Beta-galactosidase response type hemicyanine near-infrared fluorescent probe, preparation method thereof and application of beta-galactosidase response type hemicyanine near-infrared fluorescent probe as tumor organ imaging probe

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a beta-galactosidase response type hemicyanine near-infrared fluorescent probe, a preparation method thereof and application of the beta-galactosidase response type hemicyanine near-infrared fluorescent probe as a tumor organ imaging probe. The near-infrared fluorescent probe provided by the invention takes a water-soluble hemicyanine skeleton as a fluorophore, so that the biocompatibility of the near-infrared fluorescent probe is improved; based on the principle that beta-galactose can be selectively cut by beta-galactosidase, a galactose structure with a self-leaving group is used as a response group, selective quick response to the beta-galactosidase is achieved, and high sensitivity to the beta-galactosidase is shown. The near-infrared fluorescent probe provided by the invention can emit near-infrared fluorescence with long wavelength, has lower background fluorescence and good penetration depth, and can be directly used for detecting organoids containing the matrigel without eliminating the matrigel, so that the organoids are prevented from being damaged or additional experimental variables are prevented from being introduced in the process of eliminating the matrigel.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and specifically relates to a β-galactosidase-responsive hemicyanine near-infrared fluorescent probe (Hcy-SO3-β-Gal), a preparation method thereof, and an application thereof as a tumor organoid imaging probe. Background Art

[0002] Ovarian cancer is a common malignant tumor of the female reproductive system. Due to its insidious early symptoms and lack of specific diagnostic markers, most patients are diagnosed at an advanced stage, resulting in a poor prognosis. Recent studies have revealed that β-galactosidase (β-gal) is abnormally overexpressed in ovarian cancer tissues, and its activity is closely associated with tumor invasion, metastasis, and drug resistance. As a key lysosomal hydrolase, β-galactosidase not only participates in the regulation of the tumor microenvironment but also may promote the malignant progression of ovarian cancer by affecting cellular metabolism and signaling pathways. Highly sensitive and specific β-galactosidase detection methods are of great significance for the early diagnosis, disease monitoring, and development of targeted treatment strategies for ovarian cancer.

[0003] Among existing β-galactosidase detection technologies, fluorescent probes have attracted significant attention due to their high sensitivity, excellent selectivity, and real-time dynamic monitoring capabilities. Compared to traditional colorimetric methods or enzyme-linked immunosorbent assays (ELISAs), fluorescent probes can detect enzyme activity in situ in living cells or tissues, avoiding sample destruction while providing higher spatiotemporal resolution.

[0004] The development of near-infrared (NIR) fluorescent probes has further improved the penetration depth and signal-to-noise ratio of detection, making them more suitable for deep tissue or in vivo imaging. However, in organoid culture, matrix gels (such as Matrigel) are widely used due to their biocompatibility and structural support, and their autofluorescence can interfere with fluorescence detection. Currently, specific fluorescent probes for β-galactosidase in the ovarian cancer microenvironment require the removal of Matrigel for detection, which can damage the organoids or introduce experimental variables. Summary of the Invention

[0005] The object of the present invention is to provide a β-galactosidase-responsive hemicyanine near-infrared fluorescent probe, a preparation method thereof, and an application thereof as a tumor organoid imaging probe. The β-galactosidase-responsive hemicyanine near-infrared fluorescent probe provided by the present invention can be directly used for the detection of organoids containing matrigel without eliminating matrigel.

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

[0007] The present invention provides a β-galactosidase-responsive hemicyanine near-infrared fluorescent probe, the structure of which is shown in Formula A:

[0008]

[0009] The present invention also provides a method for preparing the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe described in the above scheme, comprising the following steps:

[0010] Compound 8, an alcoholysis catalyst, and methanol are mixed to perform an alcoholysis reaction to obtain the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe; the structure of compound 8 is shown in Formula VIII:

[0011]

[0012] Preferably, the molar ratio of the compound 8 to the alcoholysis catalyst is 1:1-5.

[0013] Preferably, the ratio of the amount of compound 8 to the volume of methanol is 1 mmol: (1-2) mL.

[0014] Preferably, the temperature at which the compound 8, the alcoholysis catalyst and methanol are mixed is -1 to 1°C.

[0015] Preferably, the temperature of the alcoholysis reaction is -1 to 1°C, and the insulation time is 1 to 1.5 hours.

[0016] Preferably, the preparation method of compound 8 comprises the following steps: mixing compound 4, compound 7, triethylamine and dichloromethane to perform a Williams reaction to obtain compound 8; the structure of compound 4 is shown in formula IV, and the structure of compound 7 is shown in formula VII:

[0017]

[0018] Preferably, the preparation method of compound 4 comprises the following steps: mixing tripotassium phosphate, resorcinol and anhydrous acetonitrile to obtain solution A, mixing compound 3, anhydrous acetonitrile and anhydrous methanol to obtain solution B, and adding solution B dropwise to solution A to carry out a cyclization reaction to obtain compound 4; the structure of compound 3 is shown in formula III:

[0019]

[0020] Preferably, the preparation method of the compound 7 comprises the following steps: mixing compound 6, phosphorus tribromide and dichloromethane to carry out a halogenation reaction to obtain the compound 7; the structure of the compound 6 is shown in Formula VI:

[0021]

[0022] The present invention also provides the use of the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe described in the above scheme or the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe obtained by the preparation method described in the above scheme as a tumor organoid imaging probe.

