 2), 4-methoxy-2-hydroxybenzaldehyde, cesium carbonate and an amide solvent are mixed (denoted as the second mixing) for a Knoevenagel condensation-substitution cyclization tandem reaction.

[0061] In the present invention, the structure of the 2-chlorocyclohexene-1-carbaldehyde is shown in Formula II:

[0062]

[0063] In the present invention, the preparation method of the 2-chlorocyclohexene-1-carbaldehyde may include the following steps: phosphorus oxychloride and a good solvent are mixed (denoted as the third mixing) to obtain a Vilsmeier reagent, and the Vilsmeier reagent and cyclohexanone are mixed to successively carry out a Vilsmeier-Haack formylation reaction and a chlorination reaction to obtain 2-chlorocyclohexene-1-carbaldehyde. In the present invention, the Vilsmeier reagent is used to carry out formylation on cyclohexanone, and then cyclohexanone is isomerized to form an enol form for chlorination reaction, and finally 2-chlorocyclohexene-1-carbaldehyde is prepared.

[0064] In the present invention, the volume molar ratio of the phosphorus oxychloride to cyclohexanone may be (7.5 - 8.1) mL : (48 - 52) mmol, specifically 8 mL : 50 mmol.

[0065] In the present invention, the good solvent may be an amide solvent; the amide solvent may be N,N-dimethylformamide (DMF); the amide solvent may further include drying before use.

[0066] In the present invention, the mass ratio of the phosphorus oxychloride to the good solvent may be (1.14 - 1.17) : 1, specifically 1.16 : 1.

[0067] In the present invention, the third mixing may be: under an ice bath condition, the good solvent is added to a reaction vessel with a syringe, phosphorus oxychloride is sucked with a pipette and dropped into the reaction vessel for stirring, and cyclohexanone is added to the reaction vessel in batches.

[0068] In the present invention, the dropping rate may be 0.04 - 0.06 mL / s, specifically 0.05 mL / s; the stirring time may be 30 min; the number of times of adding in batches may be 3 - 4 times.

[0069] In the present invention, the temperature of the Vilsmeier - Haack formylation reaction and the chlorination reaction may be 0 - -2 °C, specifically 0 °C, and the heat preservation time may be 3.5 - 4.5 h, specifically 4 h; the Vilsmeier - Haack formylation reaction and the chlorination reaction may be carried out under stirring conditions; the Vilsmeier - Haack formylation reaction and the chlorination reaction may be carried out in a protective atmosphere; the protective atmosphere may be nitrogen.

[0070] In the present invention, after the chlorination reaction, it may further include adding the obtained reaction system to ice water and successively carrying out neutralization, extraction, separation of the organic phase, water washing, drying and distillation.

[0071] In the present invention, the target pH value of the neutralization may be 7; the reagent for neutralization may be saturated sodium carbonate solution; the reagent for extraction may be ethyl acetate; the equipment for separating the organic phase may be a separating funnel; the drying may be drying with anhydrous sodium sulfate; the temperature of the distillation may be 40 - 45 °C, the vacuum degree may be -0.07 - -0.09 MPa, and the heat preservation time may be 15.5 - 16.5 h.

[0072] In the present invention, the molar ratio of 2 - chlorocyclohexene - 1 - carbaldehyde to 4 - methoxy - 2 - hydroxybenzaldehyde may be (3.1 - 3.2):(1.9 - 2.1), specifically 3:2.

[0073] In the present invention, the molar ratio of 2 - chlorocyclohexene - 1 - carbaldehyde to cesium carbonate may be (0.9 - 1.1):(1.9 - 2.1), specifically 1:2. In the present invention, cesium carbonate is added to achieve the target transformation by promoting β - elimination or nucleophilic substitution.

[0074] In the present invention, the amide solvent may include N,N - dimethylformamide (DMF); before using the amide solvent, it may further include drying.

[0075] In the present invention, the molar volume ratio of 2 - chlorocyclohexene - 1 - carbaldehyde to the amide solvent may be (2.9 - 3.1) mol:(3.95 - 4.05) L, specifically 3 mol:4 L.

