A fluorescent probe targeting lysosome and a preparation method and application thereof

Abstract

The application belongs to the field of fluorescent probes, and relates to a lysosome-targeting fluorescent probe and a preparation method and application thereof. An intermediate is prepared by mixing an aldehyde compound, 5-amino fluorescein and 7-bromoheptanoic acid, and then performing a reaction; the intermediate is mixed with morpholine, and then a reaction is performed, so that the lysosome-targeting fluorescent probe is obtained. The application belongs to the construction of a complex molecular system with flexible and modular Ugi four-component reaction, adopts a one-pot method, and is synthesized by a framework template conjugation method, and the yield of the product is between 60% and 80%. The fluorescent probe prepared in the application can target lysosomes, and can be used as a lysosome-targeting imaging agent for living cancer cells. The fluorescent probe containing ferrocene prepared in the application can target lysosomes, and can be used as a lysosome-targeting imaging agent and a therapeutic agent for living cancer cells. The fluorescent probe containing ferrocene prepared in the application can effectively inhibit the growth of Hela and other cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes, and relates to a fluorescent probe targeting lysosomes, its preparation method and application. Background Technology

[0002] The development of advanced fluorescent materials with multiple functional properties has facilitated advancements in bioimaging, molecular biology, biochemistry, and biomedicine. The water-soluble fluorescent dyes or fluorophores used are not only signaling units but also serve as local probes, potentially even participating in the regulation of biochemical processes. They enable imaging of specific molecules in living cells or tissues, greatly enhancing the ability to intervene in diseases. In recent years, the development of several therapeutic systems has enabled simultaneous imaging diagnosis and treatment. Many applications in these systems require fluorophores with multiple functions, such as generating signals and regulating through specific biochemical events, target binding, or therapeutic activity. Therefore, the preparation of fluorophores with excellent optical properties, targeting capabilities, and specific functional properties is a crucial step in their application for disease identification or treatment.

[0003] However, the synthesis and modification of such multifunctional fluorophores remains a challenging task. Many traditional strategies rely on linear synthesis, where the parent framework (usually the fluorophore) is progressively expanded by adding functional groups to allow for biorecognition (targeting), signal modulation, or biochemical interactions, such as through drug delivery units. Utilizing this strategy, several multifunctional fluorescent probes (fluorescent-on probes) have been developed for bioimaging and diagnostics. For example, fluorescent molecular probes with near-infrared II emission and high brightness are used for high signal-to-noise ratio imaging-guided diagnostics based on cyanine dye frameworks. Another example involves fluorescent acetyldane dyes, which are coupled with morpholine as a target group. These modifications require separate and complex synthetic procedures, both starting from the same fluorophore, to obtain probes capable of imaging HClO in lysosomes. Many other similar strategies have applications in biology. It should be emphasized that extensive optimization of reaction conditions is required before obtaining functional dyes, which hinders (or at least delays) the application of these materials beyond basic research. Summary of the Invention

[0004] The technical problem this invention aims to solve is to address the shortcomings of existing technologies by providing a fluorescent probe targeting lysosomes, its preparation method, and its applications. This application provides a modular synthetic framework that connects the fluorophore and all desired functional groups. Importantly, the preparation method provided in this application offers a more direct reaction to this scaffold compared to linear methods. It can provide functional fluorophores in several synthetic steps and allows for specific functional exchanges within the framework. This application introduces an "editable" platform for scaffold synthesis and customized targeting of fluorescent dyes via the Ugi 4-component reaction (U-4CR), which can be easily extended to other dye classes and functions. U-4CR provides excellent scaffold diversity to obtain complex chromophore structures; it combines four reaction partners in an atomically efficient manner in a single step and reaction vessel without the need for a catalyst.

