Organic organelle-targeted fluorescent probe for specific detection of tumor cells as well as preparation and application of organelle-targeted fluorescent probe

Abstract

The invention discloses an organelle-targeted fluorescent probe for specific detection of tumor cells as well as preparation and application of the organelle-targeted fluorescent probe, and the specific structural formula of the fluorescent probe is as follows: BCY-DP shows obvious specific targeting ability of tumor cell mitochondria and has been applied to ONOO-selective imaging in living cell mitochondria; results show that the BCY-DP has high selectivity on ONOO <-> in tumor cells, can successfully distinguish the ovarian cancer cells from normal ovarian cells, and can be used for diagnosis of cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of organic fluorescent probes, specifically relating to a cell organelle-targeting fluorescent probe for the specific detection of tumor cells, its preparation, and its application. Background Technology

[0002] Rapid diagnosis and precise treatment of tumors are crucial for improving cancer patient survival rates. Traditional tumor detection methods (such as tissue biopsy, CT, and MRI) have drawbacks including high invasiveness, limited resolution, and inability to monitor tumor metabolism in real time. Fluorescence imaging technology, with its advantages of high sensitivity, real-time visualization, and non-invasiveness, has shown great potential in tumor diagnosis and intraoperative navigation. Therefore, there is an urgent need to develop effective tumor probes to distinguish various tumor cells from normal cells.

[0003] The tumor microenvironment (TME) possesses unique biochemical characteristics, such as low pH, hypoxia, and high concentrations of reactive oxygen species (ROS / RNS). Among these, peroxynitrite (ONOO) is particularly prominent. - As a key member of reactive nitrogen and reactive oxygen species, it acts within living systems through superoxide radicals (O2). ·- The rapid reaction between α-oxalate and nitric oxide (NO) produces nitric oxide with strong oxidizing and nitrifying capabilities. It is an important physiological activator and signaling molecule, helping to maintain redox homeostasis in organisms. -- ONOO levels are significantly elevated in various malignant tumors (such as ovarian cancer, liver cancer, and colorectal cancer) and are positively correlated with tumor malignancy. Therefore, developing a method to specifically detect ONOO in tumor cells is crucial. - Fluorescent probes not only aid in tumor diagnosis but can also be used to assess tumor progression, drug efficacy, and prognostic monitoring. Existing ONOO... - Most fluorescent probes lack tumor cell selectivity. Due to the high heterogeneity of tumor tissue, ordinary probes are difficult to accumulate in tumor areas, resulting in background signals in normal tissues and reducing the detection signal-to-noise ratio. Mitochondria, as the cell's energy factories and the main source of ROS / RNS, exhibit stronger oxidative stress in tumor cell mitochondria compared to normal cells: tumor cell mitochondria continuously produce large amounts of O2. ·- And NO, thus quickly generating ONOO - Furthermore, tumor cell mitochondria possess high membrane potential, which facilitates the selective enrichment of positively charged molecules; this can reduce probe retention in normal tissues and improve the signal-to-noise ratio. Therefore, developing a probe that specifically targets tumor cell mitochondria and accurately detects ONOO is crucial. - Fluorescent probes have important clinical value. Summary of the Invention

[0004] This invention is the first to construct a method that can specifically target tumor cells while simultaneously monitoring mitochondrial ONOO. - The fluorescent probe BCY-DP is used to distinguish between tumor cells and normal cells. A biotin structure is introduced into the BCY-DP structure. Biotin receptors are overexpressed in many tumor cells but underexpressed in normal cells, thus achieving probe enrichment in tumor cells. BCY-DP contains a hemicyanine derivative as a fluorophore and diphenyl phosphate (DP) as an ONOO. - BCY-DP, a sensitive group, exhibits significant specific targeting ability to tumor cell mitochondria and has been applied to ONOO in living cell mitochondria. - Selective imaging; results showed that BCY-DP targeted ONOO in tumor cells. - It has high selectivity and can successfully distinguish between ovarian cancer cells and normal ovarian cells, and can be used for the diagnosis of cancer cells.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A fluorescent probe targeting organelles for the specific detection of tumor cells, the specific structural formula of which is as follows:

[0007]

[0008] As a preferred embodiment of the present invention, the fluorescent probe BCY-DP is enriched in tumor cells, targets mitochondria within the cells, and is effective against ONOO. - Selective.

[0009] This invention also provides a method for preparing the above-mentioned BCY-DP fluorescent probe. This method uses hemicyanine dye and diphenylphosphochloride as reactants and reacts them at room temperature and pressure. The intermediate product obtained is then reacted with biotin-azide to finally obtain BCY-DP. The chemical structural formula of the hemicyanine dye is as follows: The chemical structural formula of the biotin-azide is:

[0010] As a preferred embodiment of the present invention, the preparation method of the BCY-DP fluorescent probe specifically includes the following steps:

[0011] Step 1. Dissolve D-biotin in an organic solvent, add N,N'-carbonyldiimidazole, and stir the solution at room temperature; add 3-azidopropylamine and continue stirring at room temperature; after the reaction is complete, add ice-cold diethyl ether to the reaction solution, filter the obtained white solid; then perform column chromatography to separate the intermediate product, namely biotin-azide.

