A functional fluorescent probe for distinguishing and strengthening ferroptosis in near-infrared region, and a preparation method and application thereof

Abstract

The application discloses a near-infrared distinguishing and iron-apoptosis-enhancing functional fluorescent probe and a preparation method and application thereof. The structural formula of the near-infrared distinguishing and iron-apoptosis-enhancing functional fluorescent probe is as follows: firstly, the traditional boronic acid ether trigger unit is improved to be a cyclic diboronic acid ester, N-methyl imidodicarboxylic acid is introduced above the phenylboronic acid to improve the response threshold, and the benzyl ether structure is abandoned and replaced by a phenyl ether structure, which also plays a role in further increasing the oxidation threshold; secondly, the response threshold is adjusted, and in combination with the strong coordination function of the released Fe(III), the cancer environment selectively triggers and releases the functional probe for iron-apoptosis enhancement. The probe disclosed by the application increases the difference in the reaction threshold between the cancer cell environment and the normal cell environment, and through twice adjustment, the fluorescence trigger difference is increased, the iron-apoptosis intermediate is released at the same time of fluorescence triggering, the cancer cells are caused to undergo iron-apoptosis, and the probe is applied to the detection of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of cancer cell detection technology, specifically relating to a near-infrared functional fluorescent probe that distinguishes and enhances ferroptosis, its preparation method, and its application. Background Technology

[0002] Hydrogen peroxide (H2O2) typically plays a crucial messenger role in regulating cell growth, proliferation, differentiation, and apoptosis. Furthermore, H2O2 participates in initiating immune responses and is directly used by the host to defend against invading pathogens. In cancer cell environments, H2O2 is present at higher concentrations than in normal cells, making it an important biomarker of the cancerous environment. Therefore, differentiating H2O2 levels in cancer cells and normal cells is highly significant for identifying the generation and changes in the cancerous environment.

[0003] Studies have shown that the concentration of H2O2 in inflamed tissues is approximately 10. The concentration of H2O2 in boric acid is typically 100 μM, reaching 100 μM–1 mM in cancer cells, but small amounts are also present in normal tissues. Scientists have designed numerous probes based on boric acid-responsive H2O2 in vivo, with very low detection limits. However, these probes are not suitable for detecting the high concentrations of H2O2 in cancer cells or for distinguishing normal cells.

[0004] Therefore, there is an urgent need to develop a fluorescent probe with a response threshold suitable for H2O2 in cancer cells. Furthermore, if the probe can further target and kill cancer cells after they have responded to the signal, it would be of even greater significance for precision medicine. Summary of the Invention

[0005] To address the problems and deficiencies in existing technologies, this invention discloses a near-infrared distinguishing and ferroptosis-enhanced functional fluorescent probe, its preparation method, and its application. By utilizing the triggering structure of H2O2 and the enhanced ferroptosis structure generated in the cancer environment, it can target and trigger the killing of cancer cells.

[0006] This invention is achieved through the following technical solution:

[0007] A near-infrared functional fluorescent probe that differentiates and enhances ferroptosis has the following structural formula:

[0008] .

[0009] The preparation method of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe of the present invention includes the following steps:

[0010] (1) Synthesis of compound 1: Under low temperature and nitrogen protection, phosphorus oxychloride was dissolved in anhydrous CH2Cl2 and slowly added dropwise to a mixed solution of anhydrous DMF and CH2Cl2. After mixing evenly, cyclohexanone was added dropwise under light protection and the reaction was carried out at low temperature in the dark. Then the temperature was slowly restored to room temperature and heated to reflux. After the reaction was completed, the mixture was placed in ice water and allowed to stand in the dark. The liquid was extracted with a mixture of dichloromethane and ethyl acetate, dried with anhydrous sodium sulfate, and rotary evaporated to obtain compound 1.

[0011] (2) Synthesis of compound 2: Iodoethane was dissolved in toluene, and a toluene solution of 2,3,3-trimethylindole was added dropwise under light-protected conditions. The mixture was heated to reflux under light-protected conditions. After the reaction was completed, the mixture was cooled to room temperature and allowed to stand. The mixture was filtered and washed with a mixture of dichloromethane and petroleum ether to obtain compound 2.