[0005] This application describes a two-step process for labeling biomacromolecules with fluorescent dyes. This method allows for the conversion of commercial fluorophores into functional probes with high photostability and metal-sensing capabilities, directly onto biological targets. The resulting probes bind three distinct molecules: a fluorophore, a signal modulator, and a unit targeting a single organelle (lysosome) or surface receptor within a living cell. The method provided in this application utilizes the redox properties of ferrocene (Fe) and its effect on organic dyes via photoinduced electron transfer (PeT) for signal modulation. Furthermore, ferrocene participates in the Fenton-like reaction stimulated by the cancer cell microenvironment, leading to the generation of hydroxyl radicals. These hydroxyl radicals are reactive oxygen species capable of inducing cell death. More specifically, in cancer cells, the presence of slightly acidic conditions triggers the initiation of the Fenton / Fenton-like reaction, leading to excessive consumption of hydrogen peroxide (H2O2) to generate hydroxyl radicals. This reaction is relatively safe for normal cells because the Fenton reaction is significantly inhibited under slightly alkaline conditions and when H2O2 levels are insufficient in the normal environment. Importantly, this application utilizes 4-UCR to generate molecular probes targeting lysosomes and leverages the redox properties of ferrocene (Fe) and its effect on organic dyes via photoinduced electron transfer (PeT) for signal modulation.

[0006] Invention Concept: This application provides a fluorescent probe that targets lysosomes, the fluorescent probe carrying three different molecules, including a fluorophore (luciferin), a signal modulator (ferrocene), and a unit (morpholine) that targets a single organelle (lysosome) or surface receptor within a living cell.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] This invention discloses a fluorescent probe targeting lysosomes, wherein the compound structure of the fluorescent probe is shown in Formula I or Formula II.

[0009] The structural formula of the compound represented by Formula I is as follows:

[0010]

[0011] The compound represented by Formula II has the following structural formula:

[0012]

[0013] Furthermore, the present invention provides a method for preparing the above-mentioned fluorescent probe targeting lysosomes, comprising the following steps:

[0014] (1) After mixing the aldehyde compound with 5-aminofluorescein, 7-bromoheptanoic acid, tert-butylisocyanate and the first organic solvent, a first reaction was carried out. After the reaction was completed, an intermediate was obtained.

[0015] (2) The intermediate obtained in step (1) is mixed with morpholine and a second organic solvent, and a second reaction is carried out. After the reaction is completed, the product is obtained.

[0016] in,

[0017] When the aldehyde compound is formaldehyde, intermediate 1 is obtained after step (1); intermediate 1 is then processed through step (2) to obtain the compound shown in formula I.

[0018] The structural formulas of intermediate 1 and the compound represented by formula I are shown below:

[0019]

[0020] When the aldehyde compound is ferrocene formaldehyde, intermediate 2 is obtained after step (1); intermediate 2 is then processed through step (2) to obtain the compound shown in formula II.

[0021] The structural formulas of intermediate 2 and the compound represented by formula II are shown below:

[0022]

[0023] In some embodiments, in step (1), the first organic solvent is any one or a combination of several of methanol, N,N-dimethylformamide and ethanol.

[0024] In some embodiments, preferably, in step (1), the first organic solvent is methanol.

[0025] In step (1), there are no special requirements for the amount of the first organic solvent used; it is sufficient to dissolve or disperse the raw material evenly.

[0026] In some embodiments, in step (1), the molar ratio of the aldehyde compound to 5-aminofluorescein, 7-bromoheptanoic acid, and tert-butylisocyanate is 1:(0.8-2.0):(0.8-2.0):(0.8-2.0).

[0027] In some embodiments, preferably, in step (1), the molar ratio of the aldehyde compound to 5-aminofluorescein, 7-bromoheptanoic acid, and tert-butylisocyanate is 1.0:1.0:1.0:1.0.

[0028] In some embodiments, in step (1), the first reaction is carried out at a temperature of 45–65°C for 48–72 hours.

[0029] In some embodiments, preferably, in step (1), the first reaction is carried out at a temperature of 50°C for 48 hours.

[0030] In some embodiments, in step (2), the second organic solvent is any one or a combination of ethanol and methanol.

[0031] In some embodiments, preferably, in step (2), the second organic solvent is ethanol.