[0012] Step 2. Dissolve 2-hydroxy-4-methoxybenzaldehyde in an organic solvent, add cesium carbonate and 2-bromocyclohex-1-ene-1-carboxaldehyde, and stir overnight at 80°C under nitrogen protection. After the reaction is complete, filter to remove insoluble matter from the system, evaporate the solvent to dryness to obtain crude product, and then perform column chromatography to obtain a yellow solid.

[0013] Step 3. Dissolve the intermediate product from Step 2 in an organic solvent, add boron tribromide under a nitrogen atmosphere and ice bath, and then stir at room temperature; after the reaction is complete, adjust the pH of the reaction solution to neutral with a saturated sodium bicarbonate aqueous solution, a large amount of solid precipitates out, wash with distilled water and filter, dry to obtain a turmeric-colored solid;

[0014] Step 4. Dissolve 1,1,2-trimethylbenzo[e]indole in an organic solvent, add 6-iodo-1-hexyne, and stir the solution at 80°C; after the reaction is complete, add ethyl acetate to the reaction solution to precipitate a large amount of solid, and filter to obtain a green solid;

[0015] Step 5. Add an organic solvent to the intermediate products obtained in Steps 3 and 4, and stir the solution at 80°C. After the reaction is complete, concentrate the solvent and then purify it by column chromatography to obtain the intermediate product, i.e., hemicyanine dye.

[0016] Step 6. Dissolve the hemicyanine dye in an organic solvent, add diphenylphosphochloride, then add triethylamine, stir the resulting solution at room temperature, concentrate the solvent after the reaction is complete, and then purify by column chromatography to obtain the intermediate product;

[0017] Step 7. Add the intermediate product obtained in Step 6, biotin-azide and copper hexafluorophosphate tetraacetonitrile to the reactor, followed by the addition of organic solvent and diisopropylethylamine, and react at room temperature; after the reaction is completed, concentrate the solvent and then purify using reverse-phase C18 to obtain BCY-DP.

[0018] As a preferred embodiment of the present invention, the organic solvent in step 1 is selected from any one of N,N-dimethylformamide, dichloromethane, acetonitrile, methanol, and ethanol, and the molar ratio of D-biotin, N,N'-carbonyldiimidazole and 3-azidopropylamine is 0.5:0.9:0.75.

[0019] As a preferred embodiment of the present invention, the organic solvent in step 2 is selected from any one of acetonitrile, dichloromethane, methanol, and ethanol, and the molar ratio of 2-hydroxy-4-methoxybenzaldehyde, 2-bromocyclohex-1-ene-1-carboxaldehyde to cesium carbonate is 11.28:14:33.9.

[0020] As a preferred embodiment of the present invention, the organic solvent in step 3 is selected from any one of dichloromethane, acetonitrile, methanol, and ethanol, and the molar ratio of the intermediate obtained in step 2 to boron tribromide is 3.3:40.

[0021] As a preferred embodiment of the present invention, the organic solvent in step 4 is selected from any one of anhydrous acetonitrile, dichloromethane, acetonitrile, n-butanol, methanol, and ethanol, and the molar ratio of 1,1,2-trimethylbenzo[e]indole to 6-iodo-1-hexyne is 2:3.

[0022] As a preferred embodiment of the present invention, the organic solvent in step 5 is selected from any one or a mixture of two or more of anhydrous toluene, n-butanol, acetonitrile, methanol, and ethanol, and the molar ratio of the intermediate product obtained in step 3 to the intermediate product obtained in step 4 is 1:2.

[0023] As a preferred embodiment of the present invention, the organic solvent mentioned in step 6 is selected from any one of dichloromethane, acetonitrile, methanol, and ethanol, and the molar ratio of hemicyanine dye to diphenylphosphine chloride is 0.85:2.38.

[0024] As a preferred embodiment of the present invention, the organic solvent in step 7 is selected from any one of acetonitrile, methanol, ethanol, n-butanol, and anhydrous toluene, and the molar ratio of the intermediate product obtained in step 6 to the biotin-azide is 0.9:0.72.

[0025] This invention uses BCY-DP to visualize cancer cells and finds that the fluorescence signal in cancer cells is significantly stronger than that in normal cells. Therefore, BCY-DP can be used as an effective probe to distinguish cancer cells from normal cells. Thus, the fluorescent probe BCY-DP can be used in the preparation of detection reagents and / or kits for cancer diagnosis.

[0026] As a preferred embodiment of the present invention, the cancers include ovarian cancer, cervical cancer, lung cancer, breast cancer, and thyroid cancer.