[0023] The present invention provides a β-galactosidase-responsive hemicyanine near-infrared fluorescent probe. The near-infrared fluorescent probe provided by the present invention uses a water-soluble hemicyanine skeleton as a fluorophore, which improves the biocompatibility of the near-infrared fluorescent probe; based on the principle that β-galactose can be selectively cut by β-galactosidase, a galactose structure with a self-leaving group is used as a response group, thereby achieving a selective and rapid response to β-galactosidase and showing high sensitivity to β-galactosidase. The near-infrared fluorescent probe provided by the present invention can emit long-wavelength near-infrared fluorescence (710nm), has low background fluorescence and good penetration depth, and is particularly targeted at the matrix gel system widely used in organoid culture. Its long-wavelength emission characteristics can effectively avoid the interference of matrix gel autofluorescence on the detection signal. The near-infrared fluorescent probe provided by the present invention can be directly used for the detection of organoids containing matrix gel without eliminating matrix gel, avoiding organoid damage caused by eliminating matrix gel or introducing additional experimental variables.

[0024] The present invention also provides a method for preparing the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe described in the above scheme. The preparation method provided by the present invention has simple steps, convenient operation, high feasibility, and is suitable for industrial production.

[0025] The present invention also provides the use of the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe described in the above scheme, or the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe obtained by the preparation method described in the above scheme, as a tumor organoid imaging probe. The near-infrared fluorescent probe provided by the present invention can be directly used to detect organoids containing Matrigel without removing Matrigel, thus avoiding damage to the organoids or the introduction of additional experimental variables caused by the Matrigel removal process. It enables non-destructive, in situ near-infrared fluorescence imaging of human ovarian cancer cells SKOV-3 and human tumor organoids, providing a more efficient detection solution that maintains sample integrity for organoid research. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1The near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention 1 HNMR spectrum;

[0028] Figure 2 The near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention 13 C spectrum;

[0029] Figure 3 This is the ultraviolet absorption diagram of the near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention;

[0030] Figure 4 This is the fluorescence image of the near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention;

[0031] Figure 5 This is the fluorescence linear graph of the near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention;

[0032] Figure 6 This is the fluorescence response graph of the near-infrared fluorescent probe Hcy-SO3-β-Gal over time;

[0033] Figure 7 This is the fluorescence response diagram of the near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention to common interfering substances;

[0034] Figure 8 is the survival rate of cells co-incubated with SKOV-3 cells containing different concentrations of the near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention;

[0035] Figure 9 This is a fluorescence imaging diagram of ovarian cancer organoids using the near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention;

[0036] Figure 10 The invention provides a synthetic route for a β-galactosidase-responsive hemicyanine near-infrared fluorescent probe. DETAILED DESCRIPTION

[0037] The present invention provides a β-galactosidase-responsive hemicyanine near-infrared fluorescent probe, the structure of which is shown in Formula A:

[0038]

[0039] The near-infrared fluorescent probe provided by the present invention uses a water-soluble hemicyanine skeleton as a fluorophore, which improves the biocompatibility of the near-infrared fluorescent probe; based on the principle that β-galactose can be selectively cut by β-galactosidase, a galactose structure with a self-leaving group is used as a response group, which realizes a selective and rapid response to β-galactosidase and shows high sensitivity to β-galactosidase. The near-infrared fluorescent probe provided by the present invention can emit long-wavelength near-infrared fluorescence (710nm), has low background fluorescence and good penetration depth, and is particularly suitable for the matrix gel system widely used in organoid culture. Its long-wavelength emission characteristics can effectively avoid the interference of matrix gel autofluorescence on the detection signal. The near-infrared fluorescent probe provided by the present invention can be directly used for the detection of organoids containing matrix gel without eliminating matrix gel, avoiding organoid damage caused by eliminating matrix gel or introducing additional experimental variables.

[0040] The present invention also provides a method for preparing the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe described in the above scheme, comprising the following steps:

[0041] Compound 8, an alcoholysis catalyst, and methanol are mixed to perform an alcoholysis reaction to obtain the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe; the structure of compound 8 is shown in Formula VIII:

[0042]

[0043] The synthetic route of the β-galactosidase responsive hemicyanine near-infrared fluorescent probe provided by the present invention is as follows: Figure 10 In the present invention, compound 8, an alcoholysis catalyst, and methanol are mixed to obtain a reaction solution. In the present invention, the alcoholysis catalyst can be an organic sodium salt; the organic sodium salt can include one or more of sodium methoxide and sodium ethoxide.