[0032] In step (2), there are no special requirements for the amount of the second organic solvent used; it is sufficient to dissolve or disperse the raw material evenly.

[0033] In step (2), the amount of morpholine used is excessive, and the amount of morpholine used is at least 10 times the molar amount of the intermediate.

[0034] In some embodiments, in step (2), the second reaction takes 24 to 48 hours.

[0035] In some embodiments, preferably, in step (2), the second reaction takes 24 hours.

[0036] In step (2), the reaction temperature of the second reaction is determined by the boiling point of the second organic solvent used, so that the second organic solvent can be refluxed.

[0037] The application of the aforementioned fluorescent probes targeting lysosomes in the preparation of tumor diagnostic reagents is also within the scope of protection of this invention.

[0038] The application of the aforementioned fluorescent probes targeting lysosomes in the preparation of tumor imaging contrast agents is also within the scope of protection of this invention. Preferably, the application of the fluorescent probes targeting lysosomes in the preparation of tumor fluorescence imaging contrast agents is also within the scope of protection of this invention.

[0039] The application of the aforementioned fluorescent probe targeting lysosomes in the preparation of antitumor drugs is also within the scope of protection of this invention. Preferably, the fluorescent probe targeting lysosomes is a compound represented by Formula II; wherein, the compound represented by Formula II has the following structural formula:

[0040]

[0041] Among them, since the compound shown in Formula II contains a ferrocene structure, the presence of slightly acidic conditions in cancer cells triggers the initiation of a Fenton / Fenton-like reaction, leading to excessive consumption of hydrogen peroxide (H2O2) to generate hydroxyl radicals, thereby eliminating / inhibiting the proliferation of cancer cells.

[0042] Specifically, the tumor is preferably a cervical cancer cell.

[0043] Beneficial effects:

[0044] (1) This invention relates to the construction of a flexible and modular complex molecular system of Ugi four-component reaction (U-4CR). It adopts a one-pot method and a framework template conjugation method for synthesis, and the yield of the product is between 60% and 80%.

[0045] (2) The method for synthesizing targeted lysosomal fluorescent probes in this invention does not require the use of catalysts or additives, is economical and green, easy to operate and has high production efficiency.

[0046] (3) The fluorescent probe prepared by the present invention can target lysosomes and can be used as a lysosome-targeting imaging agent for live cancer cells.

[0047] (4) The fluorescent probe containing ferrocene prepared in this invention can target lysosomes and can be used as a lysosome-targeting imaging agent and therapeutic agent for live cancer cells.

[0048] (5) The fluorescent probe containing ferrocene prepared in this invention can effectively inhibit the growth of cancer cells such as HeLa. Attached Figure Description

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0050] Figure 1 The 1H NMR spectrum of intermediate 1 prepared in Example 1.

[0051] Figure 2 The image shows the carbon NMR spectrum of intermediate 1 prepared in Example 1.

[0052] Figure 3 The mass spectrum is that of intermediate 1 prepared in Example 1.

[0053] Figure 4 The image shows the 1H NMR spectrum of compound I prepared in Example 2.

[0054] Figure 5 The image shows the carbon NMR spectrum of compound I prepared in Example 2.

[0055] Figure 6 This is the mass spectrum of compound I prepared in Example 2.

[0056] Figure 7 The 1H NMR spectrum of intermediate 2 prepared in Example 3.

[0057] Figure 8 The image shows the carbon NMR spectrum of intermediate 2 prepared in Example 3.

[0058] Figure 9 The mass spectrum of intermediate 2 prepared in Example 3.

[0059] Figure 10 The image shows the 1H NMR spectrum of compound II prepared in Example 4.

[0060] Figure 11 The image shows the carbon NMR spectrum of compound II prepared in Example 4.

[0061] Figure 12 The mass spectrum of compound II prepared in Example 4 is shown.

[0062] Figure 13 This is the spectrum of the maximum absorption wavelength detected by the ultraviolet absorption spectrum of compound I.