[0012] (3) Synthesis of compound 3: Compound 2 was mixed with sodium acetate and acetic anhydride was added and stirred thoroughly. A solution of acetic anhydride of compound 1 was added dropwise. The resulting mixture was heated to reflux under light-protected conditions. After the reaction was completed, the mixture was concentrated under reduced pressure and washed successively with saturated sodium bicarbonate solution, water, and dichloromethane-methanol mixture to obtain compound 3.

[0013] (4) Synthesis of near-infrared distinguishing and ferroptosis-enhanced functional fluorescent probe: Under nitrogen protection, compound 4, anhydrous dimethylformamide and DMF were mixed, sodium hydride was added, and the mixture was stirred at low temperature. Then, a DMF solution containing compound 3 and dimethylformamide was added, and the mixture was heated to room temperature for reaction. The reaction solution was crudely distilled under reduced pressure and purified by silica gel column chromatography using CH2Cl2-CH3OH mixture as eluent to obtain the near-infrared distinguishing and ferroptosis-enhanced functional fluorescent probe.

[0014] The reaction formula is as follows:

[0015] .

[0016] Further, in step (1), the molar ratio of phosphorus oxychloride to cyclohexanone is 3:1; in step (2), the molar ratio of iodoethane to 2,3,3-trimethylindole is 13:21; in step (3), the molar ratio of compound 2 to compound 1 is 2:1; and in step (4), the molar ratio of compound 4 to compound 3 is 2:1.

[0017] Furthermore, in step (1), the heating and reflux reaction time is 13h; in step (2), the heating and reflux reaction time is 24h; in step (3), the gradient of the temperature rise is 10℃ / h, and the heating and reflux reaction time is 6h; in step (4), the reaction time after heating to room temperature is 5h.

[0018] Further, in step (1), the volume ratio of dichloromethane to ethyl acetate in the dichloromethane-ethyl acetate mixture is 3:1; in step (2), the volume ratio of dichloromethane to petroleum ether in the dichloromethane-petroleum ether mixture is 1:7; in step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 5:1; in step (4), the volume ratio of CH2Cl2 to CH3OH in the CH2Cl2-CH3OH mixture is 15:1, and the silica gel column has a mesh size of 200~300 mesh.

[0019] Furthermore, the ratio of DMF to CH2Cl2 in step (1) is (); the molar ratio of compound 2 to sodium acetate in step (3) is 2:1.

[0020] Furthermore, the low temperature mentioned is -10°C.

[0021] In this invention, the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe is used in the preparation of tumor detection reagents.

[0022] This invention first improves the traditional borate ether triggering unit by converting it into a cyclic diboronic acid ester. N-methyliminodiacetic acid is introduced above phenylboronic acid to increase its response threshold. Furthermore, the commonly used benzyl ether structure is replaced with a phenyl ether structure below, which also further upregulates the oxidation threshold. Secondly, the response threshold is adjusted, and combined with its strong coordination function with Fe(III) after release, a functional probe that selectively triggers and releases ferroptosis-enhancing components in the cancer environment is achieved.

[0023] Using an N-heterocyclic octyl borate ester as the key component of the response reduces the sensitivity of boric acid triggering, effectively distinguishing between high and low concentrations. Unlike classic boric acids, this octyl ring is only effectively triggered at higher concentrations, thus enabling accurate monitoring of H2O2 in cancer cells. Furthermore, N-methyliminodiacetic acid forms a stable complex with Fe(III), causing a decrease in the iron electrode potential, thereby enhancing the reducing power of Fe(II), which in turn promotes the Fenton reaction with H2O2, releasing hydroxyl radicals that induce apoptosis in cells. ② The p-phenyl ether structure assembled at the meso-position of cyanine can selectively undergo cascade elimination under high concentrations of H2O2, further consolidating the fluorescence triggering threshold and ensuring differentiation of the cancer environment. The design and coordination of the various molecular components allow the product to respond to cellular oxidative stress, distinguishing between H2O2 oxidative stress in normal and cancer cell environments while simultaneously exhibiting a fluorescence response. The released groups during this process can induce apoptosis in cancer cells, killing them.

