Cancer cell targeted hypochlorous acid ratio type fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a cancer cell targeted hypochlorous acid ratio type fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of hypochlorous acid ratio type fluorescent probes. According to the technical scheme, the cancer cell targeted hypochlorous acid ratiometric fluorescent probe is characterized in that the structural formula of the cancer cell targeted hypochlorous acid ratiometric fluorescent probe is shown in the specification. The invention further specifically discloses a preparation method of the cancer cell targeted hypochlorous acid ratiometric fluorescent probe and application of the cancer cell targeted hypochlorous acid ratiometric fluorescent probe to selective detection of hypochlorous acid in a water environment and a biological cell system. The ratio-type fluorescent probe has the advantages of ratio-type fluorescence emission, good selectivity, high sensitivity, excellent cancer cell targeting capability and the like.

Description

Technical Field

[0001] This invention belongs to the field of hypochlorous acid ratiometric fluorescent probe technology, specifically relating to a hypochlorous acid ratiometric fluorescent probe for cancer cell targeting, its preparation method, and its application. Background Technology

[0002] In recent years, the incidence and mortality rates of cancer have remained high. Early diagnosis and timely treatment of cancer can significantly improve survival rates. Compared to normal cells, cancer cells have multiple overexpressed receptors on their surface. Biotin, a type of B vitamin, is recognized by most cancer cell lines, such as lung cancer, breast cancer, ovarian cancer, and cervical cancer, which overexpress a receptor (avidin) that recognizes biotin. Avidin is considered an important target for cancer diagnosis and treatment due to its specific binding to biotin. Furthermore, biotin has a relatively simple biochemical structure, low molecular weight, and contains an easily modifiable active functional group -COOH, making it a promising candidate for constructing novel fluorescent probes targeting tumor cells. Reactive oxygen species (ROS) play a crucial role in various biological processes. Due to the extremely rapid respiration rate at tumor sites, HClO is often overexpressed in cancer cells. Therefore, developing a sensitive and accurate fluorescent probe for detecting HClO in vivo is essential for cancer diagnosis and treatment.

[0003] Fluorescent probes, with their advantages of high sensitivity, ease of operation, and non-invasiveness, have become effective tools for monitoring bioactive substances in organisms. To date, researchers have developed numerous fluorescent probes for hypochlorous acid. However, most of these probes achieve detection through changes in a single emission wavelength, and their response signals are easily affected by instrument factors, probe concentration, and microenvironmental variables. In contrast, ratiometric fluorescent probes provide built-in calibration by simultaneously determining the intensity ratio of two emission wavelengths, minimizing these interferences and obtaining more accurate results. Furthermore, an ideal HClO ratiometric fluorescent probe should have a large emission shift. This type of probe avoids overlap between the two emission peaks, obtaining a purer dual-emission peak signal and a more precise dual-emission peak intensity ratio, thereby achieving accurate detection of hypochlorous acid in living organisms. Summary of the Invention

[0004] This invention addresses the problems and current status of hypochlorous acid ratiometric fluorescent probes by providing a hypochlorous acid ratiometric fluorescent probe for cancer cell targeting. This fluorescent probe uses biotin as a cancer cell targeting group for imaging HClO within cancer cells. This fluorescent probe has advantages such as ratiometric fluorescence emission, good selectivity, high sensitivity, and excellent cancer cell targeting ability.

[0005] The present invention also provides a method for preparing the above-mentioned hypochlorous acid ratiometric fluorescent probe for targeting cancer cells and its application in selectively detecting hypochlorous acid in aquatic environments and biological cell systems.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a hypochlorous acid ratiometric fluorescent probe for targeting cancer cells, the structural formula of which is as follows:

[0007]

[0008] The fluorescent probe exhibits ratiometric fluorescence emission, selective detection of hypochlorous acid, and the ability to target cancer cells.