[0063] Figure 14 This is the spectrum showing the detection of the maximum absorption wavelength under ultraviolet absorption spectroscopy for compound II.

[0064] Figure 15 This is a comparison of the fluorescence intensities of compound I and compound II.

[0065] Figure 16 Confocal imaging and colocalization effects of compound I, compound II, and the commercially available lysosomal probe Lyso-Tracker.

[0066] Figure 17 The graph shows the cell viability of cancer cells after treatment with different concentrations of compound I and compound II; among them, Figure 17 A shows the cell activity of compound I in inhibiting cancer cells. Figure 17 B shows the cell activity of compound II in inhibiting cancer cells. Detailed Implementation

[0067] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0068] Example 1: Synthesis of Intermediate 1

[0069]

[0070] 5-Aminofluorescein (0.347 g, 1.0 mmol) and formaldehyde (0.030 g, 1.0 mmol) were dissolved in methanol (5.0 mL), followed by the addition of 7-bromoheptanoic acid (0.209 g, 1.0 mmol) and tert-butylisocyanate (0.083 g, 1.0 mmol) to obtain a reaction solution. The reaction solution was stirred at 50 °C for 48 hours. After the reaction was completed, the reaction solution was concentrated under vacuum to obtain a crude product. The crude oil was subjected to column chromatography (eluting with dichloromethane and methanol, gradient elution, V...) 二氯甲烷 V 甲醇 =50:1, V 二氯甲烷 V 甲醇 =25:1 and V 二氯甲烷 V 甲醇 The mixture was purified at a ratio of 12:1 to obtain intermediate 1, 0.478 g, with a yield of 73.5%.

[0071] Intermediate 1 is carried out 1 H-NMR and 13 C-NMR and HRMS mass spectrometry characterization were performed. For specific 1H NMR spectra, please refer to [link to relevant documentation]. Figure 1 The carbon NMR spectrum is shown below. Figure 2 HRMS mass spectra can be found in [link to HRMS mass spectra]. Figure 3 .in, Figure 3 Mid-mass spectrometry peaks, Found: [M+H] + =651.0434.

[0072] Example 2: Synthesis of Compound I

[0073]

[0074] Intermediate 1 (0.110 g, 0.169 mmol, prepared in Example 1) and morpholine (0.33 mL, 3.79 mmol) were dissolved in ethanol (7.0 mL) and refluxed at 75–85 °C for 24 hours. After the reaction was complete, the reaction solution was cooled to room temperature and concentrated under vacuum to obtain the crude product. The crude product was subjected to column chromatography (eluting with dichloromethane and methanol, gradient elution, V... 二氯甲烷 V 甲醇 =20:1, V 二氯甲烷 V 甲醇 =10:1, V 二氯甲烷 V 甲醇 The mixture was purified by a ratio of 6:1 to obtain 0.082 g of pure compound I, with a yield of 73.7%.

[0075] Compound I proceeds 1 H-NMR and 13 C-NMR and HRMS mass spectrometry characterization, with specific 1H NMR spectra shown in [link to HRMS mass spectra]. Figure 4 The carbon NMR spectrum is shown below. Figure 5 HRMS mass spectra can be found in [link to HRMS mass spectra]. Figure 6 .in, Figure 6 Mid-mass spectrometry peaks, Found: [M+H] + =658.3121.

[0076] Example 3: Synthesis of Intermediate 2

[0077]

[0078] 5-Aminofluorescein (0.695 g, 2.0 mmol) and ferrocene formaldehyde (0.428 g, 2.0 mmol) were dissolved in methanol (10.0 mL), followed by the addition of 7-bromoheptanoic acid (0.418 g, 2.0 mmol) and tert-butylisocyanate (0.166 g, 2.0 mmol) to obtain a reaction solution. The reaction solution was stirred at 50 °C for 48 hours. After the reaction was completed, ethyl acetate (40 mL) was added to the reaction solution, and the organic layer was washed with water (3 x 20 mL). The aqueous layer of the mixture was extracted with ethyl acetate (2 x 20 mL). The organic components were then mixed, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain pure intermediate 2, a brownish-yellow powder, 1.179 g, with a yield of 72%.