[0076] In the present invention, the second mixing may be: adding an amide solvent into a reaction vessel with a syringe, and then adding 2-chlorocyclohexene-1-carbaldehyde, 4-methoxy-2-hydroxybenzaldehyde and cesium carbonate into the reaction vessel.

[0077] In the present invention, the reaction vessel may be a pressure-resistant bottle.

[0078] In the present invention, the temperature of the Knoevenagel condensation-substitution cyclization tandem reaction may be 23 to 27 °C, specifically 25 °C, and the heat preservation time may be 15.5 to 16.5 h, specifically 16 h; the Knoevenagel condensation-substitution cyclization tandem reaction may be carried out under stirring conditions.

[0079] In the present invention, after the Knoevenagel condensation-substitution cyclization tandem reaction, it may further include successively performing solid-liquid separation, collecting the filtrate, washing with water, extraction, collecting the organic phase, drying and purification on the obtained system.

[0080] In the present invention, the solid-liquid separation may be filtration; the extraction may be alternately carried out with dichloromethane and ethyl acetate; the drying may include successively performing drying with anhydrous NaSO4 and rotary evaporation; the temperature of the rotary evaporation may be 40 to 45 °C, the vacuum degree may be -0.07 to -0.09 MPa, and the heat preservation time may be 15.5 to 16.5 h, specifically 16 h; the purification may be silica gel column chromatography separation; the reagents used for the silica gel column chromatography separation include petroleum ether and ethyl acetate; the volume ratio of the petroleum ether to the ethyl acetate may be 2:1.

[0081] In the present invention, the molar ratio of boron tribromide to compound 3 may be (19.9 to 20.1):(0.9 to 1.1), specifically 20:1.

[0082] In the present invention, the substituted hydrocarbon solvent may be a halogenated hydrocarbon; the halogenated hydrocarbon may be a halogenated methane; the halogenated methane may be dichloromethane.

[0083] In the present invention, the mass ratio of boron tribromide to the substituted hydrocarbon solvent may be (1.9 to 2.0):(16 to 25), specifically 1.95:24, 1.95:21 or 1.95:17.

[0084] In the present invention, the first mixing may be: dropping boron tribromide into a solution of compound 3 under an ice bath condition; the solution of compound 3 includes compound 3 and the substituted hydrocarbon solvent.

[0085] In the present invention, the dropping rate may be 50 to 60 drops / min, specifically 55 drops / min.

[0086] In the present invention, the temperature of the hydroxylation reaction can be 23-27°C, specifically 24°C, 25°C or 26°C, and the insulation time can be 33-37h, specifically 36h; the hydroxylation reaction can be carried out under stirring conditions.

[0087] In the present invention, after the hydroxylation reaction, the obtained system may be mixed with ice water and then solid-liquid separation and purification may be performed in sequence; the solid-liquid separation may be filtration; the purification may be chromatographic column separation; the eluent used for the chromatographic column separation includes dichloromethane and methanol; the volume ratio of dichloromethane to methanol may be 20:1.

[0088] In the present invention, the molar ratio of compound 1 to compound 4 may be (1.4-1.6):(0.9-1.1), specifically 1.5:1.

[0089] In the present invention, the alcohol solvent may be a C1-C5 alcohol; and the C1-C5 alcohol may be ethanol.

[0090] In the present invention, the mass ratio of the compound 1 to the alcohol solvent may be (1.085-1.090):(8.590-10.257), specifically 1.089:9.863.

[0091] In the present invention, the molar ratio of the compound 1 to piperidine can be (3.870-3.875):(0.246-0.492), specifically 2.458:0.369. In the present invention, piperidine is added, and piperidine is used as a catalyst to capture the α-hydrogen of the methylene group to form a nucleophilic reagent to attack the carbonyl carbon.

[0092] In the present invention, the reflux reaction time may be 11 to 13 hours, specifically 12 hours.