[0027] To investigate the targeting ability of BCY-DP, SKOV3 cells were co-incubated with BCY-DP and MTG for 30 minutes, respectively. Fluorescence images were captured using CLSM. The results showed that the Pearson coefficient (PCC) of BCY-DP and MTG was as high as 0.91, demonstrating its excellent mitochondrial targeting ability. Therefore, the fluorescent probe BCY-DP can be used in the preparation of detection reagents and / or kits for specifically labeling intracellular mitochondria.

[0028] The fluorescent probe BCY-DP provided by this invention is for ONOO - It exhibits high selectivity and is unaffected by interference from other reactive small molecules (ROS / RNS / RSS), therefore this probe can also be used in the preparation of probes for detecting ONOO. - Application in modified detection reagents and / or kits.

[0029] Advantages and beneficial effects of the present invention:

[0030] (1) This invention is the first to construct a BCY-DP fluorescent probe. The tumor-targeting group biotin is introduced into the BCY-DP structure to achieve probe enrichment in tumor cells, which has great potential in cancer diagnosis and imaging compared with other probes; in addition, BCY-DP selects diphenyl phosphate (DP) as ONOO - The sensitive groups make it sensitive to ONOO - With high selectivity and unaffected by other active small molecules (ROS / RNS / RSS), BCY-DP exhibits significant specific targeting ability to tumor cell mitochondria and has been applied to ONOO in living cell mitochondria. - Selective imaging and experimental results have demonstrated that this probe can effectively distinguish between ovarian cancer cells and normal ovarian cells, and can be used for the diagnosis of cancer cells; it also has advantages such as low cytotoxicity and excellent biocompatibility.

[0031] (2) The fluorescence intensity of BCY-DP provided by the present invention remains stable from pH 4.0 to pH 9.0, and can work in a wide pH range, making it suitable for imaging of physiological pH values.

[0032] (3) The fluorescent probe BCY-DP provided by the present invention can be used to distinguish between cancer cells and normal cells, and therefore it can be used in the preparation of detection reagents and / or kits for cancer diagnosis; in addition, due to its superior mitochondrial targeting ability, the probe can also be used in the preparation of detection reagents and / or kits for specifically labeling mitochondria in cells.

[0033] (4) The fluorescent probe BCY-DP provided by this invention for ONOO - With high selectivity, this probe can also be used in the preparation of probes for detecting intracellular ONOO. - Application in modified detection reagents and / or kits. Attached Figure Description

[0034] Figure 1 The response mechanism for BCY-DP and ONOO-.

[0035] Figure 2 The synthetic route for BCY-DP is shown below.

[0036] Figure 3 In (a) of the mixture, ONOO was added to PBS / MeOH (1 / 9, v / v, pH 7.4). - (a) Fluorescence emission spectrum of BCY-DP (10.0 μM) after (0-40 μM) excitation at 690 nm; (b) Fluorescence intensity (F) between 0-40 μM. 725nm ) and ONOO -Linear relationship of concentration; (c) BCY-DP (10.0 μM) and 40 μM ONOO - Fluorescence kinetics analysis over 60 min; (d) Fluorescence response of BCY-DP (10.0 μM) to each analyte, excited at 690 nm.

[0037] Figure 4 For BCY-DP (10μM) and BCY-DP (10μM) with ONOO - Absorption spectrum of a (40 μM) mixed solution in PBS / MeOH (1 / 9, v / v, pH 7.4); Inset: Absorption spectrum of ONOO under natural light. - Changes in BCY-DP solution.

[0038] Figure 5 To add ONOO under different pH conditions - Fluorescence spectrum after (40 μM).

[0039] Figure 6 For BCY-DP, BCY-DP and ONOO - LC-MS analysis of the mixed solution; where A is the LC-MS of BCY-DP, and B is the LC-MS of BCY-DP and ONOO. - LC-MS of mixed solutions.

[0040] Figure 7 Cell viability of SKOV3 cells treated with different concentrations of BCY-DP (0, 10.0, 20.0, 30.0, 40.0 and 50.0 μM).

[0041] Figure 8 Fluorescence images of 10 μM BCY-DP and 1 μM MTG co-incubated in SKOV3 cells and Pearson colocalization correlation; BCY-DP: λex = 640 nm, λem = 650-750 nm; MTG: λex = 488 nm, λem = 500-550 nm; Scale bar: 10 μm.

[0042] Figure 9Fluorescence images of SKOV3 cells incubated with BCY-DP (10 μM); (a) SKOV3 cells treated with BCY-DP (10 μM) for 30 min as a control; (b) SKOV3 cells pretreated with SIN-1 (100 μM) for 2 h, followed by treatment with BCY-DP (10 μM) for 30 min; (c) SKOV3 cells pretreated with UA (500 μM) and SIN-1 (100 μM) for 2 h, followed by treatment with BCY-DP (10 μM) for 30 min; (d) LPS (1 μg / mL) SKOV3 cells were pretreated with NAC (2 mM) and LPS (1 μg / mL) and INF-γ (10 ng / mL) for 24 h, and then treated with BCY-DP (10 μM) for 30 min; (e) SKOV3 cells were pretreated with NAC (2 mM) and LPS (1 μg / mL) and INF-γ (10 ng / mL) for 24 h, and then treated with BCY-DP (10 μM) for 30 min; (f) Mean fluorescence intensity (±SD, n=3) of images in ae; BCY-DP: λex=640nm, λem=650-750nm; Scale bar: 20 μm.

