Mitochondrial targeting fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a mitochondrial targeting fluorescent probe and a preparation method and application thereof.The mitochondrial targeting fluorescent probe has the structural general formula shown in the formula (I), and the preparation method comprises the step that in the presence of a basic catalyst, the mitochondrial targeting fluorescent probe is prepared. And carrying out Knoevenagel condensation reaction on the nitrogen-containing heterocyclic ring onium iodide and 4-(di (2-chloroethyl) amino)-2-fluorobenzaldehyde, thereby obtaining the compound. The mitochondrial targeting fluorescent probe has mitochondrial targeting, fluorescence imaging capability and antitumor activity, can be used for preparation of tumor cell mitochondrial imaging reagents and antitumor drugs, provides a new strategy for tumor diagnosis and treatment, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a mitochondrial-targeted fluorescent probe and a preparation method and application thereof. Background Art

[0002] Mitochondria, the "energy factories" of cells, are not only key regulators of the intrinsic apoptosis pathway but also control the activation of apoptotic effector mechanisms by regulating the transport of apoptotic proteins from the intermitochondrial membrane space to the cytosol. They play a crucial role in various forms of non-apoptotic cell death, particularly necrosis (regulated necrosis). Alterations in their function and properties are particularly pronounced during tumorigenesis, making the study of mitochondrial-targeted drugs of great significance. 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 in targeted tumor therapy, and their high sensitivity and timeliness have driven the development of biomedical research. Studying the time, location, and mechanism of drug accumulation in cells requires specific fluorescent dyes, making their application in the biomedical field essential. The mitochondrial double membrane structure creates a -180 mV potential difference between the inner and outer membranes, which drives ATP synthesis and substance uptake. Taking advantage of this property, a commonly used targeting approach is to attract positively charged groups on drug molecules (such as pyridinium and indole salts) to the negative charge of the inner membrane, allowing them to enter the mitochondrial matrix driven by the electric field of the inner mitochondrial membrane potential.

[0004] Current cancer treatment drugs include nitrogen mustards, pyrimidines, platinums, and porphyrins. Nitrogen mustards, as important anti-tumor drugs, act by forming active iminium ions or carbonium ions in the body, becoming strong alkylating agents that covalently bind to electron-rich groups in biomolecules, causing DNA fragmentation or inactivation, thereby inhibiting tumor cell division or causing cell death. The structure of nitrogen mustard drugs is composed of an alkylating moiety (functional group with anti-tumor activity) and a carrier moiety (which improves pharmacokinetic properties).

[0005] However, there are currently few anti-tumor drugs with mitochondrial targeting, such as paclitaxel, doxorubicin, camptothecin, etc. These drugs usually have multiple targets and side effects. Long-term use may also lead to multidrug resistance in tumor cells, reducing or ineffective chemotherapy effects. Summary of the Invention

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

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

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

[0009]

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

[0011] Wherein R2 is an alkyl group having less than 6 carbon atoms.

[0012] Preferably, R2 is methyl or ethyl.

[0013] Preferably, the mitochondria-targeted fluorescent probe comprises a compound with the following structure:

[0014]

[0015] To solve the above technical problems, the present invention also provides a method for preparing the mitochondrial-targeted fluorescent probe, which is specifically as follows:

[0016] In the presence of a basic catalyst, a nitrogen-containing heterocyclic onium iodide and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde undergo a Knoevenagel condensation reaction to obtain a compound of the general structural formula shown in formula (I), which is a mitochondrial-targeted fluorescent probe;

[0017] The nitrogen-containing heterocyclic onium iodide is Any one of the following, wherein R2 is an alkyl group having less than 6 carbon atoms.

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

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

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

[0021] The alkyl iodide is an alkyl iodide having less than 6 carbon atoms, preferably methyl iodide or ethyl iodide.

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

[0023] At 0-5℃, POCI3 was slowly added into DMF to form Vilsmeier reagent; 2,2'-((3-fluorophenyl)azanediyl)diethanol (CAS: 323-60-4) was added dropwise, and the reaction was carried out at 80-120℃ for 8-12 hours; after the reaction was completed, the reaction was cooled to room temperature, poured into ice water, the pH was adjusted to neutral, and then filtration and purification were carried out.

[0024] The application of the mitochondrial targeting fluorescent probe in the preparation of a tumor cell mitochondrial imaging reagent.

[0025] The application of the mitochondrial targeting fluorescent probe in the preparation of an antitumor drug.

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

[0027] 1. Good targeting: the mitochondrial targeting fluorescent probe compound can be accurately positioned in the mitochondrial region of cells through the interaction of the positively charged group with the mitochondrial membrane potential, such as CXL95 and CXL97, which exhibit good mitochondrial targeting in HeLa cell experiments.

[0028] 2. Excellent fluorescence performance: the mitochondrial targeting fluorescent probe compound has specific excitation and emission wavelengths in pure DMSO solution, the maximum ultraviolet absorption is about 500 nm, and the maximum emission wavelength is between 550-650 nm, which can be used for fluorescence imaging.