[0044] In the present invention, the molar ratio of the compound 8 to the alcoholysis catalyst may be 1:1 to 5, specifically 1:2 or 1:2.5.

[0045] In the present invention, the preparation method of compound 8 may include the following steps: mixing compound 4, compound 7, triethylamine and dichloromethane to perform a Williams reaction to obtain compound 8; the structure of compound 4 is shown in Formula IV, and the structure of compound 7 is shown in Formula VII:

[0046]

[0047] In the present invention, the preparation method of the compound 4 may include the following steps: mixing tripotassium phosphate, resorcinol and anhydrous acetonitrile (denoted as mixture D) to obtain solution A, mixing compound 3, anhydrous acetonitrile and anhydrous methanol (denoted as mixture E) to obtain solution B, and adding the solution B dropwise to the solution A to carry out a cyclization reaction to obtain compound 4; the structure of the compound 3 is shown in Formula III:

[0048]

[0049] In the present invention, the molar ratio of tripotassium phosphate to resorcinol may be 1:1-2, specifically 1:1.5.

[0050] In the present invention, the mass ratio of tripotassium phosphate to anhydrous acetonitrile can be 50 to 60: 1, specifically 55: 1. The present invention uses tripotassium phosphate as a base and anhydrous acetonitrile as a solvent to ensure the progress of the reaction.

[0051] In the present invention, the mixing D can be carried out in a protective atmosphere; the protective atmosphere can be nitrogen; the temperature of the mixing D can be 25-27° C. (room temperature), the mixing time can be 20 min; the mixing D can be stirring mixing.

[0052] In the present invention, the preparation method of compound 3 may include the following steps: mixing compound 1, compound 2, an organic base and acetic anhydride to perform a Knoevenagel condensation reaction to obtain compound 3; the structure of compound 1 is shown in formula I, and the structure of compound 2 is shown in formula II:

[0053]

[0054] In the present invention, the preparation method of compound 1 may include the following steps: mixing phosphorus oxychloride, N,N-dimethylformamide and dichloromethane (denoted as mixture F) to obtain a Vilsmeier reagent, mixing the Vilsmeier reagent with cyclohexanone (denoted as mixture G) to perform a Vilsmeier-Haack reaction to obtain compound 1.

[0055] In the present invention, the N,N-dimethylformamide may be dehydrated before use; the volume ratio of the phosphorus oxychloride to N,N-dimethylformamide may be 3:4-6, specifically 3:5.

[0056] In the present invention, the dichloromethane may be anhydrous dichloromethane; the volume ratio of the phosphorus oxychloride to dichloromethane may be 3:4-6, specifically 3:5.

[0057] In the present invention, the mixing F can be carried out in a protective atmosphere; the protective atmosphere can be nitrogen; the mixing F can be: phosphorus oxychloride is dropped into N,N-dimethylformamide and dichloromethane and stirred; the dropping rate can be 30 to 40 drops / minute, specifically 35 drops / minute; the stirring time can be 30 minutes.

[0058] In the present invention, the volume ratio of phosphorus oxychloride to the amount of cyclohexanone may be (2-2.5) mL:10 mmol, specifically 2.4 mL:10 mmol.

[0059] In the present invention, the mixing G may be: adding cyclohexanone to the Vilsmeier reagent in batches; the number of batches may be 2 to 4 times, specifically 3 times, and the interval time between each batch may be 30 to 60 seconds, specifically 45 seconds.

[0060] In the present invention, the temperature of the Vilsmeier-Haack reaction can be 80-85° C., specifically 82° C., and the insulation time can be 4-5 h, specifically 4.5 h.

[0061] In the present invention, after the Vilsmeier-Haack reaction, the obtained reaction solution may be mixed with ice water (denoted as mixed H), followed by extraction, washing and drying in sequence; the mixed H may be stirring mixing; the mixing time may be 12 hours; the extraction reagent may be petroleum ether; the washing reagent may be petroleum ether; the drying may be freeze-drying; the freeze-drying temperature may be -45 to -60°C, and the insulation time may be 10 to 12 hours.

[0062] In the present invention, the preparation method of the compound 2 may include the following steps: mixing 2,3,3-trimethyl-3H-indole and 1,3-propane sultone and performing a reflux reaction.

[0063] In the present invention, the molar ratio of the 2,3,3-trimethyl-3H-indole to 1,3-propane sultone may be 1:1-2, specifically 1:1.5.

[0064] In the present invention, the reflux reaction time can be 20 to 24 hours, specifically 22 hours.

[0065] In the present invention, after the reflux reaction, the obtained product may be cooled and then subjected to solid-liquid separation, washing and drying in sequence; the cooling may be natural cooling to room temperature; the solid-liquid separation may be filtration; and the washing reagent may be acetone.

[0066] In the present invention, the molar ratio of compound 1 to compound 2 may be 1:1.5 to 2.5, specifically 1:2.

[0067] In the present invention, the organic base includes one or more of sodium acetate, ethanol-sodium acetate and ethanol-piperidine; the molar ratio of the compound 1 to the organic base can be 1:1.5-2.5, specifically 1:2.