[0043] Figure 10 Fluorescence images of different ovarian cell lines incubated with BCY-DP (10 μM); (a) Images of IOSE-80, SKOVE, A2780, and ID8 cells after co-incubation with BCY-DP (10.0 μM) for 30 min; (b) Mean fluorescence intensity (±SD, n=3) of images in a. BCY-DP: λex=640nm, λem=650-750nm; Scale bar: 20 μm. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not specifically specified, conventional techniques can be referred to. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0045] Example 1: Preparation of fluorescent probe BCY-DP

[0046]

[0047] Synthesis of Compound 1: D-biotin (122 mg, 0.5 mmol) was dissolved in DMF (3 mL) at 55 °C. After cooling to room temperature, a DMF (0.6 mL) solution of N,N'-carbonyldiimidazole (146 mg, 0.9 mmol) was slowly added, and the mixture was stirred at room temperature for 3 h. A DMF (1.2 mL) solution of 3-azidopropylamine (75 mg, 0.75 mmol) was added dropwise, and the mixture was stirred at this temperature for 12 h. After the reaction was complete, ice-cold diethyl ether was added to the reaction mixture, and the resulting white solid was filtered. The crude product was separated by silica gel column chromatography (dichloromethane:methanol = 20:1, v / v), and a white solid (120 mg) was collected. The obtained solid was dissolved in dichloromethane and methanol, washed with 1 M sodium hydroxide aqueous solution, and the organic phase was collected. The organic phase was then washed with 20 mL of saturated sodium chloride aqueous solution, and the solvent was removed under reduced pressure to give 63 mg of white solid, with a yield of 39%.

[0048] 1 H NMR (400MHz, DMSO-d6) δ7.84(t,J=4.5Hz,1H),6.41(s,1H),6.35(s,1H),4.31(dd,J=7.3,4.7Hz,1H),4.18-4.09(m,1H),3.35(d,J=6.8Hz,2H),3.09( q,J=6.5Hz,3H),2.82(dd,J=12.4,5.1Hz,1H),2.58(d,J=12.5Hz,1H),2.06 (t,J=7.4Hz,2H),1.67-1.58(m,3H),1.56-1.39(m,3H),1.36-1.26(m,2H). 13 C NMR(150MHz,DMSO-d6)δ172.59,163.24,61.54,59.70,55.89,48.93,39.98,36.21,35.65,28.93,28.68,28.50,25.73.HR-MSm / z:[M+H] + Calculated for C 13 H 22 N6O2S,326.1525; found,327.1591.

[0049]

[0050] Synthesis of Compound 2: 2-Hydroxy-4-methoxybenzaldehyde (1.72 g, 11.28 mmol) was dissolved in a 150 mL round-bottom flask containing 40 mL of acetonitrile. Cesium carbonate (10.1 g, 33.9 mmol) and 2-bromocyclohexyl-1-en-1-carboxaldehyde (8 g, 14 mmol) were then added. The mixture was stirred overnight at 80 °C under nitrogen protection. After the reaction was completed as monitored by TLC, the reaction solution was cooled to room temperature, and insoluble matter was removed by filtration. The solvent was evaporated under reduced pressure to obtain the crude product. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 20:1, v / v) to give 1.2 g of a yellow solid, with a yield of 47%.

[0051] 1 H NMR(400MHz,Chloroform-d)δ10.32(s,1H),7.08(d,J=9.0Hz,1H),6.65(d,J=6.0Hz, 3H), 3.84 (s, 3H), 2.56 (t, J = 6.3Hz, 2H), 2.44 (t, J = 6.0Hz, 2H), 1.71 (p, J = 6.2Hz, 2H). 13 CNMR(150MHz,DMSO-d6)δ182.71,156.61,155.99,148.57,122.71,122.12,122.11,121.75,10 9.90,107.76,107.75,106.04,95.75,95.73,72.58,72.36,72.15,50.89,25.15,16.76,15.63.

[0052]

[0053] Synthesis of compound 3: Compound 2 (800 mg, 3.3 mmol) was dissolved in anhydrous dichloromethane (12 mL) under a nitrogen atmosphere and in an ice bath. Boron tribromide (20 mL, 2 mol / L in dichloromethane) was added, and the reaction was carried out at ambient temperature for 8 h. The pH of the reaction solution was adjusted to neutral by saturated sodium bicarbonate aqueous solution, and a large amount of solid precipitated out. The solid was washed with distilled water, filtered, and dried to give 720 mg of turmeric-colored solid, with a yield of 96%.