[0093] In the present invention, after the reflux reaction, the obtained reaction system may be cooled, desolventized and purified in sequence; the final cooling temperature may be 20 to 35° C.; the desolventization may be spin drying; the spin drying temperature may be 40 to 45° C., and the vacuum degree may be -0.07 to -0.09 MPa; the purification may be chromatographic column separation; the eluent used for the chromatographic column separation may include dichloromethane and methanol; the volume ratio of dichloromethane to methanol may be 100:(8 to 10).

[0094] In the present invention, the mass ratio of the indole-hemicyanine to pentafluorophenylsulfonyl chloride can be (0.9-1.1):(1-1.1), specifically 0.9:1, 1:1, 1.1:1, 0.9:1.05, 1:1.05, 1.1:1.05, 0.9:1.1 or 1:1.1.

[0095] In the present invention, the acid-binding agent may include triethylamine; the mass ratio of the indole-hemi-cyanine to the acid-binding agent may be 1:(0.40 - 0.45), specifically 1:0.40, 1:0.41, 1:0.42, 1:0.43, 1:0.44 or 1:0.45.

[0096] After mixing, the present invention subjects the reaction solution to a hydroxyl substitution reaction to obtain the small molecule near-infrared probe responsive to hydrogen peroxide. In the present invention, the temperature of the hydroxyl substitution reaction may be 23 - 27 °C, specifically 25 °C, and the heat preservation time may be 4.5 - 5.5 h, specifically 5 h; the hydroxyl substitution reaction may be carried out in a protective atmosphere; the protective atmosphere may be nitrogen; the hydroxyl substitution reaction may be carried out in an anhydrous halogenated alkane solvent; the halogenated alkane solvent may be dichloromethane (DCM).

[0097] In the present invention, after the hydroxyl substitution reaction, it may further include purifying the obtained reaction product; the purification may be silica gel column chromatography separation; the eluent used for the silica gel column chromatography separation may include dichloromethane and methanol; the volume ratio of dichloromethane to methanol may be 100:8.

[0098] The present invention also provides the application of the small molecule near-infrared probe responsive to hydrogen peroxide described in the above solution or the small molecule near-infrared probe responsive to hydrogen peroxide obtained by the preparation method described in the above solution as a tumor organoid imaging probe.

[0099] The small molecule near-infrared probe responsive to hydrogen peroxide provided by the present invention can qualitatively detect hydrogen peroxide, including in vivo hydrogen peroxide or in vitro hydrogen peroxide, by combining with a fluorescence spectrometer, and can achieve fluorescence imaging of human ovarian cancer cells SKOV-3 or organoids, and is used as a tumor organoid imaging probe.

[0100] In the present invention, when detecting in vivo hydrogen peroxide, the method of the application may include the following steps: mixing Hcy-SO3-5F with dimethyl sulfoxide to obtain a probe mother liquor, adding the probe mother liquor to cells or organoids for incubation, and then performing fluorescence imaging with a confocal microscope.

[0101] In the present invention, the molar concentration of the probe mother liquor may be 0.9 - 1.1 mM, specifically 1 mM.

[0102] In the present invention, the incubation time may be 55 min - 65 min, specifically 1 h.

[0103] In the present invention, when detecting hydrogen peroxide in vitro, the method applied may include the following steps: mixing Hcy-SO3-5F with dimethyl sulfoxide to obtain a probe mother liquor, adding the probe mother liquor to the solution to be tested, testing the fluorescence spectrum of the solution to be tested with a fluorescence spectrometer, and observing the change in the fluorescence intensity of the solution to be tested before and after adding Hcy-SO3-5F.

[0104] In the present invention, the molar concentration of the probe mother liquor may be 0.9 - 1.1 mM, specifically 1 mM.

[0105] In the present invention, the molar concentration of the solution to be tested may not be greater than 200 μM, specifically 50 μM or 120 μM; the volume ratio of the probe mother liquor to the solution to be tested may be (9 - 10):(1000 - 1010), specifically 9:1000, 9.5:1000, 10:1000, 9:1005, 9.5:1005, 10:1005, 9:1010, 9.5:1010 or 10:1010.