[0009] The preparation method of the hypochlorous acid ratiometric fluorescent probe for cancer cell targeting described in this invention includes the following specific preparation steps:

[0010] Step S1: Add 1.145 g of 2-methoxyphenthiazide, 1.36 g of 1-bromobutane, 0.4 g of NaOH, 12 mg of KI, and 10 mL of DMSO to a 500 mL three-necked flask. React at 95 °C for 6 h under nitrogen protection. After the reaction is complete, cool to room temperature, add 300 mL of water to the mixture, extract three times with dichloromethane, and dry the dichloromethane extract with anhydrous Na2SO4. Filter the dried dichloromethane extract and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain a yellow oily compound 1 with a yield of 63%. The eluent used in the purification process is a 1:1 volume ratio of petroleum ether / dichloromethane mixture. The corresponding synthetic route is as follows:

[0011]

[0012] Step S2: Add 220 μL PCOCl3 to a 100 mL three-necked flask and protect it with nitrogen. Then, under ice bath conditions, slowly add 0.2 mL DMF to the three-necked flask and stir until homogeneous. Then, dissolve 570 mg of compound 1 in 2 mL DMF and add it dropwise to the three-necked flask. React at 60 °C for 4 h. After the reaction is complete, pour the reaction solution into 100 mL of ice water and let it stand. Neutralize the resulting reaction solution with saturated NaHCO3, then extract the reaction solution with dichloromethane and dry it with anhydrous Na2SO4. Filter the dichloromethane extract and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain a yellow solid compound 2 with a yield of 70.3%. The eluent used in the purification process is a 5:1 (v / v) mixture of petroleum ether and ethyl acetate. The corresponding synthetic route is as follows:

[0013]

[0014] Step S3: Add 67.5 mg of aluminum powder and 5 mL of acetonitrile to a 100 mL three-necked flask and stir until homogeneous at room temperature. Then add 394 mg of iodine and stir under nitrogen protection until the solution turns yellow. Next, dissolve 313 mg of compound 2 in 5 mL of acetonitrile and add it dropwise to the three-necked flask. Reflux the reaction system for 6 h. After the reaction is complete, cool the reaction solution to room temperature and pour it into 80 mL of ice water. Then extract the reaction solution three times with ethyl acetate. Dry the combined ethyl acetate phases with anhydrous Na2SO4. Filter the dried ethyl acetate extract and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain a yellow oily compound 3 with a yield of 54%. The eluent used in the purification process is a 15:1 (v / v) mixture of petroleum ether and ethyl acetate. The corresponding synthetic route is:

[0015]

[0016] Step S4: Add 299 mg of compound 3 and 200 mg of diethyl malonate to 50 mL of ethanol, then add 120 μL of piperazine dropwise to the reaction solution. Reflux the reaction system for 2 h, remove the solvent under reduced pressure to obtain the residue, and purify the residue by silica gel column chromatography to obtain an orange solid compound 4 with a yield of 86%. The eluent used in the purification process was a 3:1 (v / v) mixture of petroleum ether and dichloromethane. The corresponding synthetic route is as follows:

[0017]

[0018] Step S5: Add 198 mg of compound 4 to 20 mL of methanol, then add 60 mg of NaOH. Reflux the reaction system for 2 h. After the reaction is complete, evaporate the solvent. Dissolve the residue in 100 mL of dichloromethane. Acidify the dichloromethane solution with 10 wt% hydrochloric acid to a pH of 3.0–4.0. Wash the mixture with water and dry it with anhydrous Na2SO4. Evaporate the dried dichloromethane phase under reduced pressure to obtain the residue. Purify the residue by silica gel column chromatography to obtain a reddish-brown solid compound 5 with a yield of 89%. The eluent used in the purification process is a 10:1 (v / v) dichloromethane / methanol mixture. The corresponding synthetic route is as follows:

[0019]

[0020] Step S6: 367 mg of compound 5, 500 mg of N-(2-aminoethyl)biotinamide, 183 mg of 4-dimethylaminopyridine (DMAP), 288 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 202 mg of 1-hydroxybenzotriazole (HOBt), and 20 mL of LDMF were added to a 100 mL round-bottom flask. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target fluorescent probe compound Biotin-HClO, with a yield of 47.6%. The eluent used in the purification process was a 10:1 (v / v) mixture of dichloromethane and methanol. The corresponding synthetic route is as follows:

[0021]

[0022] The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe described in this invention in the selective detection of hypochlorous acid.