[0054] 1 H NMR (400MHz, DMSO-d6) δ10.13(s,1H),7.08(d,J=8.2Hz,1H),6.89(s,1H),6.47(d,J=8.0Hz,2H),2.28(t,J=6.0Hz,2H),1.60(p,J=6.0Hz,2H). 13C NMR (150MHz, DMSO-d6) δ186.08,161.23,153.62,128.79,128.46,111.08,29.41,21.78,20.60.

[0055]

[0056] Synthesis of compound 4: 1,1,2-trimethylbenzo[e]indole (2.1 g, 10 mmol) and anhydrous acetonitrile (20 mL) were added to a 35 mL pressure flask, followed by the addition of 6-iodo-1-hexyne (1.96 mL, 15 mmol). The mixture was placed in an oil bath at 80 °C and reacted for 48 h. After the reaction was completed, the reaction system was cooled to room temperature, and ethyl acetate was added to precipitate a large amount of solid. The solid was filtered under reduced pressure to give 3.49 g of green solid, with a yield of 83%.

[0057] 1 H NMR(400MHz,Chloroform-d)δ8.15-8.04(m,3H),7.86(d,J=8.9Hz,1H),7.78-7.65(m,2H),4.89(t,J=8.0Hz,2H),3.25 (s,3H),2.37(td,J=6.7,2.6Hz,2H),2.18(p,J=7.9Hz,2H),1.99(t,J=2.6Hz,1H),1.90(s,6H),1.80(p,J=6.8Hz,2H).

[0058]

[0059] Synthesis of compound 5: Compound 3 (228 mg, 1 mmol) and compound 4 (835 mg, 2 mmol) were added sequentially to a 25 mL pear-shaped flask, followed by toluene (2 mL) and n-butanol (4.6 mL); the mixture was stirred at 80 °C for 48 h; after the reaction was completed by TLC monitoring, the solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 120:1, v / v) to obtain 540 mg of blue solid, with a yield of 86%.

[0060] 1H NMR(400MHz,DMSO-d6)δ8.70(d,J=14.9Hz,1H),8.36(d,J=8.5Hz,1H),8.17(dd,J=19.5,8.6Hz,2H),7.95(d,J=8.9Hz,1H),7.74(t,J=7.3Hz,1H),7.62(t,J=7.5Hz,1H),7.53(s,1H),7.49(d,J=8.5Hz,1H),6.96(d,J=2.2Hz,1H),6.87(dd,J=8.5,2.3Hz,1H),6.56(d,J=15.0Hz,1H),4.54(t,J=7.4Hz,2H),2.81(t,J=2.6Hz,1H),2.77-2.68(m,4H),2.29(td,J=7.0,2.7Hz,2H),2.02(s,6H),1.94(t,J=7.5Hz,2H),1.86(t,J=6.0Hz,2H),1.64(p,J=7.1Hz,2H). 13 C NMR(150MHz,DMSO-d6)δ178.63,162.16,161.23,154.69,144.30,139.56,135.91,134.50,132.64,131.19,130.51,129.68,128.55,127.61,126.40,126.36,122.94,114.98,114.92,114.23,112.85,103.62,102.53,84.49,72.19,60.22,52.32,44.82,28.80,27.79,27.16,25.47,24.06,20.50,17.85.HR-MSm / z:[M+H] + calculated for C 35 H 34 NO2 + ,500.2584;found,500.2582.

[0061]

[0062] Synthesis of compound 6: Compound 5 (535 mg, 0.85 mmol) was dissolved in dichloromethane (18 mL) at 0 °C, followed by the addition of compound diphenylphosphochloride (455 μL, 2.38 mmol) and then triethylamine (236 μL, 1.7 mmol). The resulting solution was stirred at room temperature for 1 h. The solvent was then concentrated under vacuum, and the residue was purified by silica gel chromatography (DCM / MeOH, 10:1, v / v) to give compound 6 as a blue solid (750 mg, 77%).

[0063] 1 H NMR (400MHz, DMSO-d6) δ8.65(d,J=15.2Hz,1H),8.44(d,J=8.6Hz,1H),8.22(dd,J=23.5,8.6Hz,2H),8.06-7.94(m,5H) ,7.80(t,J=7.7Hz,1H),7.71-7.65(m,3H),7.65-7.58(m,4H),7.50(d,J=8.5Hz,1H),7.40(s,1H),7.33(s,1H),7.24(d ,J=8.5Hz,1H),6.71(d,J=15.2Hz,1H),4.63(t,J=7.4Hz,2H),3.09(q,J=7.4Hz,2H),2.82(s,1H),2.70(t,J=6.1Hz,4H ),2.32-2.25(m,2H),2.03(s,6H),2.00-1.89(m,3H),1.88-1.79(m,2H),1.70-1.60(m,2H).HRMS(ESI-TOF):calcd.for C 47 H 43 NO3P[M] + 700.2975; found 700.2981.