[0068] In the present invention, the ratio of the amount of compound 1 to the volume of acetic anhydride can be 1 mmol:(5-6) mL, specifically 1 mmol:5.5 mL.

[0069] In the present invention, the Knoevenagel condensation reaction can be carried out under stirring conditions; the temperature of the Knoevenagel condensation reaction can be 60-65°C, specifically 62°C, and the insulation time can be 6-7h, specifically 6.5h.

[0070] In the present invention, after the Knoevenagel condensation reaction, the obtained product may be sequentially subjected to dissolution, solvent removal, redissolution, precipitation, solid-liquid separation, and solid drying; the dissolution solvent may include dichloromethane and methanol; the solvent removal may be rotary evaporation; the rotary evaporation temperature may be 45-55° C., and the insulation time may be 5-25 minutes; the redissolution solvent may be methanol; the precipitation reagent may be methyl tert-butyl ether; and the solid-liquid separation may be centrifugation to remove the supernatant.

[0071] In the present invention, the molar ratio of tripotassium phosphate to compound 3 may be 2:1 to 1.5, specifically 2:1.2.

[0072] In the present invention, the ratio of the mass of the compound 3 to the total mass of anhydrous acetonitrile and anhydrous methanol can be 10 mg:(1-2) mL, specifically 10 mg:1.5 mL.

[0073] In the present invention, the volume ratio of anhydrous acetonitrile to anhydrous methanol can be 2:1.

[0074] In the present invention, the mixing E can be carried out in a protective atmosphere; the protective atmosphere can be nitrogen; the temperature of the mixing E can be 25-27°C, specifically 26°C, the mixing time can be 20-30 minutes, specifically 25 minutes; the mixing E can be stirred mixing.

[0075] In the present invention, the dropping rate may be 30 to 40 drops / minute, specifically 35 drops / minute.

[0076] In the present invention, the cyclization reaction temperature can be 80-85° C., specifically 82° C., and the insulation time can be 5-6 h, specifically 5.5 h.

[0077] In the present invention, the cyclization reaction may further include removing the solvent from the obtained product and then purifying it; the solvent removal may be rotary evaporation; the temperature of the rotary evaporation may be 45-55° C., and the insulation time may be 5-25 min; the purification may be column chromatography separation; the eluent for the column chromatography separation may include dichloromethane and methanol; the volume ratio of dichloromethane to methanol may be 9:1.

[0078] In the present invention, the preparation method of the compound 7 may include the following steps: mixing compound 6, phosphorus tribromide and dichloromethane (denoted as mixture A) and performing a halogenation reaction to obtain the compound 7; the structure of the compound 6 is shown in Formula VI:

[0079]

[0080] In the present invention, the preparation method of the compound 6 may include the following steps: mixing the compound 5, a reducing agent and tetrahydrofuran (denoted as mixture B) for reduction reaction to obtain the compound 6; the structure of the compound 5 is shown in Formula V:

[0081]

[0082] In the present invention, the preparation method of compound 5 may include the following steps: mixing p-hydroxybenzaldehyde, tetraacetyl brominated galactose, a phase transfer catalyst, sodium hydroxide, water and dichloromethane (denoted as mixture C) for glycosidation reaction to obtain compound 5.

[0083] In the present invention, the molar ratio of p-hydroxybenzaldehyde to tetraacetyl brominated galactose may be 1:2-3, specifically 1:2.5.

[0084] In the present invention, the phase transfer catalyst may be tetrabutylammonium bromide; the molar ratio of p-hydroxybenzaldehyde to the phase transfer catalyst may be 1:1-2, specifically 1:1.5.

[0085] In the present invention, the ratio of the amount of p-hydroxybenzaldehyde to the volume of dichloromethane can be 2 mmol: (25-30) mL, specifically 2 mmol: 27 mL.

[0086] In the present invention, the mass ratio of sodium hydroxide to water can be 4-6:94-96, specifically 5:95. In the present invention, deprotonation is achieved by adding sodium hydroxide.

[0087] In the present invention, the ratio of the total volume of the sodium hydroxide and water to the volume of dichloromethane can be 1:5-6, specifically 1:5.5.

[0088] In the present invention, the mixture C can be: dissolving p-hydroxybenzaldehyde, tetraacetyl brominated galactose and a phase transfer catalyst in dichloromethane to obtain a premixed solution, and then mixing the premixed solution with sodium hydroxide and water. The present invention adopts the above mixing method to better dissolve the raw materials.

[0089] In the present invention, the temperature of the glycosidation reaction can be 40-45° C., specifically 42° C., and the insulation time can be 4-5 h, specifically 4.5 h.

[0090] In the present invention, after the glycosidation reaction, the obtained product may be extracted, desolventized and purified in sequence; the extraction reagent may be dichloromethane; the desolventizing may be rotary evaporation; the rotary evaporation temperature may be 45-55° C., and the insulation time may be 5-25 min; the purification may be column chromatography separation; the eluent for the column chromatography separation may include petroleum ether and ethyl acetate; the volume ratio of petroleum ether to ethyl acetate may be 2:1.