[0023] The present invention describes the application of a cancer cell-targeting hypochlorous acid ratiometric fluorescent probe for the selective detection of hypochlorous acid in aqueous solutions and biological cell systems. This fluorescent probe can detect hypochlorous acid in solutions containing ONOO. - ,H2O2,KO2,TBHP,ROO·,·OH, 1 O2, GSSG, Cl - NO3 - CO3 2- K + Na + Mg 2+ Ca 2+ S 2- Selective detection of hypochlorous acid in PBS buffer solutions containing Cys, Hcy, and GSH.

[0024] The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe described in this invention in the preparation of hypochlorous acid fluorescent detection reagents in aqueous solutions.

[0025] The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe described in this invention in the preparation of a reagent for detecting hypochlorous acid in aqueous solution using visible absorption spectroscopy.

[0026] The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe described in this invention in the preparation of cancer cell hypochlorous acid fluorescence imaging detection reagents.

[0027] Compared with existing technologies, this invention has the following advantages and beneficial effects: (1) The synthesis of the ratiometric fluorescent probe in this invention is relatively easy, and the post-processing is relatively simple; (2) The ratiometric fluorescent probe in this invention achieves high selectivity, high sensitivity, and rapid detection of hypochlorous acid, and has the ability to resist interference from other molecules in living organisms; (3) The ratiometric fluorescent probe in this invention has near-excellent cancer cell targeting ability and ratiometric emission, and can be applied to the imaging detection of hypochlorous acid in liver tissue. The ratiometric fluorescent probe in this invention obtains more accurate detection results and imaging effects by targeting cancer cells and ratiometric fluorescence signals. Therefore, the ratiometric fluorescent probe in this invention has broad application prospects in the field of HClO detection in cancer cells, and is of great significance for the study of the mechanism of action of HClO in the physiological and pathological processes of organisms. Attached Figure Description

[0028] Figure 1 The fluorescence spectra of the fluorescent probe compound Biotin-HClO prepared in Example 1 after the addition of different concentrations of hypochlorous acid are shown.

[0029] Figure 2 This is the UV-Vis absorption spectrum of the fluorescent probe compound Biotin-HClO prepared in Example 1 after the addition of different concentrations of hypochlorous acid.

[0030] Figure 3 The graph shows the relationship between the fluorescence intensity ratio of the fluorescent probe compound Biotin-HClO prepared in Example 1 and the concentration of hypochlorous acid (0-600 μM). The inset shows the linear relationship between the fluorescence intensity ratio of the fluorescent probe compound Biotin-HClO at emission wavelengths of 515 nm and 637 nm and the concentration of hypochlorite (0-100 μM).

[0031] Figure 4 The fluorescence response of the fluorescent probe compound Biotin-HClO prepared in Example 1 to different species is as follows: (1) PBS; (2) 100 μM ONOO. - ; (3) 100μM H2O2; (4) 100μM KO2; (5) 100μM TBHP; (6) 100μM ROO·; (7) 100μM·OH; (8) 100μM 1 O2; (9)500μM GSSG; (10)500μM Cl - (11) 500μM NO3 - (12) 500 μM CO3 2- (13) 1mM K + (14) 1mM Na + (15) 1mM Mg2+ (16) 1mM Ca 2+ (17) 100 μM S 2- ; (18) 200μMCys; (19) 50μMHcy; (20) 1mM GSH; (21) 100μM ClO - .

[0032] Figure 5 This shows the response of the fluorescent probe compound Biotin-HClO prepared in Example 1 to hypochlorous acid under different pH conditions.

[0033] Figure 6 This study examines the targeting ability of the fluorescent probe compound Biotin-HClO prepared in Example 1 on cancer cells.