[0024] Beneficial effects

[0025] This invention, through structural innovation of the probe and optimization of the preparation method, creates a functional fluorescent probe that distinguishes between near-infrared and enhances ferroptosis. This increases the difference in reaction threshold between the cancer cell environment and the normal cell environment. Furthermore, through two adjustments, it further increases the difference in fluorescence triggering, achieving simultaneous release of ferroptosis intermediates during fluorescence triggering, thereby inducing ferroptosis in cancer cells. Attached Figure Description

[0026] Figure 1 The image shows the nuclear magnetic resonance (HNMR) spectrum of compound 3.

[0027] Figure 2 This is the mass spectrum of compound 3;

[0028] Figure 3 Nuclear magnetic resonance (HNMR) spectra of functional fluorescent probes for near-infrared differentiation and ferroptosis enhancement;

[0029] Figure 4 Mass spectra of functional fluorescent probes that are near-infrared distinguishable and enhanced by ferroptosis;

[0030] Figure 5 The functional fluorescent probe for near-infrared differentiation and ferroptosis enhancement and its mass spectrum after reaction with H2O2;

[0031] Figure 6 Fluorescence spectra of functional fluorescent probes for near-infrared differentiation and ferroptosis enhancement at different concentrations of H2O2;

[0032] Figure 7 Functional fluorescent probe CDI and conventional molecular probe CDB, used to differentiate near-infrared apoptosis and enhance ferroptosis, were tested at different concentrations of H2O2. I 675 / I 840 and I 675 / I 845 The ratio change, excitation wavelength: 560 nm

[0033] Figure 8 Selectivity test results of a functional fluorescent probe for near-infrared differentiation and ferroptosis enhancement against H2O2;

[0034] Figure 9 This is a dynamic fluorescence imaging image of HeLa cells;

[0035] Figure 10 Confocal imaging for detecting exogenous and endogenous H2O2 in cells using a functional fluorescent probe that distinguishes between near-infrared and enhances ferroptosis. a: red channel b: purple channel, scale bar: 20 μM;

[0036] Figure 11Laser confocal imaging and flow cytometry images of functional fluorescent probes for near-infrared differentiation and ferroptosis enhancement; Left: CDI (10 μM), CDI (10 μM) + H2O2 (100 μM) and CDI (10 μM) + H2O2 (100 μM) + Fe 3+ (5mM) Confocal imaging after 2 h of co-culture with HeLa cells; a: Purple channel; b: Yellow channel; c: Red channel, scale bar: 20 μM; Right: a: CDI (10 μM), b: CDI (10 μM) + H2O2 (100 μM) + Fe 3+ (5 mM) Flow cytometry image of HeLa cells after 2 h of co-culture.

[0037] Figure 12 Imaging analysis of functional fluorescent probes for near-infrared differentiation and ferroptosis enhancement in mouse liver tissue; where A is an image of mouse liver tissue after being cultured with probes CDI (10 μM), H2O2 (0 μM; 50 μM; 100 μM) and collected by confocal microscopy, a: green channel, b: red channel, c: overlay image; B is an image of mouse liver tissue after being cultured with probes CDI (10 μM), H2O2 (100 μM) and Fe... 3+ (0 mM; 2.5 mM; 5 mM) Images collected by confocal microscopy after culture treatment. a: Blue channel, b: Red channel, c: Overlay image. Scale bar: 50 μM. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0039] The near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probes (CDIs) in the following examples were dissolved in PBS buffer (pH=7.4, 5% DMSO, 0.01 M).

