A mitochondrion-targeting fluorescent probe and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a kind of mitochondrion targeted fluorescent probe and its preparation method and application, wherein the mitochondrion targeted fluorescent probe has the structure general formula as shown in formula (I), its preparation method is that nitrogen-containing heterocyclic onium iodide and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde occur Knoevenagel condensation reaction to be prepared in the presence of basic catalyst.The mitochondrion targeted fluorescent probe of the application has mitochondrion targeting, fluorescence imaging capacity and anti-tumor activity, and can be used as tumor cell mitochondrion imaging reagent and anti-tumor drug preparation, provides new strategy for tumor diagnosis and treatment, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mitochondrial-targeting fluorescent probe, its preparation method, and its application. Background Technology

[0002] Mitochondria, as the cell's "energy factory," are not only key regulators of the intrinsic pathway of apoptosis, but also control the activation of apoptosis effector mechanisms by regulating the transport of apoptotic proteins from the intermembrane space between mitochondria to the cytosol. They play a crucial role in various forms of non-apoptotic cell death, particularly in necrotic cells (regulatory necrosis). Alterations in their function and properties are particularly significant in tumorigenesis, making research on mitochondrial-targeted drugs of great importance. Mitochondrial defects or abnormalities can affect cell growth, metabolism, and proliferation, and can also trigger neuronal death, age-related degenerative diseases, and cancer.

[0003] In recent years, fluorescent molecular probes have become an important tool for targeted cancer therapy, with their high sensitivity and timeliness driving the development of biomedical research. Studying the accumulation time, location, and mechanism of action of drugs in cells requires specific fluorescent dyes, making the application of fluorescent molecular probes in the biomedical field essential. The mitochondrial double membrane structure creates a potential difference of -180mV across the inner membrane, driving ATP synthesis and nutrient uptake. Utilizing this property, positively charged groups on drug molecules (such as pyridinium salts and indole salts) are attracted by the negative charge of the inner membrane, allowing the drug to enter the mitochondrial matrix under the influence of the inner membrane potential's electric field. This is a commonly used targeting method.

[0004] Currently, drugs used to treat tumors include nitrogen mustards, pyrimidines, platinum-based drugs, and porphyrins. Nitrogen mustards, as important antitumor drugs, work by forming active imine or carbocation ions in the body, becoming strong alkylating agents. These ions covalently bind to electron-rich groups of biomolecules, causing DNA breakage or loss of activity, thereby inhibiting tumor cell division or causing cell death. The structure of nitrogen mustard drugs consists of an alkylation moiety (the antitumor active functional group) and a carrier moiety (which improves pharmacokinetic properties).

[0005] However, there are currently few anti-tumor drugs that target mitochondria, such as paclitaxel, doxorubicin, and camptothecin. These drugs usually have multiple targets and side effects, and long-term use may lead to multidrug resistance in tumor cells, reducing or eliminating the effectiveness of chemotherapy. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a mitochondrial-targeting fluorescent probe with high mitochondrial targeting, good fluorescence performance and anti-tumor activity, and to provide a method for preparing the probe, as well as its application in mitochondrial imaging of tumor cells and anti-tumor therapy, so as to solve the problems of existing anti-tumor drugs such as multiple targets, large side effects and easy development of drug resistance.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A mitochondrial-targeting fluorescent probe has the general structural formula shown in formula (I):

[0009]

[0010] In equation (I), R1 is One of the groups;

[0011] R2 is an alkyl group with fewer than 6 carbon atoms.

[0012] Preferably, R2 is methyl or ethyl.

[0013] Preferably, the mitochondrial-targeting fluorescent probe comprises compounds with the following structures:

[0014]

[0015] To address the aforementioned technical problems, this invention also provides a method for preparing this mitochondrial-targeting fluorescent probe, specifically as follows:

[0016] In the presence of an alkaline catalyst, nitrogen-containing heterocyclic iodides undergo a Knoevenagel condensation reaction with 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde to obtain a compound with the general structural formula shown in formula (I), which is a mitochondrial-targeting fluorescent probe.

[0017] The nitrogen-containing heterocyclic iodide is Any one of them, wherein R2 is an alkyl group with fewer than 6 carbon atoms.

[0018] Preferably, the alkaline catalyst is piperidine, pyridine, or triethylamine.