[0034] Figure 7 This is a fluorescence imaging image of exogenous hypochlorous acid in cancer cells using the fluorescent probe compound Biotin-HClO prepared in Example 1.

[0035] Figure 8 This is a fluorescence imaging image of the fluorescent probe compound Biotin-HClO prepared in Example 1 on endogenous hypochlorous acid in cancer cells. Detailed Implementation

[0036] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0037] Example 1

[0038] Synthesis of the fluorescent probe compound Biotin-HClO

[0039] (1) Synthesis of Compound 1

[0040] 1.145 g of 2-methoxyphenthiazide, 1.36 g of 1-bromobutane, 0.4 g of NaOH, 12 mg of KI, and 10 mL of DMSO were added to a 500 mL three-necked flask. The mixture was reacted at 95 °C for 6 h under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and 300 mL of water was added. The mixture was extracted three times with dichloromethane (100 mL × 3), and the resulting dichloromethane extracts were dried over anhydrous Na₂SO₄. The dried dichloromethane extracts were filtered and the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography to give 10.90 g of a yellow oily compound, with a yield of 63%. The eluent used in the purification process was petroleum ether / dichloromethane at a volume ratio of 1:1. The synthetic route is as follows:

[0041]

[0042] (2) Synthesis of compound 2

[0043] 220 μL of POCl3 was added to a 100 mL three-necked flask under nitrogen protection. Then, 0.2 mL of DMF was slowly added to the flask under ice bath conditions, and the mixture was stirred for 15 min. Subsequently, 570 mg of compound 1 was dissolved in 2 mL of DMF and added dropwise to the three-necked flask, and the reaction was carried out at 60 °C for 4 h. After the reaction was complete, the reaction solution was poured into 100 mL of ice water and left overnight. The resulting solution was neutralized with saturated NaHCO3, then extracted with dichloromethane (100 mL × 3), and dried over anhydrous Na2SO4. The dichloromethane extract was filtered and the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 20.44 g of a yellow solid compound, with a yield of 70.3%. The eluent used in the purification process was petroleum ether / ethyl acetate in a volume ratio of 5:1. The synthetic route is as follows:

[0044]

[0045] (3) Synthesis of compound 3

[0046] Add 67.5 mg of aluminum powder and 5 mL of acetonitrile to a 100 mL three-necked flask and stir at room temperature for 5 min. Then add 394 mg of iodine and stir under nitrogen protection until the solution turns yellow. Next, dissolve 313 mg of compound 2 in 5 mL of acetonitrile and add it dropwise to the three-necked flask. Reflux the reaction mixture for 6 h. After the reaction is complete, cool the reaction solution to room temperature and pour it into 80 mL of ice water. Extract the mixture three times with ethyl acetate (100 mL × 3), and dry the combined ethyl acetate phases with anhydrous Na₂SO₄. Filter the dried ethyl acetate extract and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain 3162 mg of a yellow oily compound, with a yield of 54%. The eluent volume ratio used in the purification process was petroleum ether / ethyl acetate = 15:1. The synthetic route is as follows:

[0047]

[0048] (4) Synthesis of compound 4

[0049] 299 mg of compound 3 and 200 mg of diethyl malonate were added to 50 mL of ethanol, followed by the dropwise addition of 120 μL of piperazine. The reaction mixture was refluxed for 2 h. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography to obtain 4339 mg of an orange solid compound, with a yield of 86%. The eluent used in the purification process was petroleum ether / dichloromethane in a volume ratio of 3:1. The synthetic route is as follows:

[0050]

[0051] (5) Synthesis of compound 5

[0052] 198 mg of compound 4 was added to 20 mL of methanol, followed by 60 mg of NaOH. The reaction mixture was refluxed for 2 h. After the reaction, the solvent was evaporated, and the residue was dissolved in 100 mL of dichloromethane. The dichloromethane solution was acidified to pH 3.0–4.0 with 10 wt% hydrochloric acid. The solution was then washed three times with water (100 mL × 3) and dried with anhydrous Na₂SO₄. The residue was evaporated under reduced pressure to obtain a dry dichloromethane phase. The residue was purified by silica gel column chromatography to give 5115 mg of a reddish-brown solid compound, with a yield of 89%. The eluent ratio used in the purification process was dichloromethane / methanol = 10:1. The synthetic route is as follows:

[0053]

[0054] (6) Synthesis of the fluorescent probe Biotin-HClO

[0055] The following ingredients were added: 367 mg of compound 5, 500 mg of N-(2-aminoethyl)biotinamide, 183 mg of 4-dimethylaminopyridine (DMAP), 288 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 202 mg of 1-

[0056] Hydroxybenzotriazole (HOBt) and 20 mL LDM were added to a 100 mL round-bottom flask and stirred at room temperature for 24 h.

[0057] After the reaction, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 302.4 mg of the target fluorescent probe compound Biotin-HClO, with a yield of 47.6%. The eluent used in the purification process was dichloromethane / methanol.

[0058] =10:1, and its synthesis route is as follows:

[0059]

[0060] Example 2

[0061] Determination of fluorescence spectra of the fluorescent probe compound Biotin-HClO reacting with hypochlorous acid of different concentrations

[0062] The fluorescent probe compound Biotin-HClO prepared in Example 1 was dissolved in N,N-dimethylformamide (DMF) to prepare a 20 μM stock solution. 2 mL of this stock solution was added to a 5 mL centrifuge tube, and different equivalents (0–600 μM) of hypochlorous acid standard solution were added. The solution was diluted to 4 mL with 20 mM PBS buffer (pH = 7.4) (DMF / PBS volume ratio 1:1). The fluorescence spectrum was measured using 440 nm excitation light and a slit width of 5 nm / 5 nm. The fluorescence spectrum is shown below. Figure 1 As shown, with the addition of hypochlorous acid, the fluorescence intensity at 515 nm continuously increases, while the fluorescence intensity at 637 nm continuously decreases. The relationship between fluorescence intensity and hypochlorous acid concentration is as follows: Figure 3 As shown, at 4.0×10 -7 ~1.0×10 -4 The fluorescence exhibits a linear relationship within the concentration range of mol / L, and the fluorescence measurement instrument used is a Hitachi F-7100 fluorescence spectrophotometer.

[0063] Example 3

[0064] Determination of UV-Vis absorption spectra of the fluorescent probe compound Biotin-HClO reacting with hypochlorous acid of different concentrations

[0065] Figure 2 The images show the UV-Vis absorption spectra of the fluorescent probe compound Biotin-HClO prepared in Example 1 after reaction with hypochlorous acid of different concentrations, with the amount of hypochlorous acid added ranging from 0 to 600 μM. Figure 2 As can be seen, the UV-Vis spectrum of the fluorescent probe Biotin-HClO exhibits a blue shift, with the maximum absorption wavelength shifting from 445 nm to 390 nm. With increasing hypochlorous acid concentration, the absorbance at 445 nm gradually decreases, while the absorbance at 390 nm gradually increases. The UV-Vis absorption spectroscopy was measured using a Cary 100 UV-Vis spectrophotometer.

[0066] Example 4

[0067] The selectivity of the fluorescent probe compound Biotin-HClO for various substances in organisms

[0068] Two mL of the fluorescent probe stock solution from Example 3 was added to a 5 mL centrifuge tube. The standard solution of the competing substance to be investigated was added, and the solution was diluted to 4 mL with PBS buffer (20 mM, pH 7.4) (DMF / PBS volume ratio 1:1). A total of 20 substances were investigated, resulting in 21 samples, the last of which was sodium hypochlorite. The fluorescence spectra were measured using 440 nm excitation light and a slit width of 5 nm / 5 nm. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that other intracellular substances affect the fluorescent probe compound Biotin-HClO in I... 515 nm / I 617 nm has almost no effect, while the addition of sodium hypochlorite solution makes the probe Biotin-HClO at I 515 nm / I 617 nm significantly enhanced.