[0091] In the present invention, the reducing agent may be sodium borohydride; the molar ratio of the compound 5 to the reducing agent may be 1:1 to 1.2, specifically 1:1.1.

[0092] In the present invention, the ratio of the amount of compound 5 to the volume of tetrahydrofuran can be 2 mmol: (40-45) mL, specifically 2 mmol: 43 mL.

[0093] In the present invention, the temperature of the mixture B may be -1 to 1°C, specifically 0°C.

[0094] In the present invention, the temperature of the reduction reaction can be 25-27° C. (room temperature), specifically 26° C., and the insulation time can be 4-5 h, specifically 4.5 h.

[0095] In the present invention, after the reduction reaction, the obtained product may be extracted and the organic layer may be dried in sequence; the extraction may be: mixing a mixed solution of saturated ammonium chloride and dichloromethane with the reaction product and then separating the two phases to obtain an aqueous phase and an organic phase, extracting the aqueous phase with dichloromethane and then combining the organic phases; the drying may be drying over anhydrous sodium sulfate.

[0096] In the present invention, the molar ratio of compound 6 to phosphorus tribromide can be 2:1 to 1.2, specifically 2:1.1.

[0097] In the present invention, the ratio of the amount of compound 6 to the volume of dichloromethane can be 1 mmol: (1-1.5) mL, specifically 1 mmol: 1.2 mL.

[0098] In the present invention, the temperature of the mixture A may be -1 to 1°C, specifically 0°C.

[0099] In the present invention, the halogenation reaction can be carried out under stirring conditions; the temperature of the halogenation reaction can be -1 to 1°C, specifically 0°C, and the insulation time can be 2 to 3 hours, specifically 2.5 hours.

[0100] In the present invention, after the halogenation reaction, the obtained product may be extracted, the organic layer may be dried and purified in sequence; the extraction may be: mixing the reaction product with saturated NaHCO3 and then extracting the aqueous layer with dichloromethane; the drying of the organic layer may be dried over anhydrous sodium sulfate; the purification may be column chromatography separation; the eluent for the column chromatography separation may include petroleum ether and ethyl acetate; the volume ratio of petroleum ether to ethyl acetate may be 1:1.

[0101] In the present invention, the molar ratio of compound 4 to compound 7 may be 1:1-2, specifically 1:1.5.

[0102] In the present invention, the molar ratio of compound 4 to triethylamine can be 1:2 to 2.5, specifically 1:2.2.

[0103] In the present invention, the ratio of the amount of compound 4 to the volume of dichloromethane can be 1 mmol: (20-25) mL, specifically 1 mmol: 22 mL.

[0104] In the present invention, the Williams reaction can be carried out under light-proof and protective atmosphere conditions; the protective atmosphere can be nitrogen; the temperature of the Williams reaction can be 25-27°C, specifically 26°C, and the insulation time can be 12-14h, specifically 13h.

[0105] In the present invention, the Williams reaction may further include purifying the obtained product; the purification may be column chromatography separation; the eluent for the column chromatography separation may include dichloromethane and methanol; the volume ratio of dichloromethane to methanol may be 9:1.

[0106] In the present invention, the ratio of the amount of compound 8 to the volume of methanol can be 1 mmol: (1-2) mL, specifically 1 mmol: 1.5 mL.

[0107] In the present invention, the temperature at which the compound 8, the alcoholysis catalyst and methanol are mixed may be -1 to 1°C, specifically 0°C.

[0108] After obtaining the reaction solution, the present invention performs an alcoholysis reaction on the reaction solution to obtain the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe. In the present invention, the alcoholysis reaction temperature can be -1 to 1°C, specifically 0°C, and the holding time can be 1 to 1.5 hours, specifically 1.3 hours.

[0109] In the present invention, the alcoholysis reaction may further include purifying the obtained product; the purification may be column chromatography separation; the eluent of the column chromatography separation may include dichloromethane and methanol; the volume ratio of dichloromethane to methanol may be 9:1.

[0110] The present invention also provides the use of the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe described in the above scheme or the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe obtained by the preparation method described in the above scheme as a tumor organoid imaging probe.

[0111] The near-infrared fluorescent probe provided by the present invention can be directly used to detect organoids containing Matrigel without removing the Matrigel, avoiding organoid damage or the introduction of additional experimental variables caused by the Matrigel removal process. It realizes non-destructive, in situ near-infrared fluorescence imaging of human ovarian cancer cells SKOV-3 and human tumor organoids, providing a more efficient detection solution that maintains sample integrity for organoid research.