[0019] Furthermore, the nitrogen-containing heterocyclic onyx iodide is prepared by reacting a nitrogen-containing heterocyclic compound with an alkyl iodide;

[0020] The nitrogen-containing heterocyclic compound is One of them;

[0021] The alkyl iodide is an alkyl iodide with fewer than 6 carbon atoms, preferably iodomethane or iodoethane.

[0022] Furthermore, the 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde is prepared by the following process:

[0023] At 0–5°C, POCl3 is slowly added dropwise to DMF to form Vilsmeier's reagent; 2,2'-((3-fluorophenyl)azonyl)diethanol (CAS: 323-60-4) is added dropwise, and the temperature is raised to 80–120°C for 8–12 hours. After the reaction is completed, the mixture is cooled to room temperature, poured into ice water, and the pH is adjusted to neutral. The mixture is then filtered and purified.

[0024] The application of the mitochondrial-targeting fluorescent probe described in this invention in the preparation of mitochondrial imaging reagents for tumor cells.

[0025] The application of the mitochondrial-targeting fluorescent probe described in this invention in the preparation of antitumor drugs.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. Good targeting: Mitochondrial-targeting fluorescent probe compounds can be precisely located in the mitochondrial region of the cell through the interaction of positively charged groups with the mitochondrial membrane potential, such as CXL95 and CXL97, which showed good mitochondrial targeting in HeLa cell experiments.

[0028] 2. Excellent fluorescence performance: Mitochondrial-targeting fluorescent probe compounds have specific excitation and emission wavelengths in pure DMSO solution, with maximum ultraviolet absorption around 500 nm and maximum emission wavelength between 550 and 650 nm, which can be used for fluorescence imaging.

[0029] 3. Significant antitumor activity: In vitro experiments showed that the mitochondrial-targeting fluorescent probe compound had a certain inhibitory effect on A549 (human non-small cell lung cancer cells), DU145 (human prostate cancer cells) and MDA-MB-231 (human breast cancer cells), and showed different inhibition rates at different concentrations.

[0030] 4. Simple preparation method: The synthetic route is reasonably designed, the reaction conditions are mild, the yield is high, and it is easy to carry out large-scale production and application. Attached Figure Description

[0031] Figure 1 These are the UV-Vis absorption (A) and fluorescence emission (B) spectra of the five compounds prepared in Examples 1-5.

[0032] Figure 2 This is a cellular fluorescence imaging image of mitochondrial fluorescence colocalization of compounds CXL95 and CXL97.

[0033] Figure 3 The bar chart shows the MTT test results of the five compounds prepared in Examples 1-5. Detailed Implementation

[0034] The following examples further illustrate the specific technical 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 content of the present invention fall within the scope of the present invention.

[0035] Example 1

[0036] The mitochondrial-targeting fluorescent probe CXL95 was prepared using the following synthetic route:

[0037]

[0038] Synthesis of 2,2'-((3-fluorophenyl)azonyl)diethanol (F-1) (commercially available products can also be used directly, CAS: 323-60-4):

[0039] Weigh CaCO3 (4.0 eq, 20.82 g) and KI (0.5 eq, 4.32 g) into a round-bottom flask, add 50 mL of pure water and stir well. Add 5 mL of 3-fluoroaniline (1.0 eq, 5.78 g) dropwise at room temperature, then add 9.08 mL of 2-bromoethanol (2.5 eq, 16.01 g) dropwise. Finally, slowly heat to 100 °C and react overnight. Filter while hot, extract with EA, concentrate and precipitate by column chromatography to obtain the oily product 2,2'-((3-fluorophenyl)azonyl)diethanol (F-1), yield 92.1%.

[0040] Synthesis of 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2):

[0041] POCl3 (25.35 g, 0.1563 mol) was slowly added dropwise to a dry round-bottom flask containing DMF (30 mL), which was pre-cooled with ice water. The mixture was stirred vigorously while 2,2'-((3-fluorophenyl)azanidinediyl)diethanol (F-1) was added dropwise. The reaction mixture was stirred at 100 °C for 10 hours. After the reaction was complete, the reaction solution was cooled to room temperature and then slowly poured into 500 mL of ice water. Saturated sodium hydroxide solution was added while stirring vigorously to adjust the pH of the solution to 7.0. A solid precipitated out. The suspension was filtered and washed three times thoroughly with distilled water, and then purified by chromatographic column (EA:PE = 1:1) to obtain the desired product 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) in 86.8% yield. 1H NMR (400MHz, CDCl3) δ10.10 (s, 1H), 7.76 (t, J = 8.6Hz, 1H), 6.53 (dd, J = 12.4Hz, 8.2Hz ,1H),6.34(dd,J=10.6Hz,8.4Hz,1H),3.84(t,J=10.6Hz,4H),3.68(t,J=8.2Hz,4H).