[0040] Example 1

[0041] The preparation of a near-infrared spectroscopy-mediated functional fluorescent probe (CDI) for ferroptosis enhancement is shown in the following reaction formula:

[0042]

[0043] The specific steps are as follows:

[0044] (1) Synthesis of compound 1: 23.5 g (153 mmol) of phosphorus oxychloride was dissolved in 20 mL of anhydrous dichloromethane (CH2Cl2) under nitrogen protection at -10 °C. The solution was slowly added dropwise to a mixed solution of 5 mL of anhydrous dimethylformamide (DMF) and 15 mL of CH2Cl2 under nitrogen atmosphere using a constant pressure dropping funnel. After stirring at -10 °C for 1 hour, 5 g (51 mmol) of cyclohexanone was added dropwise under light protection. The mixture was stirred at low temperature under light protection for another hour. After the mixture was slowly restored to room temperature, it was heated to reflux at 150 °C for 13 hours. The liquid after the reaction was completed was poured into a beaker containing ice and allowed to stand in the dark. The solution was extracted with 20 mL of dichloromethane-ethyl acetate mixture (v:v=3:1), dried with anhydrous sodium sulfate, and then rotary evaporated to obtain compound 1.

[0045] (2) Synthesis of compound 2: 6 g (39 mmol) of iodoethane was dissolved in 30 mL of toluene. A solution of 2,3,3-trimethylindole (10 g (63 mmol)) dissolved in 20 mL of toluene was added dropwise under light-protected conditions. The mixture was heated to reflux at 80 °C for 24 hours in the dark. After the reaction was completed, the mixture was cooled to room temperature and allowed to stand for 3 hours. The mixture was then filtered and washed with dichloromethane-petroleum ether (v:v=1:7; 3×20 mL) to obtain pink compound 2.

[0046] (3) Synthesis of compound 3: 7.3 g (23.2 mmol) of compound 2 was mixed with 1.0 g (11.6 mmol) of sodium acetate and 30 mL of acetic anhydride was added. The mixture was stirred thoroughly for 1 hour. Then, 2 g (11.6 mmol) of compound 1 in acetic anhydride (20 mL) solution was added dropwise. The resulting mixture was placed under light-protected conditions and heated gradually (10 °C / h) to 80 °C and refluxed for 6 hours. After the reaction was completed, the mixture was concentrated to 1 / 3 volume under reduced pressure. The mixture was washed successively with 20 mL of saturated sodium bicarbonate solution and 20 mL of water. Finally, it was washed with a mixture of dichloromethane and methanol (v:v=5:1; 20 mL*3 times) to obtain compound 3. The nuclear magnetic resonance (NMR) spectrum of compound 3 is shown below. Figure 1 As shown, the mass spectrum is as follows Figure 2 As shown;

[0047] (4) Synthesis of a near-infrared distinguishing and ferroptosis-enhanced functional fluorescent probe: Under nitrogen protection, 0.15 g (0.6 mmol) of compound 4, 8 mL of anhydrous dimethylformamide, and 2 mL of DMF were added to a three-necked flask. After thorough mixing, 0.015 g (0.6 mmol) of sodium hydride was added to the system, and the mixture was stirred at -10 °C for 50 minutes. 3 mL of a DMF solution containing 0.26 g (0.3 mmol) of compound 3 and 2 mL of dimethylformamide was added to the resulting system. The temperature was gradually raised to room temperature and the reaction was carried out for 5 hours. The reactants were distilled under reduced pressure to 1 / 3 volume, and then reacted with CH2Cl2:CH3OH ( V : V The eluent (15:1 ratio) was purified by silica gel column chromatography (200-300 mesh) to obtain a near-infrared differentiated and ferroptosis-enhanced functional fluorescent probe, the NMR spectrum of which is shown below. Figure 3 As shown, the high-resolution mass spectrum is as follows: Figure 4 As shown.

[0048] Example 2

[0049] The molecular mechanism of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1 in response to H2O2 was verified by mass spectrometry.

[0050] 20 μL of CDI (10 μM) solution was diluted to 1.5 mL with chromatographic grade HPLC acetonitrile solution, and the products of its reaction with H2O2 were detected by mass spectrometry. The results are as follows: Figure 5 As shown, after CDI reacts with H2O2, a new fluorescent group and a mass spectrometry peak of MDA-sodium diacetate ions were detected.