[0069] Example 5

[0070] Effect of solution pH on the fluorescence response of the fluorescent probe compound Biotin-HClO to hypochlorous acid

[0071] The effect of pH on the fluorescence intensity ratio was investigated in two cases: one with the blank fluorescent probe compound Biotin-HClO and the other with Biotin-HClO + hypochlorous acid (100 μM). The results are as follows: Figure 5 As shown, within the pH range of 5–9, pH has virtually no effect on the fluorescence intensity ratio of the blank fluorescent probe compound Biotin-HClO. However, in the presence of sodium hypochlorite (100 μM), within the pH range of 5–7.4, the fluorescence intensity ratio of the detection system is almost unaffected by pH, indicating that the fluorescent probe responds well to hypochlorous acid at this pH. Figure 5 As can be seen, the fluorescent probe compound Biotin-HClO showed a good response to hypochlorous acid within the pH range of 5 to 9.

[0072] Example 6

[0073] Investigation of the targeting ability of the fluorescent probe compound Biotin-HClO on cancer cells

[0074] First, such as Figure 6 As shown in Figure a, the fluorescent probe compound Biotin-HClO (10 μM) was incubated with normal RAW264.7 cells and HeLa cells for half an hour, followed by washing three times with Duchenne phosphate-buffered saline (DPBS) buffer. Subsequently, NaClO solution (final concentration 300 μM) was added, and after 30 minutes, the cells were washed three times with DPBS buffer. The cells were then imaged under a laser confocal microscope. Figure 6As shown in Figure a, under the same conditions and NaClO concentration, the fluorescence intensity of HeLa cells was significantly stronger than that of RAW 264.7 cells. This result indicates that HeLa cells have a significantly stronger uptake capacity for the fluorescent probe compound Biotin-HClO than RAW264.7 cells. This is because HeLa cells have a more abundant expression of biotin receptors on their surface, which facilitates their uptake of the biotin-containing probe Biotin-HClO. Furthermore, the uptake capacity of different cancer cells for the probe was further investigated, such as... Figure 6 As shown in image b. The first row of images shows the incubation of HeLa, HepG2, and MCF-7 cancer cells with the fluorescent probe compound Biotin-HClO (10 μM) for 1 hour, followed by washing three times with Duchenne phosphate-buffered saline (DPBS) buffer, and then imaging under a laser confocal microscope. The second row of images shows the incubation of HeLa, HepG2, and MCF-7 cancer cells with biotin (1 mM) for 1 hour, followed by incubation with the fluorescent probe compound Biotin-HClO (10 μM) for another hour, and then imaging under a laser confocal microscope. According to relevant literature, all three cancer cell types contain endogenous hypochlorous acid, which can illuminate the green channel of the probe. Comparing the two images, it is evident that the fluorescence intensity of cancer cells not pre-incubated with biotin is significantly stronger than that of those pre-incubated with biotin. This result indicates that pre-incubated cancer cells, due to the occupancy of biotin receptors on their surface by biotin, have a reduced number of biotin groups on the probe binding to biotin receptors on the cancer cell surface, thus leading to a decreased probe uptake capacity. These results demonstrate that the fluorescent probe compound Biotin-HClO exhibits good selectivity for cancer cells and demonstrates good targeting ability. The fluorescence imaging conditions were set as follows: λ ex =405nm, fluorescence emission band set to 500-550nm.

[0075] Example 7

[0076] Imaging of exogenous hypochlorous acid in cancer cells using the fluorescent probe compound Biotin-HClO

[0077] First, such as Figure 7As shown, the first row of images shows HeLa cells incubated with the fluorescent probe compound Biotin-HClO (10 μM) for half an hour, washed three times with Duchenne phosphate-buffered saline (DPBS), and then imaged under a laser confocal microscope. The second row of images shows the same process: HeLa cells incubated with the fluorescent probe compound Biotin-HClO (10 μM) for half an hour, washed three times with Duchenne phosphate-buffered saline (DPBS), then incubated with NaClO solution (300 μM) for half an hour, followed by imaged under a laser confocal microscope. The images show that with the addition of exogenous hypochlorous acid, the fluorescence in the long-wavelength red channel decreases, while the fluorescence in the short-wavelength green channel increases. These results indicate that the fluorescent probe compound Biotin-HClO can effectively ratiometrically detect exogenous hypochlorous acid in cancer cells. The fluorescence imaging conditions were set as follows: λ ex =405nm, the green fluorescence emission band is set to 500-550nm, and the red fluorescence emission band is set to 625-750nm.