[0042] Synthesis of 1,2,3,3-tetramethyl-3H-indole-1-onium(4-1):

[0043] 2,3,3-Trimethyl-3H-indole (1.0 eq, 1.59 g) and iodomethane (CH3I, 1.2 eq, 1.69 g) were dissolved in 100 mL of toluene, and the mixture was slowly heated to reflux and reacted for 10 hours. TLC monitoring was performed. After the reaction was complete, the solution in the reaction system was evaporated to dryness using a rotary evaporator under vacuum. Upon addition of petroleum ether, a solid was produced. Filtration yielded 1.51 g of a pink solid, with a yield of 86.78%. 1 H NMR (400MHz, CDCl3) δ7.28 (d, J = 8.7Hz, 1H), 7.12 (d, J = 9.2Hz, 1H), 6.82-6.42 (m, 2H), 2.85 (s, 3H), 1.56 (s, 6H), 1.43 (s, 3H).

[0044] Synthesis of (E)-2-(4-(bis(2-chloroethyl)amino)-2-fluorostyryl)-1,3,3-trimethyl-3H-indole-1-iodide (CXL95):

[0045] Under nitrogen atmosphere, 1,2,3,3-tetramethyl-3H-indole-1-onium(4-1) (1.0 eq, 100 mg) and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL), and piperidine (0.01 eq) was added. The reaction mixture was stirred at 60 °C for 8 hours. After the reaction was complete, the red reaction solution was concentrated, and the product was purified by column chromatography to obtain a red solid, which was the mitochondrial-targeting fluorescent probe CXL95, with a yield of 78.6%. 1H NMR (400MHz, CDCl3) δ9.13(t,J=8.6Hz,1H),8.33(d,J=4.6Hz,1H),8.05(d,J=12.2Hz,1H),7.58-7.48(m,4H),6.83(dd,J=12. 4Hz,8.6Hz,1H),6.40(dd,J=10.6Hz,4.2Hz,1H),4.43(s,3H),3.90(t,J=8.4Hz,4H),3.72(t,J=8.2Hz,4H),1.79(s,6H).MSm / z found:419.14.

[0046] Example 2

[0047] The mitochondrial-targeting fluorescent probe CXL96 was prepared using the following synthetic route:

[0048]

[0049] Synthesis of 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium(4-2):

[0050] 2,3,3-Trimethyl-3H-indole (1.0 eq, 1.59 g) and iodoethane (CH3CH2I, 1.2 eq, 1.86 g) were dissolved in 50 mL of toluene, and the mixture was slowly heated to reflux and reacted for 10 hours. TLC monitoring was performed. After the reaction was complete, the reaction solution was evaporated to dryness under vacuum. The addition of petroleum ether resulted in the formation of a solid, which was filtered to give 1.49 g of a red solid, with a yield of 79.26%. 1 H NMR(400MHz, DMSO-d6)δ8.17(d,J=8.6Hz,1H),7.76(d,J=8.4Hz,1H),7.12-6.72(m, 2H), 3.53 (dd, J = 8.2Hz, 4.7Hz, 2H), 2.34 (s, 3H), 1.77 (s, 6H), 1.51 (t, J = 8.6Hz, 3H).

[0051] Synthesis of (E)-2-(4-(bis(2-chloroethyl)amino)-2-fluorostyryl)-1-ethyl-3,3-dimethyl-3H-indole-1-onium iodide (CXL96):

[0052] Under nitrogen atmosphere, 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium(4-2) (1.0 eq, 105 mg) and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL), and piperidine (0.01 eq) was added. The reaction mixture was stirred at 60 °C for 8 hours. After the reaction was complete, the red reaction solution was concentrated, and the product was purified by column chromatography to obtain a red solid, which was the mitochondrial-targeting fluorescent probe CXL96, with a yield of 45.7%. 1 HNMR (400MHz, DMSO-d6) δ8.97(t,J=10.6Hz,1H),8.32(d,J=12.2Hz,1H),7.75(d,J=8.4Hz,1H),7.57-7.50(m,4H),6.84(t,J= 10.6Hz,1H),6.42(d,J=8.4Hz,1H),4.96(dd,J=10.6Hz,8.6Hz,2H),3.95-3.72(m,8H),1.80(s,6H),1.61(t,J=8.2Hz,3H).MS m / zfound:433.16.