[0064]

[0065] Synthesis of compound BCY-DP: Compound 6 (750 mg, 0.9 mmol), biotin-azide (236 mg, 0.72 mmol), and copper hexafluorophosphate tetraacetonitrile (113 mg, 0.3 mmol) were added to a round-bottom flask, followed by the addition of acetonitrile (20 mL) and diisopropylethylamine (105 μL). The reaction was carried out at room temperature for 24 hours. The solvent was then concentrated under vacuum and purified using a reverse-phase C18 reactor (water / acetonitrile, 50:50, v / v) to give compound BCY-DP, a blue solid (300 mg, 28.9%).

[0066] 1H NMR (400MHz, CD3OD) δ8.79 (d, J = 15.1Hz, 1H), 8.46-8.38 (m, 1H), 8.20-8.07 (m, 2H), 8.05-7.93 (m, 3H), 7.86-7 .73(m,3H),7.72-7.47(m,7H),7.43-7.34(m,2H),7.24-7.11(m,2H),6.63(d,J=14.7Hz,1H),5.40-5.21(m,1H) ,4.68-4.49(m,3H),4.49-4.40(m,1H),4.40-4.32(m,2H),4.30-4.19(m,1H),3.21-3.06(m,4H),2.96-2.78(m, 3H),2.78-2.57(m,4H),2.32-1.85(m,16H),1.76-1.48(m,5H),1.01-0.79(m,2H).HRMS(ESI-TOF):calcd.forC 60 H 65 IN7O5PS[M] + 1153.3550; found 1153.3557.

[0067] Example 2: BCY-DP to ONOO - Spectral response, selectivity and pH stability

[0068] In this embodiment, the effect of BCY-DP on ONOO was examined. - The spectral response performance of BCY-DP in the PBS / MeOH (1 / 9, v / v, pH 7.4) system is as follows (specific experimental procedures are described in detail below). Figure 4 As shown, through Figure 4 It can be seen that adding ONOO - Subsequently, BCY-DP exhibited a red shift, and the solution color changed from blue to blue-green.

[0069] Figure 3 a depiction of BCY-DP (10μM) and ONOO at an excitation wavelength of 690nm. - The fluorescence spectrum of the response (0 μM-40.0 μM) in PBS / MeOH (1 / 9, v / v, pH 7.4) solution was obtained by... Figure 3 As can be seen, BCY-DP itself has almost no fluorescence signal; with the addition of 40.0 μM ONOO - Subsequently, significant fluorescence enhancement was observed at 725 nm; furthermore, the fluorescence intensity (F) of BCY-DP was [not specified]. 725 nmIt also showed similarity to ONOO in the 0-40.0 μM range. - Good linear relationship ( Figure 3 b) The detection limit is as low as 43.9 nM; Figure 3 c illustrates the differences between BCY-DP (10.0 μM) and 40 μM ONOO. - Fluorescence kinetic analysis within 60 min, via Figure 3 c shows that BCY-DP and ONOO - The response reached saturation within 30 minutes.

[0070] This embodiment also investigated the selectivity of BCY-DP in PBS / MeOH (1 / 9, v / v, pH 7.4) (specific experimental procedures are as described in existing methods and will not be detailed here; the analytes investigated included H₂O₂, TBHP, NaOCl, ·OH, and O₂). ·- , 1 O2, NO, ONOO - NO2 - Fe 2+ Fe 3+ Cu 2+ Ca 2+ Zn 2+ Mg 2+ K + Na + , Glycine, Leucine, Cysteine, Lysine, Arginine, Histidine, Phenylalanine), the results are as follows Figure 3 As shown in d, add ONOO - Subsequently, the fluorescence intensity of BCY-DP increased rapidly by more than 5 times; in contrast, the fluorescence intensity changes induced by other analytes such as reactive oxygen species, reactive nitrogen species, and amino acids were negligible, confirming that BCY-DP inhibits the fluorescence intensity of ONOO. - High selectivity.

[0071] This embodiment considers the presence or absence of ONOO. - The pH effect of BCY-DP under certain conditions was evaluated (specific experimental procedures are described in detail below, referring to existing methods), and the results are as follows: Figure 5 As shown, the fluorescence intensity of BCY-RDP remained stable in the pH range of 4.0 to 9.0; in ONOO - In its presence, BCY-DP exhibits a significant fluorescence on-off response in the pH range of 4.0 to 9.0, indicating that BCY-DP can operate over a wide pH range and is suitable for imaging applications at physiological pH values.

[0072] To explore the effects of BCY-DP on ONOO- The response mechanism in this embodiment combines BCY-DP with ONOO. - Mass spectrometry analysis was performed after the treatment, and the results are as follows: Figure 6 As shown; in relation to ONOO - After the reaction, a mass peak of partially free BCY-DP was observed at m / z = 1026.56 (calculated value of BCY-DP: 1026.45). Additionally, m / z = 826.58 corresponds to [BCY-DP + ONOO]. - The new peak (calculated value 826.41) was observed. Based on these observations, it can be inferred that BCY-DP and ONOO... - The fluorescence reaction may be achieved through Figure 1 The route shown is as follows: ONOO - The strong nucleophilic substitution promotes the cleavage of the DP group to generate the phenoxy anion intermediate, releasing the hemicyanine fluorophore BCY, thereby significantly enhancing ONOO. - Fluorescence-dependent.