[0078] Example 8

[0079] Imaging of endogenous hypochlorous acid in cancer cells using the fluorescent probe compound Biotin-HClO

[0080] First, such as Figure 7 As shown, the first row of images shows HeLa cells incubated with the fluorescent probe compound Biotin-HClO (10 μM) for half an hour, washed three times with Duchenne phosphate-buffered saline (DPBS) buffer, and then imaged under a laser confocal microscope. The second row of images shows cells incubated with the fluorescent probe compound Biotin-HClO (10 μM) and Cl... - After incubating HeLa cells with 2.0 mM and MPO (myeloperoxidase, 1 U / mL) for half an hour, the cells were washed three times with Duchenne phosphate-buffered saline (DPBS) buffer. Then, H2O2 (200 μM) was added to the cells and incubated for another half hour. The cells were then imaged under a laser confocal microscope. The third row of images shows the fluorescent probe compound Biotin-HClO (10 μM), Cl... -After incubating HeLa cells with 2.0 mM MPO (myeloperoxidase, 1 U / mL) for half an hour, the cells were washed three times with Duchenne phosphate-buffered saline (DPBS) buffer. Then, 500 μM H2O2 was added to the cells and incubated for another half hour. The cells were then imaged under a laser confocal microscope. The results show that with increasing H2O2 concentration, the concentration of endogenous hypochlorous acid produced under myeloperoxidase catalysis gradually increases, the fluorescence of the long-wavelength red channel in cell imaging gradually decreases, and the fluorescence of the short-wavelength green channel gradually increases. These results indicate that the fluorescent probe compound Biotin-HClO can effectively detect endogenous hypochlorous acid in cancer cells using a ratiometric method. The fluorescence imaging conditions were set as follows: λ ex =405nm, the green fluorescence emission band is set to 500-550nm, and the red fluorescence emission band is set to 625-750nm.

[0081] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A hypochlorous acid ratiometric fluorescent probe for targeting cancer cells, characterized in that... The structural formula of the fluorescent probe is: The fluorescent probe exhibits ratiometric fluorescence emission, selective detection of hypochlorous acid, and the ability to target cancer cells.