[0079] Intermediate 2 is carried out 1 H-NMR and 13 C-NMR and HRMS mass spectrometry characterization were performed. For specific 1H NMR spectra, please refer to [link to relevant documentation]. Figure 7 The carbon NMR spectrum is shown below. Figure 8 HRMS mass spectra can be found in [link to HRMS mass spectra]. Figure 9 .in, Figure 9 Mid-mass spectrometry peaks, Found: [M+H] + =835.1088.

[0080] Example 4: Synthesis of Compound II

[0081]

[0082] Intermediate 2 (0.050 g, 0.06 mmol, prepared in Example 3) and morpholine (0.25 mL, 2.87 mmol) were dissolved in ethanol (5.0 mL) and refluxed at 75–85 °C for 24 hours. After the reaction was complete, the reaction solution was cooled to room temperature and concentrated under vacuum to obtain the crude product. The crude product was subjected to column chromatography (eluting with dichloromethane and methanol, gradient elution, V... 二氯甲烷 V甲醇 =25:1, V 二氯甲烷 V 甲醇 =15:1, V 二氯甲烷 V 甲醇 The mixture was purified by a ratio of 1:18 to obtain 0.035 g of pure compound II, with a yield of 69.3%.

[0083] Compound II proceeds 1 H-NMR and 13 C-NMR and HRMS mass spectrometry characterization were performed. For specific 1H NMR spectra, please refer to [link to relevant documentation]. Figure 10 The carbon NMR spectrum is shown below. Figure 11 HRMS mass spectra can be found in [link to HRMS mass spectra]. Figure 12 .in, Figure 12 Mid-mass spectrometry peaks, Found: [M+H] + =842.3132.

[0084] Example 5: Effect of the presence or absence of ferrocene on the optical properties of compounds I and II

[0085] (1) Maximum absorption wavelength of ultraviolet absorption spectrum

[0086] Compound I (prepared in Example 2) and Compound II (prepared in Example 4) were prepared into 10 mM stock solutions I and II using DMSO, respectively. A small amount of each stock solution was taken and diluted to 3 mL with PBS (pH = 7.4, 10 mmol / L) (the solution concentration was about 1 μM). The solutions were then tested in a UV-Vis spectrophotometer.

[0087] Test results are as follows Figure 13 , Figure 14 As shown in the figure, the maximum absorption wavelength of compound I under ultraviolet absorption spectrum is 489 nm, and the maximum absorption wavelength of compound II under ultraviolet absorption spectrum is 477 nm.

[0088] (2) Fluorescence intensity test

[0089] Compound I (prepared in Example 2) and Compound II (prepared in Example 4) were respectively prepared into 10 mM stock solutions I and II using DMSO. Small amounts of each stock solution were then diluted with PBS (pH = 7.4, 10 mmol / L) to 3 mL (solution concentration approximately 1 μM) and placed in a fluorescence spectrometer for testing. The excitation wavelengths were set to 489 nm and 477 nm, respectively, according to the maximum absorption wavelength in the ultraviolet absorption spectrum. Figure 13 , Figure 14 The maximum absorption wavelength in the UV absorption spectra of the two compounds was detected. Unless otherwise specified, the slit width was set to 2 nm / 2 nm, and fluorescence intensity was measured. The detection results are as follows: Figure 15As shown in the figure, by utilizing the redox properties of ferrocene (Fe) and its influence on organic dyes through photoinduced electron transfer (PeT) for signal modulation, the maximum absorption wavelengths of both compound I and compound II under ultraviolet absorption spectra remained almost unchanged. However, the fluorescence of probe compound II containing ferrocene was suppressed, while the fluorescence of probe compound I without ferrocene was normal.