[0106] In the present invention, the excitation wavelength of the fluorescence spectrometer may be 680 nm; the change in fluorescence intensity refers to the change in the fluorescence peak at 720 nm in the fluorescence spectrum; hydrogen peroxide in the solution to be tested can cause a change in the fluorescence spectrum of the fluorescent probe. By observing the change in the fluorescence spectrum in the fluorescence spectrometer, the content of hydrogen peroxide in the solution can be judged, so as to perform quantitative detection. The linear relationship curve between the concentration (X) of hydrogen peroxide and the fluorescence intensity (Y) is as follows:

[0107] Y = 4.57497×X + 100.70512, R 2 = 0.99079.

[0108] When there is no hydrogen peroxide in the solution to be tested, there is no fluorescence emission peak at the maximum excitation wavelength of 680 nm. When there is hydrogen peroxide in the solution to be tested, at the maximum excitation wavelength of 680 nm, the maximum fluorescence emission wavelength is 720 nm.

[0109] To further illustrate the present invention, the solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0110] Example 1 Synthesis of a Small Molecule Near-Infrared Probe Responsive to Hydrogen Peroxide

[0111] (1) Synthesis of Compound 1:

[0112]

[0113] 2.3.3-Trimethyl-3H-indole and 1,3-propane sultone were added into a pressure-resistant bottle in a molar ratio of 1:1. Without solvent and under N2 protection, the mixture was heated at 120 °C for 24 h. After the reaction, it was cooled to room temperature (25 °C), and the solvent was removed under reduced pressure at 45 °C and -0.08 MPa. The residue was precipitated with petroleum ether to obtain Compound 1.

[0114] (2) Synthesis of Compound 2:

[0115]

[0116] 8 mL of dry DMF was added into a two-necked flask. Under ice bath and N2 protection, 7.7 mL of phosphorus oxychloride was added dropwise into the two-necked flask at a rate of 0.05 mL / s. After ice bath for 30 min, 50 mmol of cyclohexanone was weighed and added into the system in three portions, and the mixture was stirred at 0 °C for 4 h. After the reaction, the reaction system was added into ice water, neutralized to pH 7 with saturated sodium carbonate solution, and the organic phase was extracted with ethyl acetate using a separatory funnel, washed with brine and dried. The solvent was rotary evaporated under reduced pressure at 43 °C and -0.08 MPa for 16 h to obtain Compound 2 as a yellow liquid.

[0117] (3) Synthesis of Compound 3:

[0118]

[0119] 15 mmol of Compound 2, 10 mmol of 4-methoxy-2-hydroxybenzaldehyde, and 30 mmol of cesium carbonate were added into a pressure-resistant bottle and dissolved in 20 mL of anhydrous DMF. The mixture was stirred at 25 °C for 16 h. After the reaction, the reaction solution was filtered, washed with water to remove DMF, and extracted alternately with dichloromethane and ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate. The organic phase was rotary evaporated under reduced pressure at 42 °C and -0.08 MPa for 16 h, and purified by silica gel column chromatography (the volume ratio of petroleum ether to ethyl acetate was 2:1) to obtain Compound 3.

[0120] (4) Synthesis of Compound 4:

[0121]

[0122] Under ice bath conditions, 0.75 mL of boron tribromide was added dropwise into a dichloromethane solution of Compound 3 (5 mmol) at a rate of 55 drops / min, and the mixture was stirred at 25 °C for 36 h. After the reaction, the reaction solution was poured into ice water, and the crude product was obtained by filtration. After filtration, the crude product was separated by column chromatography, and the eluent was dichloromethane and methanol with a volume ratio of 20:1 to obtain Compound 4.

[0123] (5) Synthesis of indole-semicyanine:

[0124]

[0125] Under N2 protection, compound 1 (1089 mg) and compound 4 (561 mg) were dissolved in 9.863 g of ethanol, and then 31.4 mg of piperidine was added and the mixture was refluxed for 12 h. After the reaction was completed, it was cooled to room temperature, and the solvent was removed by rotary evaporation at 42 °C and -0.08 MPa. The crude product was separated by column chromatography, and the eluent was dichloromethane and methanol with a volume ratio of 100:9 to obtain indole-semi-cyanine, denoted as compound 5.