[0073] Example 3: Cell Culture and Cytotoxicity Assay

[0074] SKOVE cells (source: cells provided by the Department of Gynecologic Oncology, First Hospital of Jilin University (Jilin University, Changchun, China)) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C under a 5% CO2 atmosphere. The cytotoxicity of BCY-DP to live SKOV3 cells was detected using the standard CCK8 assay. Approximately 1×10⁶ cells were added to 100 μL of cell culture medium. 4 Cells were seeded at 100 cells / well in 96-well microplates, then replaced with fresh medium (RPMI 1640 medium) containing different concentrations of BCY-DP (0, 10, 20, 30, 40, 50 μM) for 24 h, with 10 μL of CCK-8 reagent added to each well. After a further 2-hour incubation, absorbance at 450 nm was measured using a microplate reader. Three parallel wells were set up for each concentration, and the average value was calculated. Cell viability is expressed relative to control cells (100% metabolic activity). Results are as follows: Figure 7 As shown, through Figure 7 It can be seen that the probe has low cytotoxicity.

[0075] Example 4: Fluorescence Imaging in Live Cells

[0076] For colocalization experiments:

[0077] SKOV3 cells were co-incubated with BCY-DP (10.0 μM) and Mito Tracker Green (MTG, a mitochondrial green fluorescent probe) (1.0 μM) for 30 minutes, respectively; fluorescence images were captured using CLSM, and the results are as follows: Figure 8 As shown; via Figure 8 As can be seen, the Pearson coefficient (PCC) of BCY-DP versus MTG is as high as 0.91, indicating that BCY-DP has excellent mitochondrial targeting ability. BCY-DP:λ ex =640nm,λ em =650-700nm; MTG:λ ex =488nm,λ em =500-550nm.

[0078] For ONOO of BCY-DP in cells - Sensitivity:

[0079] SKOV3 cells were incubated with BCY-DP (10.0 μM) for 30 minutes before imaging as a control. SKOV3 cells were first incubated with SIN-1 (lincidomin, an ONOO... - Pretreatment with donor (100 μM) for 2 h, followed by BCY-DP (10 μM) treatment for 30 min, and imaging showed significant fluorescence enhancement. Subsequently, UA (urea, a highly efficient ONOO) was used. - SKOV3 cells were pretreated with a scavenger (500 μM) and SIN-1 (100 μM) for 2 h, followed by incubation with BCY-DP (10 μM) for 30 min before imaging. The fluorescence was significantly suppressed. These results indicate that BCY-DP can be used for in situ detection of endogenous ONOO. - Changes were observed. SKOV3 cells were pretreated with LPS (lipopolysaccharide, 1 μg / mL) and INF-γ (interferon-γ, 10 ng / mL) for 24 h, followed by BCY-DP (10 μM) treatment for 30 min before imaging; a significant increase in fluorescence was also observed. Adding NAC (N-acetylcysteine, 2 mM) to the aforementioned system significantly reduced the fluorescence emission of cells incubated with BCY-DP (10 μM). This further demonstrates that BCY-DP can be used for in situ detection of endogenous ONOO. - The changes.

[0080] Visualizing cancer cells using BCY-DP:

[0081] Normal ovarian cells (IOSE-80) and ovarian cancer cells (SKOV3, A2780, and ID8) were incubated with BCY-DP (10.0 μM) for 30 minutes. All cells were provided by the Department of Gynecologic Oncology, First Hospital of Jilin University (Jilin University, Changchun, China). The fluorescence signal in cancer cells was significantly stronger than that in normal cells, with a fluorescence enhancement of more than 5 times. Figure 10 This result is due to the overexpression of biotin receptors in tumor cells and the high levels of ONOO in tumor cell mitochondria. - This result demonstrates that BCY-DP can be used as an effective probe to distinguish cancer cells from normal cells.

[0082] The above-described specific examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention. Therefore, the scope of protection of this invention should be determined by the scope of the claims.

Claims

1. A fluorescent probe targeting organelles for the specific detection of tumor cells, characterized in that, The specific structural formula of the fluorescent probe is as follows:

2. The organelle-targeting fluorescent probe for specific detection of tumor cells according to claim 1, characterized in that, The fluorescent probe BCY-DP is enriched in tumor cells, targets intracellular mitochondria, and is effective against ONOO. - Selective.