[0029] 3. Significant antitumor activity: in vitro experiments show that the mitochondrial targeting fluorescent probe compound has 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 exhibits different inhibition rates at different concentrations.

[0030] 4. Simple preparation method: the synthesis route is reasonably designed, the reaction conditions are mild, the yield is high, and it is convenient for large-scale production and application. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the ultraviolet-visible absorption (A) and fluorescence emission (B) spectrum of the five compounds prepared in Example 1-5.

[0032] Figure 2 is the cell fluorescence imaging diagram of the mitochondrial fluorescence co-localization of compounds CXL95 and CXL97.

[0033] Figure 3 is the column chart of the MTT experiment results of the five compounds prepared in Example 1-5. DETAILED DESCRIPTION

[0034] The specific technical contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0035] Example 1

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

[0037]

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

[0039] CaCO3 (4.0eq, 20.82g) and KI (0.5eq, 4.32g) were weighed into a round-bottom flask, 50mL of pure water was added and stirred evenly, 5mL of 3-fluoroaniline (1.0eq, 5.78g) was added dropwise at room temperature, and then 9.08mL of 2-bromoethanol (2.5eq, 16.01g) was added dropwise. Finally, the temperature was slowly raised to 100°C and the reaction was allowed to proceed overnight; the mixture was filtered while hot, extracted with EA, and concentrated by column chromatography to obtain an oily product 2,2'-((3-fluorophenyl)azanediyl)diethanol (F-1) with a yield of 92.1%.

[0040] Synthesis of 4-(bis(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) and pre-cooled with ice water. The mixture was vigorously stirred and 2,2'-((3-fluorophenyl)azanediyl)diethanol (F-1) was added dropwise. The reaction mixture was stirred at 100 ° C for 10 hours. After the reaction was completed, the reaction solution was cooled to room temperature and then slowly poured into 500 mL of ice water. Saturated sodium hydroxide solution was added under vigorous stirring to adjust the pH of the solution to 7.0. Solids precipitated, and the suspension was filtered and washed three times with distilled water. It was then purified by column chromatography (EA:PE = 1:1) to obtain the desired product 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) with a yield of 86.8%. 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-indol-1-ium (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. The temperature was slowly raised to reflux and the reaction was allowed to proceed for 10 hours. TLC monitoring was performed. After completion of the reaction, the solution was evaporated to dryness using a rotary evaporator under vacuum. Petroleum ether was added to produce a solid, which was filtered to obtain 1.51 g of a pink solid in an 86.78% yield. 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-fluorophenyl)-1,3,3-trimethyl-3H-indole-1-iodide (CXL95):

[0045] Under nitrogen, 1,2,3,3-tetramethyl-3H-indol-1-ium (4-1) (1.0 eq, 100 mg) and 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL). Piperidine (0.01 eq) was added, and the reaction solution was stirred at 60°C for 8 hours. After completion of the reaction, the red reaction solution was concentrated and the product was purified by column chromatography to obtain a red solid, the mitochondrial-targeted 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-targeted fluorescent probe CXL96 was prepared using the following synthetic route:

[0048]

[0049] Synthesis of 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium (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. The temperature was slowly raised to reflux and the reaction was allowed to react for 10 hours. TLC monitoring was performed. After completion of the reaction, the reaction solution was vacuum dried. Petroleum ether was added to produce a solid, which was filtered to obtain 1.49 g of a red solid in 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-fluorophenyl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide (CXL96):

[0052] Under nitrogen, 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium (4-2) (1.0 eq, 105 mg) and 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL). Piperidine (0.01 eq) was added, and the reaction solution was stirred at 60°C for 8 hours. After completion of the reaction, the red reaction solution was concentrated and the product was purified by column chromatography to obtain a red solid, the mitochondrial-targeted 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-targeted fluorescent probe CXL97 was prepared using the following synthetic route:

[0055]

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

[0057] 1,1,2-Trimethyl-1H-benzindole (1.0 eq, 2.09 g) and iodoethane (1.2 eq, 1.86 g) were dissolved in 80 mL of toluene, slowly heated to reflux, and reacted for 10 hours. TLC monitoring was performed. After completion of the reaction, the reaction solution was vacuum-dried. Petroleum ether was added to produce a solid, which was filtered to obtain a red solid. The solid was washed twice with anhydrous ether to obtain 1.90 g of the final product, 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-fluorophenylvinyl)-3-ethyl-1,1-dimethyl-1H-benzo[E]indole-3-iodide (CXL97):

[0059] Under nitrogen, 3-ethyl-1,1,2-trimethyl-1H-benzindole-3-ium (4-3) (1.0 eq, 121 mg) and 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL). Piperidine (0.01 eq) was added, and the reaction solution was stirred at 60°C for 8 hours. After completion of the reaction, the red reaction solution was concentrated, and the product was purified by column chromatography to obtain a red solid, the mitochondrial-targeted 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-targeted fluorescent probe CXL98 was prepared using the following synthetic route:

[0062]