[0126] (6) Synthesis of compound Hcy-SO3-5F:

[0127]

[0128] Under N2 protection, compound 5 (77.3 mg), pentafluorophenylsulfonyl chloride (80.0 mg), and triethylamine (44.8 μL) were added to a 25 mL pressure-resistant flask and dissolved in anhydrous DCM (4.5 mL), and stirred at 25 °C for 5 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and separation and purification were carried out by silica gel column chromatography (the eluent was dichloromethane and methanol, and the volume ratio of dichloromethane:methanol was 100:8) to obtain Hcy-SO3-5F as a purple solid, and its 1 1H NMR spectrum and 19 19F NMR spectrum were detected, and the results are as Figure 1 and Figure 2 shown.

[0129] 1 1H NMR detection results: 1 1H NMR (400 MHz, DMSO) δ 8.54 (d, J = 15.2 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.81 - 7.74 (m, 1H), 7.61 - 7.49 (m, 4H), 7.32 (s, 1H), 7.14 (dd, J = 8.4, 2.4 Hz, 1H), 6.96 (d, J = 15.3 Hz, 1H), 4.67 (t, J = 7.8 Hz, 2H), 2.71 (d, J = 5.8 Hz, 5H), 2.60 (t, J = 6.4 Hz, 3H), 2.10 (t, J = 7.6 Hz, 2H), 1.99 (dt, J = 14.0, 6.9 Hz, 2H).

[0130] 19 19F NMR detection results: 19 19F NMR (377 MHz, DMSO) δ -135.28 (dt, J = 20.7, 10.3 Hz), -142.23 (tt, J = 23.2, 8.3 Hz), -157.89 - -158.12 (m).

[0131] According to Figure 1 and Figure 2 it can be seen that the Hcy-SO3-5F target molecule of the present invention has been successfully synthesized.

[0132] Test Example 1 Absorbance changes of the probe Hcy-SO3-5F before and after reaction with hydrogen peroxide

[0133] Take the probe Hcy-SO3-5F prepared in Example 1 and dissolve it in DMSO to obtain a 1 mM probe stock solution; prepare a hydrogen peroxide stock solution with a concentration of 50 mM and dilute it separately; take two centrifuge tubes, add 20 μL of the probe stock solution, 980 μL of acetonitrile solution and 980 μL of PBS buffer solution (concentration 100 mmol / L, pH = 7.4) to each centrifuge tube. Add 20 μL of a 20 mM hydrogen peroxide solution to one of them, and add no hydrogen peroxide solution to the other, and use an equal amount of PBS buffer solution as a blank control. Finally, the probe concentration is 10 μM, the hydrogen peroxide concentrations are 0, 50, 100 and 200 μM, and the DMSO content is 1%. First, measure the absorption spectrum to observe the absorbance changes of Hcy-SO3-5F before and after reaction with hydrogen peroxide. The results are as Figure 3 shown.

[0134] According to Figure 3 it can be seen that the maximum absorption wavelength redshifts from 600 nm to 693 nm.

[0135] Test Example 2 Fluorescence changes of the probe Hcy-SO3-5F before and after reaction with hydrogen peroxide

[0136] Take the probe Hcy-SO3-5F prepared in Example 1 and dissolve it in DMSO to obtain a 1 mM probe stock solution; prepare a hydrogen peroxide stock solution with a concentration of 50 mM and dilute it separately; take four centrifuge tubes, add 20 μL of the probe stock solution, 980 μL of acetonitrile solution and 980 μL of PBS buffer solution (concentration 100 mmol / L, pH = 7.4) to each centrifuge tube. Then add 20 μL of hydrogen peroxide solutions with concentrations of 50 mM, 20 mM, 5 mM respectively, and add no hydrogen peroxide solution to one of them, and use an equal amount of PBS buffer solution as a blank control. Finally, the probe concentration is 10 μM, the hydrogen peroxide concentrations are 200 μM, 150 μM, 100 μM, 50 μM, 20 μM, 0 μM, and the DMSO content is 1%. First, measure the absorption spectrum to observe the absorbance changes of Hcy-SO3-5F before and after reaction with hydrogen peroxide. The results are as Figure 4 shown.