[0112] In order to further illustrate the present invention, the scheme of the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0113] Example 1

[0114] (1) Under nitrogen protection, 2.4 mL of phosphorus oxychloride was added dropwise to 4 mL of dry N,N-dimethylformamide and 4 mL of anhydrous dichloromethane at a rate of 30 drops / min and stirred for 30 min to obtain Vilsmeier reagent. Subsequently, 10 mmol of cyclohexanone was added to the Vilsmeier reagent in three batches, with an interval of 45 s between each batch. The Vilsmeier-Haack reaction was carried out at 80°C for 4 h. After the reaction, the reaction solution was poured into ice water and stirred for 12 h. The mixture was extracted with petroleum ether, washed with petroleum ether, and freeze-dried at -50°C for 11 h to obtain compound 1.

[0115] (2) 2,3,3-Trimethyl-3H-indole and 1,3-propane sultone were refluxed at 120°C in a molar ratio of 1:1 for 20 h. After the reaction, the mixture was cooled to room temperature and filtered to obtain a purple solid, which was washed with acetone and dried to obtain compound 2.

[0116] (3) Compound 1 (1 mmol), compound 2, and sodium acetate were added to 5.5 mL of acetic anhydride in a molar ratio of 1:2:2, and the mixture was stirred at 60°C for Knoevenagel condensation reaction for 6 h. After the reaction, dichloromethane and methanol were added to dissolve the mixture, and the mixture was rotary evaporated at 50°C for 15 min. Methanol was added to dissolve the solid, and methyl tert-butyl ether was added to precipitate the mixture. The supernatant was removed by centrifugation and dried to obtain compound 3.

[0117] (4) Under nitrogen protection, potassium phosphate (K3PO4) and resorcinol were added to 6 mL of anhydrous acetonitrile in a molar ratio of 1:1 (the mass ratio of potassium phosphate and anhydrous acetonitrile was 55:1), and stirred at room temperature at 27°C for 20 min to obtain solution A. Then, compound 3 (the molar ratio of K3PO4 and compound 3 was 2:1) was dissolved in anhydrous acetonitrile and anhydrous methanol in a volume ratio of 2:1 (the mass ratio of compound 3 to the total mass of anhydrous acetonitrile and anhydrous methanol was 10 mg:1.5 mL) under nitrogen protection, and stirred at 27°C for 25 min to obtain solution B. Under nitrogen protection, solution B was added dropwise to solution A at a rate of 30 drops / min, and the cyclization reaction was carried out at 80°C for 5 h. After the reaction was completed, the mixture was rotary evaporated at 50°C for 15 min, and the crude product was separated by column chromatography using dichloromethane and methanol in a volume ratio of 9:1 as the eluent to obtain compound 4.

[0118] (5) p-Hydroxybenzaldehyde, tetraacetyl bromogalactose, and tetrabutylammonium bromide were dissolved in dichloromethane in a molar ratio of 1:2:1 (the ratio of p-hydroxybenzaldehyde to dichloromethane was 2 mmol:25 mL) to obtain a mixture. A 5% sodium hydroxide solution (the volume ratio of sodium hydroxide solution to dichloromethane was 1:5.5) was added to the mixture, and the mixture was subjected to glycosidation reaction at 40°C for 4 h. After the reaction, the mixture was extracted with dichloromethane and rotary evaporated at 50°C for 15 min. The crude product was separated by column chromatography using a 2:1 volume ratio of petroleum ether to ethyl acetate as the eluent to obtain compound 5.

[0119] (6) Compound 5 was added to tetrahydrofuran at 0°C in a ratio of 2 mmol to 40 mL. Sodium borohydride was then added at a molar ratio of 1:1 to compound 5, and the reaction was carried out at room temperature at 26°C for 4 h. After the reaction, a mixed solution of saturated ammonium chloride and dichloromethane was added, the two phases were separated, the aqueous layer was extracted with dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate to obtain compound 6.

[0120] (7) Compound 6 was added to dichloromethane at a ratio of 1 mmol:1 mL. Phosphorus tribromide was added at 0°C at a molar ratio of 2:1. The mixture was stirred at 0°C for 2 h for halogenation. After the reaction, saturated sodium bicarbonate (NaHCO) was added. The aqueous layer was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The crude product was separated by column chromatography using a 1:1 volume ratio of petroleum ether to ethyl acetate as the eluent to obtain compound 7.

[0121] (8) Under nitrogen, compound 4, compound 7, triethylamine, and dichloromethane were mixed in a ratio of 1 mmol:2 mmol:2 mmol:2 mmol:2 mmol:22 mL for a Williams reaction at room temperature at 26°C in the dark for 12 h. The crude product was separated by column chromatography using dichloromethane and methanol in a 9:1 volume ratio as the eluent to obtain compound 8.

[0122] (9) Compound 8 and sodium methoxide were added to methanol at a molar ratio of 1:2 (1 mmol:1.5 mL) at 0°C, and the alcoholysis reaction was carried out at 0°C for 1 h. The crude product was separated by column chromatography using a 9:1 volume ratio of dichloromethane to methanol as the eluent to obtain a β-galactosidase-responsive hemicyanine near-infrared fluorescent probe, designated Hcy-SO3-β-Gal.