3. The method for preparing the BCY-DP fluorescent probe according to claim 1, characterized in that, This method uses hemicyanine dye and diphenylphosphine chloride as reactants under ambient temperature and pressure. The intermediate product is then reacted with biotin-azide to finally obtain BCY-DP. The chemical structural formula of the hemicyanine dye is as follows: The chemical structural formula of the biotin-azide is:

4. The method for preparing the BCY-DP fluorescent probe according to claim 3, characterized in that, The method specifically includes the following steps: Step 1. Dissolve D-biotin in an organic solvent, add N,N'-carbonyldiimidazole, and stir the solution at room temperature; add 3-azidopropylamine and continue stirring at room temperature; after the reaction is complete, add ice-cold diethyl ether to the reaction solution, filter the obtained white solid; then perform column chromatography to separate the intermediate product, namely biotin-azide. Step 2. Dissolve 2-hydroxy-4-methoxybenzaldehyde in an organic solvent, add cesium carbonate and 2-bromocyclohex-1-ene-1-carboxaldehyde, and stir overnight at 80°C under nitrogen protection. After the reaction is complete, filter to remove insoluble matter from the system, and then perform column chromatography to obtain a yellow solid. Step 3. Dissolve the intermediate product from Step 2 in an organic solvent, add boron tribromide under a nitrogen atmosphere and ice bath, and then stir at room temperature; after the reaction is complete, adjust the pH of the reaction solution to neutral with a saturated sodium bicarbonate aqueous solution, a large amount of solid precipitates out, wash with distilled water and filter, dry to obtain a turmeric-colored solid; Step 4. Dissolve 1,1,2-trimethylbenzo[e]indole in an organic solvent, add 6-iodo-1-hexyne, and stir the solution at 80°C; after the reaction is complete, add ethyl acetate to the reaction solution to precipitate a large amount of solid, and filter to obtain a green solid; Step 5. Add an organic solvent to the intermediate products obtained in Steps 3 and 4, and stir the solution at 80°C. After the reaction is complete, concentrate the solvent and then purify it by column chromatography to obtain the intermediate product, i.e., hemicyanine dye. Step 6. Dissolve the hemicyanine dye in an organic solvent, add diphenylphosphochloride, then add triethylamine, stir the resulting solution at room temperature, concentrate the solvent after the reaction is complete, and then purify by column chromatography to obtain the intermediate product; Step 7. Add the intermediate product obtained in Step 6, biotin-azide and copper hexafluorophosphate tetraacetonitrile to the reactor, followed by the addition of organic solvent and diisopropylethylamine, and react at room temperature; after the reaction is completed, concentrate the solvent and then purify using reverse-phase C18 to obtain BCY-DP.

5. The method for preparing the BCY-DP fluorescent probe according to claim 4, characterized in that, The organic solvent in step 1 is selected from any one of N,N-dimethylformamide, dichloromethane, acetonitrile, methanol, and ethanol, and the molar ratio of D-biotin, N,N'-carbonyldiimidazole, and 3-azidopropylamine is 0.5:0.9:0.75; the organic solvent in step 2 is selected from any one of acetonitrile, dichloromethane, methanol, and ethanol, and the molar ratio of 2-hydroxy-4-methoxybenzaldehyde, 2-bromocyclohex-1-ene-1-carboxaldehyde, and cesium carbonate is 11.28:14:33.9; the organic solvent in step 3 is selected from dichloromethane and acetonitrile. The intermediate obtained in step 2 is selected from any one of methanol, ethanol, and acetonitrile, and the molar ratio of the intermediate obtained in step 2 to boron tribromide is 3.3:40; the organic solvent in step 4 is selected from any one of anhydrous acetonitrile, dichloromethane, acetonitrile, n-butanol, methanol, ethanol, and the molar ratio of 1,1,2-trimethylbenzo[e]indole to 6-iodo-1-hexyne is 2:3; the organic solvent in step 5 is selected from any one or a mixture of two or more of anhydrous toluene, n-butanol, acetonitrile, methanol, ethanol, and the molar ratio of the intermediate obtained in step 3 to the intermediate obtained in step 4 is 1:

2.

6. The method for preparing the BCY-DP fluorescent probe according to claim 4, characterized in that, The organic solvent mentioned in step 6 is selected from any one of dichloromethane, acetonitrile, methanol, and ethanol, and the molar ratio of hemicyanine dye to diphenylphosphine chloride is 0.85:2.38; the organic solvent mentioned in step 7 is selected from any one of acetonitrile, methanol, ethanol, n-butanol, and anhydrous toluene, and the molar ratio of the intermediate product obtained in step 6 to biotin-azide is 0.9:0.

72.

7. The fluorescent probe BCY-DP as described in claim 1 is used in the preparation of detection reagents and / or kits for cancer diagnosis.

8. The application according to claim 7, characterized in that, The cancers mentioned include ovarian cancer, cervical cancer, lung cancer, breast cancer, and thyroid cancer.

9. The use of the fluorescent probe BCY-DP as described in claim 1 in the preparation of detection reagents and / or kits for specifically labeling intracellular mitochondria.

10. The fluorescent probe BCY-DP as described in claim 1 is used in the preparation of a probe for detecting ONOO. - Application in modified detection reagents and / or kits.

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