[0051] Example 3

[0052] The response experiment of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1 to H2O2;

[0053] The probe CDI (10 μM) was dissolved in a mixture of PBS (pH=7.4, 10 mM PBS) and 5% DMSO. Different concentrations of H2O2 (0, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) were added to the solution, and the fluorescence spectra were measured. The fluorescence spectra at different concentrations of H2O2 are shown below. Figure 6 As shown, Figure 6 This indicates that CDI has strong fluorescence emission at 840 nm. When it reacts with H2O2, a new fluorescence emission peak appears at 675 nm, while the fluorescence emission at 840 nm decreases with increasing H2O2 concentration, showing a ratio-dependent change.

[0054] Example 4

[0055] The triggering sites of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1 were investigated in response to different H2O2.

[0056] Both the CDI probe and the conventional molecular CDB probe were used to detect H2O2 at an excitation wavelength of 560 nm, with a slit width of 10 nm / 10 nm. For fluorescence spectroscopy, both the CDI and CDB probes were at a concentration of 10 μM, and the solvent was a mixture of PBS buffer solution (pH=7.4, 10 mM PBS) and 5% DMSO. The changes in the fluorescence ratio intensity of H2O2 at different concentrations of CDI and CDB probes were observed, and the results are as follows: Figure 7 As shown, Figure 7 As shown, the fluorescence ratio change trend of CDB at lower H2O2 concentrations is greater than that of CDI, proving that the fluorescent probe CDB can be effectively triggered by lower H2O2 concentrations, while CDI requires concentrations greater than 30 μM to be effectively triggered. Therefore, this high-threshold response mechanism of CDI enhances the accuracy of cancer cell detection.

[0057] Example 5

[0058] Selectivity study of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1;

[0059] Due to the complexity of the biological environment, to demonstrate the high selectivity of the probe CDI for H2O2, it is necessary to examine whether other species introduce interference. The fluorescence ratio (IL) of CDI (10 μM) to other substances was detected in a mixture of PBS buffer (pH=7.4; 10 mM) and 5% DMSO. 675 / I 840 ), λ ex = 560 nm. The response of probe CDI to some biologically relevant reactive oxygen species, reactive nitrogen species, and other biological signals was detected, and the results are shown in 8(a), where 1. blank; 2. NaClO (500 μM); 3. O2 • (500 μM); 4. ONOO - (500 μM); 5. 1 O2(500 μM); 6. t-BOOH (500 μM); 7.NaNO2(500 μM); 8. NO 2- (500 μM); 9. NO 3-(500 μM); 10. NO; 11. Glucose (10 mM); 12. L-isoleucine (1 mM); 13. L-gltamate (1 mM); 14. GSH (1 mM); 15. Cys (1 mM); 16. NaHS (1 mM), the response results to metal ions are shown in Figure 8(b), where 1. Ca 2+ (20 mM); 2. Mg 2 + (20 mM); 3. Cr 3+ (0.1 mM); 4. K + (20 mM); 5. Ba 2+ (20 mM); 6. Mn 2+ (20 mM); 7.Zn 2+ (20 mM); 8. Cu 2+ (1 mM); 9. Hg 2+ (20 mM), from Figure 8 As shown, the fluorescence ratio intensity of the added H2O2 was significantly higher than that of other interfering ions. Therefore, the selectivity experiment demonstrates that the probe CDI exhibits good selectivity for H2O2, making it suitable for application in complex biological samples.

[0060] Example 6

[0061] Experiments on the cell permeability of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1 in HeLa;

[0062] To ensure the CDI probe is suitable for fluorescence imaging in cells and tissues, a kinetic experiment was designed. Cells were incubated in fresh culture medium with CDI (10 μM) and H2O2 (100 μM), respectively, and then imaged under a laser confocal microscope. Excitation wavelength: 560 nm, green channel (600-700 nm) was collected. Results are shown below. Figure 9 As shown, by Figure 9 It can be seen that the probe CDI exhibits significant fluorescence changes within 35 min, demonstrating good membrane permeability and rapid response.