[0123] β-galactosidase-responsive hemicyanine near-infrared fluorescent probe 1 HNMR spectra and 13 C spectrum analysis, the results are as follows Figure 1 and Figure 2 shown.

[0124] according to Figure 1 and Figure 2 It can be seen that the present invention successfully prepared the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe.

[0125] Test Example 1

[0126] Changes in the UV absorption spectrum of the probe Hcy-SO3-β-Gal before and after the reaction with β-galactosidase:

[0127] The probe Hcy-SO3-β-Gal prepared in Example 1 was dissolved in dimethyl sulfoxide to prepare a 1 mM probe mother solution; 400 U / mL of β-galactose mother solution was prepared by adding β-galactose and PBS buffer solution (concentration 100 mM, pH 7.4); two 4.0 mL centrifuge tubes were taken, 1.99 mL of PBS buffer solution (concentration 100 mM, pH 7.4) was added to one, and then 10 μL of 1 mM probe mother solution was added; 1.98 mL of phosphate buffer solution was added to the other, and then 10 μL of 1 mM β-galactose mother solution was added, and finally 10 μL of 1.0 mM probe mother solution was added; the two centrifuge tubes were incubated at 37°C for 5 min, and the absorption spectra were tested respectively. The results are as follows: Figure 3 shown.

[0128] according to Figure 3 It can be seen that the original absorption double peak disappears and a new peak appears, and the maximum absorption wavelength red-shifts from 600 nm to 693 nm, indicating that the chemical structure of the Hcy-SO3-β-Gal probe molecule changes due to the specific hydrolysis of the β-galactosidase bond in the Hcy-SO3-β-Gal probe, proving that the Hcy-SO3-β-Gal probe has the ability to respond to β-galactosidase.

[0129] Test Example 2

[0130] Fluorescence spectrum changes of the probe Hcy-SO3-β-Gal before and after reaction with different concentrations of β-galactosidase:

[0131] The probe Hcy-SO3-β-Gal prepared in Example 1 was dissolved in dimethyl sulfoxide to prepare a 1 mM probe stock solution; β-galactose was prepared into a β-galactose stock solution with a concentration of 200 U / mL and diluted to the following required concentrations; 1980 μL of PBS buffer solution (concentration 100 mM, pH 7.4) was added to each 4.0 mL centrifuge tube, and 10 μL of 200 U / mL, 160 U / mL, and 80 U / mL of each were added. , 40U / mL, 20U / mL, 10U / mL β-galactose solution, one without β-galactose solution, replaced with an equal amount of PBS as a blank control, and finally added 10μL of 1.0mM probe mother solution; the final probe concentration was 5μM, and β-galactose was 1U / mL, 0.8U / mL, 0.4U / mL, 0.2U / mL, 0.1U / mL, 0.05U / mL, 0U / mL, respectively. The fluorescence spectra were tested, and the results are as follows Figure 4 and Figure 5 shown.

[0132] according to Figure 4It can be seen that when there is no β-galactose, the system has almost no fluorescence. After adding β-galactose, the fluorescence intensity gradually increases with the increase of β-galactose concentration.

[0133] according to Figure 5 It can be seen that the linear curve fitting the relationship between β-galactose concentration and fluorescence intensity is Y=2408.40602X+96.78701, R 2 =0.994. According to the formula for calculating the lower limit of detection (LOD) = k × Sb / S, the lowest detection limit was calculated to be 0.00105 U / mL.

[0134] Test Example 3

[0135] The fluorescence intensity spectrum of the probe Hcy-SO3-β-Gal at 710 nm changes with different reaction times of β-galactosidase:

[0136] The probe Hcy-SO3-β-Gal prepared in Example 1 was dissolved in DMSO to prepare a 1 mM probe mother solution; β-galactose was prepared into a β-galactose mother solution with a concentration of 400 U / mL; 1980 μL of PBS buffer solution (concentration 100 mM, pH 7.4) was added to a 4.0 mL centrifuge tube, and then 10 μL of 400 U / mL β-galactose solution was added, and finally 10 μL of 1.0 mM probe mother solution was added; the final probe concentration was 5 μM, and the β-galactose was 2 U / mL. The fluorescence spectrum of the fluorescence spectrum was tested at 37°C over time using an excitation wavelength of 675 nm. The results are as follows: Figure 6 shown.

[0137] according to Figure 6 It can be seen that as time increases, the fluorescence at 710 nm gradually increases and reaches equilibrium at 5 min.

[0138] Test Example 4

[0139] The probe Hcy-SO3-β-Gal prepared in Example 1 was subjected to interference test. The interference substances were GSH, H2O2, HClO, O2 ·- The test method is as follows: take a 4.0mL centrifuge tube, add 10μL of interfering substance solution, add 1980μL of PBS, mix well, then add 10μL of 1.0mM probe stock solution, the final probe concentration is 5μM, incubate at 37℃ for 5min. The fluorescence intensity of the test probe at 710nm after reacting with the above interfering substances is as follows Figure 7 shown.