[0053] Example 3

[0054] The mitochondrial-targeting fluorescent probe CXL97 was prepared using the following synthetic route:

[0055]

[0056] Synthesis of 3-ethyl-1,1,2-trimethyl-1H-benzoindole-3-onium(4-3):

[0057] 1,1,2-Trimethyl-1H-benzoindole (1.0 eq, 2.09 g) and iodoethane (1.2 eq, 1.86 g) were dissolved in 80 mL of toluene, and the mixture was slowly heated to reflux and reacted for 10 hours. TLC monitoring was performed. After the reaction was complete, the reaction solution was evaporated to dryness under vacuum. Upon addition of petroleum ether, a solid was produced. The solid was filtered to obtain a red solid, which was washed twice with anhydrous diethyl ether. The final product was 1.90 g, with a yield of 79.80%. 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=8.8Hz,1H),8.31(d,J=12.2Hz,1H),8.23(d,J=8.0Hz,1H),8.16(d,J=8.6 Hz,1H),7.82-7.72(m,2H),4.63(dd,J=12.2Hz,4.6Hz,2H),2.94(s,3H),1.77(s,6H),1.16(t,J=8.6Hz,3H).

[0058] Synthesis of (E)-2-(4-(bis(2-chloroethyl)amino)-2-fluorostyryl)-3-ethyl-1,1-dimethyl-1H-benzo[E]indole-3-iodide (CXL97):

[0059] Under nitrogen atmosphere, 3-ethyl-1,1,2-trimethyl-1H-benzoindole-3-onium(4-3) (1.0 eq, 121 mg) and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL), and piperidine (0.01 eq) was added. The reaction mixture was stirred at 60 °C for 8 hours. After the reaction was complete, the red reaction solution was concentrated, and the product was purified by column chromatography to obtain a red solid, which was the mitochondrial-targeting fluorescent probe CXL97, with a yield of 53.3%. 1 HNMR (400MHz, DMSO-d6) δ8.41(d,J=10.6Hz,1H),8.35-8.19(m,4H),8.08(d,J=4.4Hz,1H),7.79(t,J=8.6Hz,1H),7.70(t,J=6.4Hz,1H),7. 44(d,J=10.6Hz,1H),6.93(d,J=8.2Hz,2H),4.72(dd,J=12.6Hz,8.4Hz,2H),4.00-3.84(m,8H),1.99(s,6H),1.49(t,J=10.2Hz,3H).MSm / z found:483.17.

[0060] Example 4

[0061] The mitochondrial-targeting fluorescent probe CXL98 was prepared using the following synthetic route:

[0062]

[0063] Synthesis of 1-ethyl-2-methylquinoline-1-onium(4-4):

[0064] 2-Methylquinoline (1.0 eq, 1.43 g) and iodoethane (1.2 eq, 1.86 g) were dissolved in 50 mL of toluene, and the mixture was slowly heated to reflux and reacted for 20 hours. TLC monitoring showed that a solid was produced after the reaction was complete. The reaction solution was filtered to obtain a white solid, which was washed twice with anhydrous diethyl ether. The final product was 1.02 g, with a yield of 59.30%. 1H NMR (400MHz, DMSO-d6) δ9.12(d,J=8.8Hz,1H),8.63(d,J=10.0Hz,1H),8.43(dd,J=12.4Hz,4.8Hz,1H),8.26-8.2 2(m,1H),8.14(d,J=8.6Hz,1H),8.00(t,J=8.4Hz,1H),5.02(q,J=12.2Hz,4.6Hz,2H),3.13(s,3H),1.54(s,3H).

[0065] Synthesis of (E)-2-(4-(bis(2-chloroethyl)amino)-2-fluorostyryl)-1-ethylquinoline-1-iodide (CXL98):

[0066] Under nitrogen atmosphere, 1-ethyl-2-methylquinoline-1-onium(4-4) (1.0 eq, 100 mg) and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL), and piperidine (0.01 eq) was added. The reaction mixture was stirred at 60 °C for 8 hours. After the reaction was complete, the red reaction solution was concentrated, and the product was purified by column chromatography to obtain a red solid, which was the mitochondrial-targeting fluorescent probe CXL98, with a yield of 69.6%. 1 HNMR(400MHz,DMSO-d6)δ8.93(d,J=6.4Hz,1H),8.61(d,J=8.4Hz,1H),8.52 (d,J=4.6Hz,1H),8.34(d,J=8.6Hz,1H),8.21-8.13(m,2H),8.03(t,J=8.2Hz ,1H),7.91(d,J=6.4Hz,1H),7.58(d,J=12.8Hz,1H),6.84-6.79(m,2H),5.0 7(dd,J=12.8Hz,8.4Hz,2H),3.91-3.81(m,8H),1.57(t,J=6.8Hz,3H).MSm / z found:417.12.