2. A method for preparing a hypochlorous acid ratiometric fluorescent probe for cancer cell targeting as described in claim 1, characterized in that... The specific preparation steps are as follows: Step S1: Add 1.145 g of 2-methoxyphenthiazide, 1.36 g of 1-bromobutane, 0.4 g of NaOH, 12 mg of KI, and 10 mL of DMSO to a 500 mL three-necked flask. React at 95 °C for 6 h under nitrogen protection. After the reaction is complete, cool to room temperature, add 300 mL of water to the mixture, extract three times with dichloromethane, and dry the dichloromethane extract with anhydrous Na2SO4. Filter the dried dichloromethane extract and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain a yellow oily compound 1 with a yield of 63%. The eluent used in the purification process is a 1:1 volume ratio of petroleum ether / dichloromethane mixture. The corresponding synthetic route is as follows: Step S2: Add 220 μL PCOCl3 to a 100 mL three-necked flask and protect it with nitrogen. Then, under ice bath conditions, slowly add 0.2 mL DMF to the three-necked flask and stir until homogeneous. Then, dissolve 570 mg of compound 1 in 2 mL DMF and add it dropwise to the three-necked flask. React at 60 °C for 4 h. After the reaction is complete, pour the reaction solution into 100 mL of ice water and let it stand. Neutralize the resulting reaction solution with saturated NaHCO3, then extract the reaction solution with dichloromethane and dry it with anhydrous Na2SO4. Filter the dichloromethane extract and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain a yellow solid compound 2 with a yield of 70.3%. The eluent used in the purification process is a 5:1 (v / v) mixture of petroleum ether and ethyl acetate. The corresponding synthetic route is as follows: Step S3: Add 67.5 mg of aluminum powder and 5 mL of acetonitrile to a 100 mL three-necked flask and stir until homogeneous at room temperature. Then add 394 mg of iodine and stir under nitrogen protection until the solution turns yellow. Next, dissolve 313 mg of compound 2 in 5 mL of acetonitrile and add it dropwise to the three-necked flask. Reflux the reaction system for 6 h. After the reaction is complete, cool the reaction solution to room temperature and pour it into 80 mL of ice water. Then extract the reaction solution three times with ethyl acetate. Dry the combined ethyl acetate phases with anhydrous Na2SO4. Filter the dried ethyl acetate extract and evaporate the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain a yellow oily compound 3 with a yield of 54%. The eluent used in the purification process is a 15:1 (v / v) mixture of petroleum ether and ethyl acetate. The corresponding synthetic route is: Step S4: Add 299 mg of compound 3 and 200 mg of diethyl malonate to 50 mL of ethanol, then add 120 μL of piperazine dropwise to the reaction solution. Reflux the reaction system for 2 h, remove the solvent under reduced pressure to obtain the residue, and purify the residue by silica gel column chromatography to obtain an orange solid compound 4 with a yield of 86%. The eluent used in the purification process was a 3:1 (v / v) mixture of petroleum ether and dichloromethane. The corresponding synthetic route is as follows: Step S5: Add 198 mg of compound 4 to 20 mL of methanol, then add 60 mg of NaOH. Reflux the reaction system for 2 h. After the reaction is complete, evaporate the solvent. Dissolve the residue in 100 mL of dichloromethane. Acidify the dichloromethane solution with 10 wt% hydrochloric acid to a pH of 3.0–4.

0. Wash the mixture with water and dry it with anhydrous Na2SO4. Evaporate the dried dichloromethane phase under reduced pressure to obtain the residue. Purify the residue by silica gel column chromatography to obtain a reddish-brown solid compound 5 with a yield of 89%. The eluent used in the purification process is a 10:1 (v / v) dichloromethane / methanol mixture. The corresponding synthetic route is as follows: Step S6: 367 mg of compound 5, 500 mg of N-(2-aminoethyl)biotinamide, 183 mg of 4-dimethylaminopyridine (DMAP), 288 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 202 mg of 1-hydroxybenzotriazole (HOBt), and 20 mL of LDMF were added to a 100 mL round-bottom flask. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target fluorescent probe compound Biotin-HClO, with a yield of 47.6%. The eluent used in the purification process was a 10:1 (v / v) mixture of dichloromethane and methanol. The corresponding synthetic route is as follows:

3. The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe of claim 1 in the selective detection of hypochlorous acid.

4. The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe of claim 1 in the selective detection of hypochlorous acid in aqueous solutions and biological cell systems, wherein the fluorescent probe can detect hypochlorous acid in solutions containing ONOO. - ,H2O2,KO2,TBHP,ROO·,·OH, 1 O2, GSSG, Cl - NO3 - CO3 2- K + Na + Mg 2+ Ca 2+ S 2- Selective detection of hypochlorous acid in PBS buffer solutions containing Cys, Hcy, and GSH.

5. The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe of claim 1 in the preparation of a hypochlorous acid fluorescent detection reagent in aqueous solution.

6. The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe of claim 1 in the preparation of a reagent for detecting hypochlorous acid in aqueous solution using visible absorption spectroscopy.

7. The application of the cancer cell-targeting hypochlorous acid ratiometric fluorescent probe of claim 1 in the preparation of a cancer cell hypochlorous acid fluorescence imaging detection reagent.

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