[0137] According to Figure 4 it can be seen that when there is no hydrogen peroxide, there is almost no fluorescence in the system. After adding hydrogen peroxide, the fluorescence intensity of the system increases, and the fluorescence is the strongest at 720 nm.

[0138] Test Example 3 Fluorescence intensity at 720 nm vs. time

[0139] Take the probe Hcy-SO3-5F prepared in Example 1 and dissolve it in DMSO to obtain a 1 mM mother liquor of the probe; prepare a mother liquor of hydrogen peroxide with a concentration of 50 mM and dilute it separately; take four centrifuge tubes, add 20 μL of the probe mother liquor, 980 μL of acetonitrile solution and 980 μL of PBS buffer solution (concentration 100 mmol / L, pH = 7.4) to each centrifuge tube, then add 20 μL of hydrogen peroxide solutions with concentrations of 50 mM, 20 mM, 5 mM respectively, and one tube without hydrogen peroxide solution, replace it with an equal amount of PBS buffer solution as a blank control. Finally, the probe concentration is 10 μM, the hydrogen peroxide concentrations are 500 μM, 200 μM, 50 μM, 0 μM, and the DMSO content is 1%. Use an excitation wavelength of 680 nm to test the fluorescence spectrum vs. time, and the results are as Figure 5 shown.

[0140] According to Figure 5 it can be seen that as time increases, the fluorescence at 720 nm gradually increases.

[0141] Test Example 4 Absorbance of probe Hcy-SO3-5F vs. time before and after reaction with hydrogen peroxide

[0142] Take the probe Hcy-SO3-5F prepared in Example 1 and dissolve it in DMSO to obtain a 1 mM mother liquor of the probe; prepare a mother liquor of hydrogen peroxide with a concentration of 50 mM and dilute it separately; take four centrifuge tubes, add 20 μL of the probe mother liquor, 980 μL of acetonitrile solution and 980 μL of PBS buffer solution (concentration 100 mmol / L, pH = 7.4) to each centrifuge tube, then add 20 μL of hydrogen peroxide solutions with different concentrations, and one tube without hydrogen peroxide solution, replace it with an equal amount of PBS buffer solution as a blank control. Finally, the probe concentration is 10 μM, the hydrogen peroxide concentrations are 200 μM, 150 μM, 100 μM, 50 μM, 20 μM, 0 μM, and the DMSO content is 1%. Use an excitation wavelength of 680 nm to test the fluorescence spectrum changes of the reaction solution of the probe with hydrogen peroxide at different concentrations, and the results are as Figure 6 shown.

[0143] According to Figure 6 it can be seen that as the concentration of hydrogen peroxide increases, the fluorescence value of the probe Hcy-SO3-5F at 720 nm gradually increases; fit the linear curve of the relationship between hydrogen peroxide concentration and fluorescence intensity, Y = 4.57497x + 100.70512, R 2 = 0.99079; according to the detection limit calculation formula: LOD = k×Sb / S, the lowest detection limit is calculated to be 0.88 μM.

[0144] Test Example 5

[0145] Take the probe Hcy-SO3-5F prepared in Example 1 and dissolve it in DMSO to obtain a 1 mM probe mother liquor; prepare a test mother liquor of hydrogen peroxide with a concentration of 50 mM and dilute it respectively; take four centrifuge tubes, add 20 μL of the probe mother liquor, 980 μL of acetonitrile solution and 980 μL of PBS buffer solution (concentration of 100 mmol / L, pH = 7.4) to each centrifuge tube, and then add 20 μL of hydrogen peroxide solutions with concentrations of 50 mM, 20 mM, 5 mM respectively, and one tube without adding hydrogen peroxide solution, replace it with an equal amount of PBS buffer solution as a blank control. Finally, the probe concentration is 10 μM, the hydrogen peroxide concentration is 500 μM, 200 μM, 50 μM, 0 μM, and the DMSO content is 1%. Use an excitation wavelength of 680 nm to test the absorption spectrum diagram changing with time, and the results are as Figure 7 shown.