[0063] Example 7

[0064] Imaging analysis of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1 in HeLa;

[0065] First, to verify whether the CDI probe can detect exogenous and endogenous H2O2, one group of HeLa cells was treated with H2O2 (0 μM, 100 μM) for 30 min, followed by treatment with CDI probe (10 μM) for 30 min, and observed under a confocal microscope. Another group of HeLa cells was first treated with the endogenous H2O2 scavenger NAC (1 mM) for 60 min, then treated with PMA (1 μg / mL) to generate endogenous H2O2 for 60 min, followed by treatment with CDI probe for 30 min, and observed under a confocal microscope. The excitation wavelength was 560 nm, and the red channel (600-700 nm) and purple channel (800-900 nm) were collected separately. The confocal images of exogenous and endogenous H2O2 detected by the CDI probe (10 μM) are shown below. Figure 10 As shown, a: red channel, b: purple channel, scale bar: 20 μM; by Figure 10 It can be seen that CDI is suitable for detecting changes in the concentration of exogenous H2O2 and endogenous H2O2 in cells.

[0066] Example 8

[0067] The near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1 was used in an apoptosis-inducing experiment in HeLa.

[0068] To verify whether the probe CDI, after releasing its complex MDA, induces apoptosis, cultured HeLa cells were added to a medium containing probe CDI (10 μM) and cultured for 30 min, followed by the addition of H2O2 (100 μM) and Fe. 3+ (5 mM) co-cultured for 2 h and observed under a confocal microscope (excitation wavelength: 710 nm, collection purple channel: 750-850 nm, excitation wavelength: 550 nm, collection yellow channel: 580-680 nm, excitation wavelength: 670 nm, collection red channel: 700-800 nm) to observe Fe 3+ To investigate whether the Fenton reaction could induce apoptosis, control experiments were conducted with either the addition of only the probe CDI or only the probe and H2O2. The results are as follows: Figure 12 As shown, Fe was added 3+ Subsequently, the fluorescence in the yellow channel gradually turned off, while the fluorescence in the red channel gradually turned on, whereas in the control experiment, there was no obvious change in the fluorescence of the red channel; by comparing control group a (only CDI added) and group b (CDI with H2O2 and Fe), 3+ It was found that Fe 3+It can induce apoptosis in the human epithelial cell cervical cancer cell line HeLa; the proportion of late apoptotic cells increased from 8.57% to 24.0% compared with the control group; the survival rate of normal living cells was also 34.6% lower than that of the control group.

[0069] Therefore, by adding Fe exogenously 3+ Experiments have shown that the MDA released by the probe CDI in response to H2O2 is capable of continuing to bind with Fe. 3+ It binds to the Fenton reaction, promoting apoptosis.

[0070] Example 9

[0071] Imaging analysis of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe (CDI) prepared in Example 1 in mouse liver tissue;

[0072] Liver tissues were obtained from healthy mice aged 6 to 8 weeks, embedded in a polyvinyl chloride (PVA) medium, and frozen at -80°C for 1 hour. After freezing, the liver tissue was sectioned (approximately 1.5 mm thick) using a cryostat. The frozen sections of mouse liver tissue were washed three times with physiological saline and used for the following experiments.

[0073] First, the sections were stained with CDI probe (10 μM) for 1 hour. Next, they were incubated with H2O2 (0 μM, 50 μM, 100 μM) for 30 min, and then washed three times with PBS (pH = 7.4, 10 mM). Finally, the fluorescence changes of the sections were scanned using a laser scanning confocal microscope (excitation wavelength: 560 nm, green channel: 600-700 nm; red channel: 800-900 nm). The results are shown below. Figure 12 As shown in (A). By Figure 12 (A) It can be seen that as the concentration of H2O2 increases, the fluorescence of the green channel increases while the fluorescence of the red channel decreases, showing a trend of ratio change.

[0074] By adding Fe 3+ The ability of the probe CDI to induce the Fenton reaction was explored using (0, 2, 5 mM) methods. First, the sections were stained with CDI (10 μM) for 1 hour. Then, they were incubated with H2O2 (100 μM) for 30 min, followed by inoculation with different concentrations of Fe. 3 + Incubate for 1 h at (0, 2, 5 mM). Finally, scan the fluorescence changes of the sections using laser scanning confocal microscopy (excitation wavelength: 550 nm, collection blue channel: 580-680 nm; excitation wavelength: 670 nm, collection red channel: 700-800 nm). The results are as follows: Figure 12 As shown in (B). Figure 12 (B) indicates that, with Fe 3+ As the concentration increases, the fluorescence in the blue channel decreases, while the fluorescence in the red channel increases, showing a trend of ratio change.