[0090] Example 6: Co-localization imaging experiment of compounds I and II in cells

[0091] (1) Confocal fluorescence imaging

[0092] First, HeLa cells were pre-cultured. Then, the old DMEM high-glucose medium was aspirated from the original culture dish, and an equal volume of PBS (pH 7.4, 10 mmol / L) was added. The culture dish was gently shaken on the table, and then the PBS (pH 7.4, 10 mmol / L) was aspirated. Next, 0.25% trypsin was added to cover the adherent cells at the bottom of the dish, and the dish was placed in a CO2 incubator for 2 minutes for digestion. The cells were observed to be rounded under a microscope. An equal volume of complete culture medium was then added, and the cell suspension was aspirated with a pipette and the bottom of the dish was agitated 2-3 times. The cell suspension was then transferred to a 15 mL centrifuge tube and centrifuged at room temperature (1200 rpm, 5 minutes), discarding the supernatant. 1 mL of fresh DMEM high-glucose medium was added to prepare a homogeneous cell suspension.

[0093] The cell suspension was transferred to confocal dishes for incubation (approximately 5 × 10⁶ cells per dish). 4 Add 1 mL of complete culture medium to each confocal dish (1 cell per dish) and incubate at 37°C for 24 hours in a CO2 incubator. Afterward, aspirate the original culture medium from the confocal dish and wash 2-3 times with PBS. Then perform the following experiments:

[0094] For the lysosomal colocalization assay, cells were treated with DMEM high-glucose medium containing different probes (10 μM, namely compound I prepared in Example 2 and compound II prepared in Example 4). The cells were incubated at 37°C for 24 hours in a CO2 incubator. After removing the probe solution, a lysosomal localization reagent (commercially available lysosomal probe Lyso-Tracker, 50 nM) was added, and the cells were incubated at 37°C for 30 minutes in a CO2 incubator.

[0095] Before imaging, cells were washed three times with PBS (pH 7.4, 10 mmol / L) and stored in PBS. Cell fluorescence was observed using a Zeiss LSM880NLO confocal microscope system equipped with a 63× / 1.15W Korr objective lens. During image acquisition, laser intensity, exposure time, and objective lens settings were maintained for all samples. Finally, the photomicrographs were analyzed using ImageJ software. To investigate the colocalization effect of our probe,

[0096] Figure 16 The three images in A, from left to right, are: an image of compound I targeting lysosomes, an image of the commercially available lysosome probe Lyso-Tracker targeting lysosomes, and an image of compound I and the commercially available lysosome probe Lyso-Tracker co-localizing lysosomes. As can be seen from the images, compound I has a good ability to target lysosomes.

[0097] Figure 16 The three images in C, from left to right, are: an image showing the effect of compound II on lysosome targeting; an image showing the effect of the commercially available lysosome probe Lyso-Tracker on lysosome targeting; and an image showing the co-localization of compound II and the commercially available lysosome probe Lyso-Tracker on lysosome targeting. As can be seen from the images, due to the quenching effect of ferrocene, the fluorescence effect of compound II is weakened compared to compound I. However, it can still achieve good co-localization with the commercially available lysosome probe Lyso-Tracker, indicating that the synthesized compound II also has good lysosome targeting ability.

[0098] Figure 16 B is a diagram showing the co-localization effect of compound I and the commercially available lysosomal probe Lyso-Tracker on lysosomes. As can be seen from the figure, compound I and the commercially available lysosomal probe Lyso-Tracker have a good co-localization effect, indicating that the synthesized compound I has a good ability to target lysosomes.

[0099] Figure 16 D is a diagram showing the co-localization effect of compound II and the commercially available lysosomal probe Lyso-Tracker on lysosomes. As can be seen from the figure, compound II and the commercially available lysosomal probe Lyso-Tracker have a good co-localization effect, indicating that the synthesized compound II has a good ability to target lysosomes.