[0140] according to Figure 7It can be seen that the fluorescence intensity of the test solution did not change significantly after adding various interfering agents, but was significantly enhanced after adding β-galactose, indicating that the probe Hcy-SO3-β-Gal has good selectivity for β-galactose.

[0141] Test Example 5

[0142] Cytotoxicity of the probe Hcy-SO3-β-Gal in ovarian cancer cells SKOV-3:

[0143] The probe Hcy-SO3-β-Gal was co-cultured with ovarian cancer cells SKOV-3, and its cytotoxicity was quantitatively evaluated by MTT colorimetry. In the experiment, probe Hcy-SO3-β-Gal solutions of different concentrations (0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 25 μM) were prepared and incubated with SKOV-3 cells (96-well plates, 2 × 10 cells per well). 4 The cells were co-incubated for 24 h to simulate the time span that the probe may encounter in actual application, so as to more accurately evaluate its long-term effects. Figure 8 shown.

[0144] according to Figure 8 It can be seen that when the concentration of the probe Hcy-SO3-β-Gal is 25μM, the survival rate of ovarian cancer cells remains above 80% after a 24-h culture period. This data shows that the probe Hcy-SO3-β-Gal exhibits low cytotoxicity to cells and also shows good biocompatibility. This finding provides an important safety basis for the application of the probe Hcy-SO3-β-Gal in the biomedical field.

[0145] Test Example 6

[0146] The fluorescence effect of the probe Hcy-SO3-β-Gal prepared in Example 1 was tested in ovarian cancer organoids. The results are as follows: Figure 9 shown. Figure 9 (B) is a fluorescence imaging image of ovarian cancer organoids using the near-infrared fluorescent probe Hcy-SO3-β-Gal of the present invention. (B) is a fluorescence imaging image of the organoids after incubation with 10 μM probe Hcy-SO3-β-Gal for 1 hour while retaining the matrix gel. (D) is a fluorescence imaging image after co-culturing the organoids with 7.5 nM camptothecin for 1 day to induce senescence while retaining the matrix gel, and then adding 10 μM probe Hcy-SO3-β-Gal and incubating for another 1 hour. (A) is a bright field imaging image of (B), and (C) is a bright field imaging image of (D).

[0147] according to Figure 9It can be seen that the experimental group with the addition of camptothecin induced organoid aging, increased the β-galactosidase content, and thus significantly enhanced the fluorescence of the organoids.

[0148] As can be seen from the above examples, the near-infrared fluorescent probe provided by the present invention has excellent biocompatibility, achieves a selective and rapid response to β-galactosidase, shows high sensitivity to β-galactosidase, can effectively avoid the interference of Matrigel autofluorescence on the detection signal, and can be directly used for the detection of organoids containing Matrigel without removing the Matrigel, avoiding organoid damage caused by the Matrigel removal process or the introduction of additional experimental variables.

[0149] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A β-galactosidase-responsive hemicyanine near-infrared fluorescent probe, characterized in that: The structure is shown in formula A:

2. The method for preparing the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe according to claim 1, characterized in that: The following steps are involved: Compound 8, an alcoholysis catalyst, and methanol are mixed to perform an alcoholysis reaction to obtain the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe; the structure of compound 8 is shown in Formula VIII:

3. The preparation method according to claim 2, characterized in that The molar ratio of the compound 8 to the alcoholysis catalyst is 1:1-5.

4. The preparation method according to claim 2, characterized in that The ratio of the amount of the compound 8 to the volume of methanol is 1 mmol: (1-2) mL.

5. The preparation method according to claim 2, characterized in that The temperature of mixing the compound 8, the alcoholysis catalyst and methanol is -1 to 1°C.

6. The preparation method according to claim 2 or 3, characterized in that The temperature of the alcoholysis reaction is -1 to 1° C., and the insulation time is 1 to 1.5 hours.

7. The preparation method according to any one of claims 2 to 5, characterized in that: The preparation method of compound 8 comprises the following steps: Compound 4, compound 7, triethylamine and dichloromethane are mixed and subjected to Williams reaction to obtain compound 8; the structure of compound 4 is shown in Formula IV, and the structure of compound 7 is shown in Formula VII:

8. The preparation method according to claim 7, characterized in that The preparation method of compound 4 comprises the following steps: Potassium phosphate, resorcinol and anhydrous acetonitrile are mixed to obtain solution A, compound 3, anhydrous acetonitrile and anhydrous methanol are mixed to obtain solution B, and the solution B is added dropwise to the solution A to carry out a cyclization reaction to obtain compound 4; the structure of the compound 3 is shown in formula III:

9. The preparation method according to claim 7, characterized in that The preparation method of the compound 7 comprises the following steps: mixing compound 6, phosphorus tribromide and dichloromethane to carry out a halogenation reaction to obtain the compound 7; the structure of the compound 6 is shown in Formula VI:

10. Use of the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe according to claim 1 or the β-galactosidase-responsive hemicyanine near-infrared fluorescent probe obtained by the preparation method according to any one of claims 2 to 9 as a tumor organoid imaging probe.

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