[0146] According to Figure 7 it can be seen that as time increases, the absorption first increases and then decreases.

[0147] Test Example 6

[0148] Take the probe Hcy-SO3-5F prepared in Example 1 and dissolve it in DMSO to obtain a 0.5 mM probe mother liquor; prepare a test mother liquor of hydrogen peroxide with a concentration of 5 mM and dilute it respectively; take eight centrifuge tubes, add 10 μL of the probe mother liquor, 500 μL of acetonitrile solution and 400 μL of PBS buffer solution (concentration of 100 mmol / L, pH = 7.4) to each centrifuge tube, and then add 100 μL of hydrogen peroxide, Na + , NaClO, K + , TBHP, GSH, O2 - solutions respectively, and one tube without adding hydrogen peroxide solution, replace it with 100 μL of PBS buffer solution as a blank control. Finally, the probe concentration is 5 μM, the hydrogen peroxide concentration is 500 μM, and the DMSO content is 1%. Use an excitation wavelength of 680 nm to test the absorption spectrum diagram, and the results are as Figure 8 shown.

[0149] According to Figure 8 it can be seen that the fluorescence intensity of the test solutions added with various ions and amino acids does not change significantly; however, the fluorescence intensity of the test solution added with hydrogen peroxide increases significantly. The experimental results show that the probe has good selectivity for hydrogen peroxide.

[0150] Test Example 7 Cytotoxicity of Probe Hcy-SO3-5F in Ovarian Cancer Cell SKOV-3

[0151] The probe Hcy-SO3-5F was co-cultured with ovarian cancer cells SKOV-3, and its cytotoxicity was quantitatively evaluated by the MTT colorimetric method. Stock solutions of Hcy-SO3-5F at different concentrations were prepared and co-incubated with SKOV-3 cells for 24 h respectively, aiming to simulate the time span that the probe might encounter in actual applications, so as to more accurately evaluate its long-term effects. The results are as Figure 9 shown.

[0152] According to Figure 9 it can be seen that when the concentration of the probe Hcy-SO3-5F reached 5 μM, after a 24-h culture period, the survival rate of ovarian cancer cells remained above 80%, indicating that the probe Hcy-SO3-5F exhibited low cytotoxicity to cells and also showed good biocompatibility. This finding provides an important safety basis for the further application of Hcy-SO3-5F in the biomedical field.

[0153] Test Example 8 Fluorescence imaging of Hcy-SO3-5F and hydrogen peroxide in human ovarian cancer cells SKOV-3

[0154] The stock solution of the probe at a concentration of 2 mM in Test Example 1 was used. Experimental group A: 10 μL of H2O2 at a concentration of 100 μM was added to a 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 stock solution of the probe was added and incubated for 1 h. Experimental group B: 10 μL of H2O2 at a concentration of 100 μM was added to a culture dish containing SKOV-3 cells, and then 10 μL of the stock solution of the probe was added and incubated for 1 h (the total volume of the culture medium was 2 mL). Control group: 10 μL of the stock solution of the probe was added to a culture dish containing SKOV-3 cells and incubated for 1 h (the total volume of the culture medium was 2 mL). Subsequently, fluorescence imaging was performed using a confocal microscope respectively. The results are as Figure 10 shown.

[0155] According to Figure 10 it can be seen that more obvious fluorescence was observed in the experimental groups compared with the control group, indicating that Hcy-SO3-5F can detect hydrogen peroxide in the cell environment by confocal microscopy.