[0067] Example 5

[0068] The mitochondrial-targeting fluorescent probe CXL99 was prepared using the following synthetic route:

[0069]

[0070] Synthesis of 2,3-dimethylbenzothiazole-3-onium(4-5):

[0071] 2-Methylbenzothiazole (1.0 eq, 1.49 g) and iodomethane (1.2 eq, 1.69 g) were dissolved in 50 mL of toluene, and the mixture was slowly heated to reflux and reacted for 24 hours. TLC monitoring showed that a solid was produced after the reaction was complete. The reaction solution was filtered to obtain a pink solid, which was washed twice with anhydrous diethyl ether. The final product was 0.98 g, with a yield of 79.04%. 1 H NMR (400MHz, DMSO-d6) δ8.47(d,J=8.4Hz,1H),8.30(d,J=8.6Hz,1H),7.90(q,J=12.4Hz,8.4Hz,1H),7.81(d,J=6.4Hz,1H),4.22(s,3H),3.20(s,3H).

[0072] Synthesis of (E)-2-(4-(bis(2-chloroethyl)amino)-2-fluorostyryl)-3-methylbenzo[d]thiazole-3-iodide (CXL99):

[0073] Under nitrogen atmosphere, 2,3-dimethylbenzothiazol-3-onium(4-5) (1.0 eq, 97 mg) and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL), and piperidine (0.01 eq) was added. The reaction mixture was stirred at 60 °C for 8 hours. After the reaction was complete, the red reaction solution was concentrated, and the product was purified by column chromatography to obtain a red solid, which was the mitochondrial-targeting fluorescent probe CXL99, with a yield of 63.7%. 1 HNMR (400MHz, DMSO-d6) δ8.34(d,J=6.4Hz,1H),8.13(t,J=10.6Hz,1H),8.06(d,J=10.2Hz,2H),7.82(t, J=10.2Hz,1H),7.71(t,J=8.4Hz,2H),6.86(m,2H),4.27(s,3H),3.93-3.83(m,8H).MSm / zfound:409.07.

[0074] Example 6: Fluorescence property determination

[0075] Solution preparation: Accurately prepare 1 mM dimethyl sulfoxide (DMSO) stock solutions for each of the five compounds (CXL95, CXL96, CXL97, CXL98, and CXL99) and store them at 4°C for use in determining the excitation and emission wavelengths. During testing, dilute to 10 μM with DMSO and determine their spectral properties at room temperature.

[0076] Experimental instruments: FluoroMax-4 fluorescence spectrophotometer (Horiba Jobin Yvon), Hitachi Pharma SpecUV-1900UV-visible spectrophotometer.

[0077] Experimental results: The measured UV-Vis absorption and fluorescence spectra are as follows: Figure 1 As shown, the maximum ultraviolet absorption of the five compounds is around 500 nm, and the maximum emission wavelength is between 550 and 650 nm, which does not reach the near-infrared emission wavelength. Specific data are shown in Table 1.

[0078] Table 1 Excitation and emission wavelengths of the compounds

[0079] Mitochondrial-targeting fluorescent probe compounds Ex(nm) Em(nm) CXL95 508 645 CXL96 509 569 CXL97 519 592 CXL98 485 617 CXL99 485 594