[0100] Example 7: Experiments on the inhibition of cancer cell proliferation by compounds I and II

[0101] Experimental Procedure: The CCK-8 cell viability assay was used for detection. HeLa cells were resuscitated, cultured for 24 hours, and then passaged. The cell suspension was added to each well of a 96-well culture dish, with approximately 1 × 10⁶ cells introduced into each well. 4 Add DMEM high-glucose culture medium to 200 μL, and place the 96-well plate in an incubator to continue culturing overnight at 37°C. Prepare different concentrations of compound I probe solution and compound II probe solution (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, compound I prepared in Example 2, compound II prepared in Example 4) using DMEM high-glucose culture medium. After all cells in the 96-well culture dish have adhered to the well wall, remove the supernatant, and then add 200 μL of probe solution containing different concentrations to each well. The negative control group only adds culture medium, and each group is repeated in 3 wells. The treated 96-well culture dishes were placed in a CO2 incubator and incubated at 37°C for 48 hours. After incubation, the original culture medium was removed, and 100 μL of culture medium containing 5% CCK-8 was added to each well. The dishes were then incubated at 37°C for 2 hours. The 96-well culture dishes were then placed in a pre-prepared microplate reader, with the wavelength set to 450 nm. The OD value (cell density) of each well was measured at this wavelength. Viability (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%, where OD value refers to optical density. The OD value of the experimental group is the absorbance value of the treated cells, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0102] For specific cancer cell inhibition effects, see Figure 17 As shown in the figure, compound I, which does not contain ferrocene, is almost non-toxic to cancer cells, while compound II, which contains ferrocene, becomes more toxic with increasing concentration and can significantly inhibit the growth of cancer cells.

[0103] This invention provides a fluorescent probe targeting lysosomes, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A fluorescent probe targeting lysosomes, characterized in that, The compound structure of the fluorescent probe is shown in Formula I or Formula II. The structural formula of the compound represented by Formula I is as follows: ； The compound represented by Formula II has the following structural formula: 。 2. The method for preparing the fluorescent probe targeting lysosomes according to claim 1, characterized in that, Includes the following steps: (1) After mixing the aldehyde compound with 5-aminofluorescein, 7-bromoheptanoic acid, tert-butylisocyanate and the first organic solvent, a first reaction was carried out. After the reaction was completed, an intermediate was obtained. (2) After mixing the intermediate obtained in step (1) with morpholine and the second organic solvent, a second reaction is carried out. After the reaction is completed, the product is obtained. in, When the aldehyde compound is formaldehyde, intermediate 1 is obtained after step (1); intermediate 1 is then processed through step (2) to obtain the compound shown in formula I. The structural formulas of intermediate 1 and the compound represented by formula I are shown below: ； When the aldehyde compound is ferrocene formaldehyde, intermediate 2 is obtained after step (1); intermediate 2 is then processed through step (2) to obtain the compound shown in formula II. The structural formulas of intermediate 2 and the compound represented by formula II are shown below: 。 3. The preparation method according to claim 2, characterized in that, In step (1), the first organic solvent is any one or a combination of several of methanol, N,N-dimethylformamide and ethanol.

4. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of the aldehyde compound to 5-aminofluorescein, 7-bromoheptanoic acid, and tert-butylisocyanate is 1:(0.8~2.0):(0.8~2.0):(0.8~2.0).

5. The preparation method according to claim 2, characterized in that, In step (1), the first reaction is carried out at a temperature of 45~65℃ for 48~72 hours.

6. The preparation method according to claim 2, characterized in that, In step (2), the second organic solvent is any one or a combination of two of ethanol and methanol.

7. The preparation method according to claim 2, characterized in that, In step (2), the second reaction takes 24 to 48 hours.

8. The use of the fluorescent probe targeting lysosomes according to claim 1 in the preparation of tumor diagnostic reagents.

9. The use of the fluorescent probe targeting lysosomes according to claim 1 in the preparation of tumor imaging contrast agents.

10. The use of the fluorescent probe targeting lysosomes according to claim 1 in the preparation of tumor fluorescence imaging contrast agents.

11. The use of the fluorescent probe targeting lysosomes according to claim 1 in the preparation of antitumor drugs, wherein, The fluorescent probe targeting the lysosome is a compound represented by Formula II.

Images