[0156] Test Example 9 Fluorescence effect of the probe Hcy-SO3-5F and hydrogen peroxide in organoids

[0157] Use the probe stock solution with a concentration of 2 mM in Test Example 1. Experimental group: Under the condition of retaining Matrigel, add 10 μL of H2O2 with a concentration of 100 μM to the culture dish containing organoids and incubate for 1 h (total volume of the culture medium is 2 mL). After 1 h, remove the original culture medium, add 1990 μL of fresh culture medium, and then add 10 μL of the probe stock solution and incubate for 1 h. Control group: Under the condition of retaining Matrigel, add 110 μL of the probe stock solution to the culture dish containing organoids and incubate for 1 h. Subsequently, fluorescence imaging was performed using a confocal microscope, and the results are as Figure 11 shown.

[0158] According to Figure 11 it can be seen that more obvious fluorescence can be observed in the experimental group compared with the control group, indicating that Hcy-SO3-5F can detect hydrogen peroxide in the organoid environment through a confocal microscope.

[0159] As can be seen from the above embodiments, the small molecule near-infrared probe responsive to hydrogen peroxide provided by the present invention has a relatively long emission wavelength, can be directly detected without removing Matrigel, exhibits high selectivity, specificity and sensitivity in hydrogen peroxide detection, has strong specific selectivity, a detection limit of 0.88 μM, good biocompatibility, good water solubility, and can resist the interference of various ions.

[0160] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, not all of them. Other embodiments can be obtained according to these embodiments without creative work, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A small molecule near-infrared probe responsive to hydrogen peroxide, characterized in that, The structure is as shown in Formula A:

2. The preparation method of the small molecule near-infrared probe responsive to hydrogen peroxide according to claim 1, characterized in that, It includes the following steps: Mix indole-hemi-cyanine, pentafluorophenylsulfonyl chloride and an acid-binding agent to carry out a hydroxyl substitution reaction to obtain the small molecule near-infrared probe responsive to hydrogen peroxide; the structure of the indole-hemi-cyanine is as shown in Formula B:

3. The preparation method according to claim 2, characterized in that, The mass ratio of the indole-hemi-cyanine to the pentafluorophenylsulfonyl chloride is (0.9 - 1.1):(1 - 1.1); the mass ratio of the indole-hemi-cyanine to the acid-binding agent is 1:(0.5 - 2).

4. The preparation method according to claim 2, characterized in that, The temperature of the hydroxyl substitution reaction is 23 - 27 °C, and the heat preservation time is 4.5 - 5.5 h. The hydroxyl substitution reaction is carried out in a protective atmosphere.

5. The preparation method according to claim 2, characterized in that, The preparation method of the indole-hemi-cyanine includes the following steps: Mix compound 1, compound 4, an alcohol solvent and piperidine to carry out a reflux reaction; the structure of compound 1 is as shown in Formula I: The structure of compound 4 is as shown in Formula IV:

6. The preparation method according to claim 5, characterized in that, The preparation method of compound 1 includes the following steps: Mix 2,3,3-trimethyl-3H-indole and 1,3-propane sultone to carry out a nucleophilic addition reaction.

7. The preparation method according to claim 5, characterized in that, The preparation method of compound 4 includes the following steps: Mix boron tribromide, compound 3 and a substituted hydrocarbon solvent to carry out a hydroxylation reaction; the structure of compound 3 is as shown in Formula III:

8. The preparation method according to claim 7, wherein, The temperature of the hydroxylation reaction is 23 - 27 °C, and the heat preservation time is 33 - 37 h. The hydroxylation reaction is carried out under stirring conditions.

9. The preparation method according to claim 7, wherein The preparation method of compound 3 includes the following steps: Mix 2-chlorocyclohexene-1-carbaldehyde, 4-methoxy-2-hydroxybenzaldehyde, cesium carbonate and an amide solvent to carry out a Knoevenagel condensation-substitution cyclization tandem reaction; the structure of 2-chlorocyclohexene-1-carbaldehyde is as shown in Formula II:

10. Application of the small molecule near-infrared probe responsive to hydrogen peroxide according to claim 1 or the small molecule near-infrared probe responsive to hydrogen peroxide obtained by the preparation method according to any one of claims 2 - 9 as a tumor organoid imaging probe.

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