[0080] Example 7 Targeted Mitochondrial Localization Experiment

[0081] Cervical cancer cells (HeLa cells) were cultured in DMEM medium (a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin) and incubated at 37°C in a 5% CO2 incubator. After culture, a certain number of cells were evenly seeded into confocal culture dishes and cultured overnight. Once the cells adhered, the culture medium was first removed from the dishes, and then the cells were washed three times with autoclaved PBS buffer. 2 mL of PBS, the test compound (2 μM), and the mitochondrial dye MitoTracker (MT, commercially available, 1 μM) were added to the confocal culture dishes, and the dishes were incubated at 37°C in a 5% CO2 incubator for 30 min. After incubation, the culture dishes were removed, the culture medium was removed, and the cells were washed three more times with PBS. 1 mL of PBS was added (to prevent cell death). Finally, live-cell imaging was performed using a laser confocal fluorescence imaging system. The maximum UV absorption of compounds CXL95 and CXL97 is around 450–500 nm, and the maximum emission wavelength is between 550–605 nm. Therefore, for the green channel (550–600 nm) of the compounds, Ex@488 nm; for the red channel (620–750 nm) of MT Deep Red, Ex@633 nm, the results are as follows. Figure 2 As shown, the signals of compounds CXL95 and CXL97 can override the signals of MT, indicating that compounds CXL95 and CXL97 can locate the mitochondrial region in the cell, i.e., they have mitochondrial targeting.

[0082] Example 8: Validation of in vitro antitumor activity

[0083] Preparation of MTT solution: Prepare a 5 mg / mL, 40 mL MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) solution. Specifically, weigh 0.2 g of MTT powder and add it to a 50 mL BD tube, then add 40 mL of physiological saline, shake, and let it dissolve overnight. The next day, sonicate for 2 min in an ultrasonic instrument, filter through a 0.22 μM biofilter in a clean bench, place in a sterile BD tube, and store in a 4°C refrigerator protected from light.

[0084] Cell culture: A549 (human non-small cell lung cancer cells), DU145 (human prostate cancer cells), and MDA-MB-231 (human breast cancer cells) were cultured in 1640 medium and DMEM medium, respectively. These cell types were incubated at 37°C in a 5% CO2 incubator. After culture, a certain number of cells were evenly seeded into 96-well plates. Once the cells adhered, compounds CXL95, CXL96, CXL97, CXL99, and CXL99 at concentration gradients (5 μM, 10 μM, 20 μM) were added, and the plates were incubated at 37°C in a 5% CO2 incubator for 72 hours. After culturing, 20 μL of 5 mg / mL MTT solution was added and the plates were incubated for 2–4 hours. The culture medium and MTT solution were then removed from the 96-well plates, and 150 μL of DMSO solution was added with gentle shaking for 10 min. Finally, the OD value of each well was measured using a microplate reader.

[0085] Experimental Results: After 72 hours of treatment with cancer cells, compounds CXL95, CXL96, CXL97, CXL99, and CXL99, at concentrations of 5 μM, 10 μM, and 20 μM, all exhibited inhibitory effects on A549 cells, DU-145 cells, and MDA-MB-231 cells, with inhibition rates as shown below. Figure 3 As shown.

[0086] 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 mitochondrial-targeting fluorescent probe, characterized in that, Compounds including those with the following structures: , , or .

2. The method for preparing the mitochondrial-targeting fluorescent probe according to claim 1, characterized in that, Specifically: A nitrogen-containing heterocyclic iodide was dissolved in freshly distilled anhydrous acetonitrile and subjected to a Knoevenagel condensation reaction in the presence of an alkaline catalyst to obtain compounds with the structures shown in CXL95, CXL96, CXL97 or CXL98, which are mitochondrial-targeting fluorescent probes. The nitrogen-containing heterocyclic iodide is , , or Any one of them.

3. The method for preparing the mitochondrial-targeting fluorescent probe according to claim 2, characterized in that, The alkaline catalyst is piperidine, pyridine, or triethylamine.

4. The method for preparing the mitochondrial-targeting fluorescent probe according to claim 2, characterized in that, The nitrogen-containing heterocyclic onyx iodide is prepared by reacting a nitrogen-containing heterocyclic compound with an alkyl iodide; The nitrogen-containing heterocyclic compound is , or One of them; The alkyl iodide is iodomethane or iodoethane.

5. The method for preparing the mitochondrial-targeting fluorescent probe according to claim 2, characterized in that, The 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde is prepared by the following process: At 0–5 °C, POCl3 is slowly added dropwise to DMF to form Vilsmeier reagent; 2,2'-((3-fluorophenyl)azonyl)diethanol is added dropwise, and the temperature is raised to 80–120 °C for 8–12 hours. After the reaction is completed, the mixture is cooled to room temperature, poured into ice water, and the pH is adjusted to neutral. After filtration and purification, 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde is obtained.

6. The use of the mitochondrial-targeting fluorescent probe according to claim 1 in the preparation of mitochondrial imaging reagents for tumor cells.

7. The use of the mitochondrial-targeting fluorescent probe according to claim 1 in the preparation of antitumor drugs.

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