[0075] Therefore, this probe can detect exogenous H2O2 and induce the Fenton reaction at the tissue level.

Claims

1. A near-infrared functional fluorescent probe for distinguishing and enhancing ferroptosis, characterized in that, The structural formula is as follows: 。 2. A method for preparing the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Synthesis of compound 1: Phosphorus oxychloride was dissolved in anhydrous CH2Cl2 at -10℃ and under nitrogen protection. The solution was slowly added dropwise to a mixed solution of anhydrous DMF and CH2Cl2. After mixing, cyclohexanone was added dropwise under light protection. The reaction was carried out at -10℃ under light protection. Then the temperature was slowly restored to room temperature and heated to reflux. After the reaction was completed, the solution was placed in ice water and allowed to stand in the dark. The liquid was extracted with a mixture of dichloromethane and ethyl acetate, dried with anhydrous sodium sulfate, and rotary evaporated to obtain compound 1. (2) Synthesis of compound 2: Iodoethane was dissolved in toluene, and a toluene solution of 2,3,3-trimethylindole was added dropwise under light-protected conditions. The mixture was heated to reflux under light-protected conditions. After the reaction was completed, the mixture was cooled to room temperature and allowed to stand. The mixture was filtered and washed with a mixture of dichloromethane and petroleum ether to obtain compound 2. (3) Synthesis of compound 3: Compound 2 was mixed with sodium acetate and acetic anhydride was added and stirred thoroughly. A solution of acetic anhydride of compound 1 was added dropwise. The resulting mixture was heated to reflux under light-protected conditions. After the reaction was completed, the mixture was concentrated under reduced pressure and washed successively with saturated sodium bicarbonate solution, water, and dichloromethane-methanol mixture to obtain compound 3. (4) Synthesis of near-infrared distinguishing and ferroptosis-enhanced functional fluorescent probe: under nitrogen protection, compound 4 and DMF were mixed, sodium hydride was added, and the mixture was stirred at -10℃. Then, a DMF solution containing compound 3 was added, and the mixture was heated to room temperature for reaction. The reaction solution was crudely distilled under reduced pressure and purified by silica gel column chromatography using CH2Cl2-CH3OH mixture as eluent to obtain the near-infrared distinguishing and ferroptosis-enhanced functional fluorescent probe. The reaction formula is as follows: 。 3. The method for preparing the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe according to claim 2, characterized in that, In step (1), the molar ratio of phosphorus oxychloride to cyclohexanone is 3:1; in step (2), the molar ratio of iodoethane to 2,3,3-trimethylindole is 13:21; in step (3), the molar ratio of compound 2 to compound 1 is 2:1; in step (4), the molar ratio of compound 4 to compound 3 is 2:

1.

4. The method for preparing the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe according to claim 2, characterized in that, In step (1), the heating and reflux reaction time is 13h; in step (2), the heating and reflux reaction time is 24h; in step (3), the gradient of the temperature rise is 10℃ / h, and the heating and reflux reaction time is 6h; in step (4), the reaction time after heating to room temperature is 5h.

5. The method for preparing the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe according to claim 2, characterized in that, In step (1), the volume ratio of dichloromethane to ethyl acetate in the dichloromethane-ethyl acetate mixture is 3:1; in step (2), the volume ratio of dichloromethane to petroleum ether in the dichloromethane-petroleum ether mixture is 1:7; in step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 5:1; in step (4), the volume ratio of CH2Cl2 to CH3OH in the CH2Cl2-CH3OH mixture is 15:1, and the silica gel column has a mesh size of 200~300 mesh.

6. The method for preparing the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe according to claim 2, characterized in that, In step (1), the volume ratio of DMF to CH2Cl2 is 6:1; in step (3), the molar ratio of compound 2 to sodium acetate is 2:

1.

7. The use of the near-infrared distinguishing and ferroptosis-enhancing functional fluorescent probe of claim 1 in the preparation of a reagent for detecting hydrogen peroxide in a tumor environment.

Images