[0063] Synthesis of 1-ethyl-2-methylquinolin-1-ium (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, slowly heated to reflux, and reacted for 20 hours. After completion of the reaction, solids were produced by TLC monitoring. The reaction solution was filtered to obtain a white solid, which was washed twice with anhydrous 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-fluorophenyl)-1-ethylquinoline-1-iodide (CXL98):

[0066] Under nitrogen, 1-ethyl-2-methylquinolin-1-ium (4-4) (1.0 eq, 100 mg) and 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL). Piperidine (0.01 eq) was added, and the reaction solution was stirred at 60°C for 8 hours. After completion of the reaction, the red reaction solution was concentrated and the product was purified by column chromatography to obtain a red solid, the mitochondrial-targeted 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-targeted fluorescent probe CXL99 was prepared using the following synthetic route:

[0069]

[0070] Synthesis of 2,3-dimethylbenzothiazol-3-ium (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, slowly heated to reflux, and reacted for 24 hours. After completion of the reaction, solids were produced by TLC monitoring. The reaction solution was filtered to obtain a pink solid, which was washed twice with anhydrous 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-fluorophenylvinyl)-3-methylbenzo[d]thiazole-3-iodide (CXL99):

[0073] Under nitrogen, 2,3-dimethylbenzothiazol-3-ium (4-5) (1.0 eq, 97 mg) and 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde (F-2) (1.0 eq, 88 mg) were dissolved in freshly distilled anhydrous acetonitrile (1.4 mmol, 10 mL). Piperidine (0.01 eq) was added, and the reaction solution 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, the mitochondrial-targeted 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: Prepare 1 mM dimethyl sulfoxide (DMSO) solutions of each of the five compounds (CXL95, CXL96, CXL97, CXL98, and CXL99) and store them in a refrigerator at 4°C until ready for excitation and emission wavelength measurement. For testing, dilute the solutions to 10 μM in DMSO and measure their spectral properties at room temperature.

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

[0077] Experimental results: The measured UV-visible absorption spectrum and fluorescence spectrum 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. The specific data are shown in Table 1.

[0078] Table 1 Excitation wavelength and emission wavelength 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 Mitochondria Localization Experiment

[0081] Cervical cancer cells (HeLa cells) were cultured in DMEM medium (containing a mixture of 10% fetal bovine serum and 1% double antibiotic (penicillin-streptomycin)) and incubated at 37°C in an incubator containing 5% CO2. After the cells were cultured, a certain number of cells were evenly plated onto confocal culture dishes and cultured overnight. After the cells attached, the culture medium was removed from the dish, followed by three washes with high-temperature, high-pressure sterilized 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 dish, and the dish was incubated in an incubator at 37°C in 5% CO2 for 30 minutes. After the culture was completed, the dish was removed, the culture medium was removed, and the cells were washed three times with PBS. 1 mL of PBS was added to prevent cell death, and live cell imaging was performed using a laser confocal fluorescence imager. The maximum UV absorption of compounds CXL95 and CXL97 is around 450-500nm, and the maximum emission wavelength is between 550-605nm. Therefore, for the green channel (550-600nm) of the compound, Ex@488nm; for the red channel (620-750nm) of MT Deep Red, Ex@633nm. The results are as follows Figure 2 As shown, the signals of compounds CXL95 and CXL97 can cover the signals of MT, indicating that compounds CXL95 and CXL97 can locate the mitochondrial region in cells, that is, they have mitochondrial targeting.

[0082] Example 8 Verification of in vitro antitumor activity

[0083] Preparation of MTT solution: prepare MTT (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyl tetrazolium bromide) solution with a concentration of 5 mg / mL and a volume of 40 mL, specifically: weigh 0.2 g of MTT powder into a 50 mL capacity BD tube, then add 40 mL of normal saline, shake and dissolve overnight; the next day, ultrasonic in an ultrasonic instrument for 2 min, filter in a super-clean bench with a 0.22 μM biological filter membrane, fill in a sterile BD tube and store in a 4°C refrigerator in the dark.

[0084] Culture of cells: 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, and the three kinds of cells were cultured and incubated in a 37°C incubator containing 5% CO2, after the cells were cultured, a certain amount of cells were uniformly spread in a 96-well plate, after the cells were attached, a certain concentration gradient (5 μM, 10 μM, 20 μM) of compounds CXL95, CXL96, CXL97, CXL99, CXL99 were added, and placed in a 37°C incubator containing 5% CO2 for 72 hours. After the culture was completed, 20 μL of MTT solution with a concentration of 5 mg / mL was added and cultured for 2-4 h, the culture medium and MTT solution in the 96-well plate were removed, 150 μL of DMSO solution was added and shaken gently for 10 min, and finally the OD value of each well was measured on an enzyme marker.

[0085] Experimental results: after 72 hours of action on cancer cells, compounds CXL95, CXL96, CXL97, CXL99 and CXL99 had a certain inhibitory effect on A549 cells, DU-145 cells and MDA-MB-231 cells at concentrations of 5 μM, 10 μM and 20 μM, and the inhibition rate was as shown in Table 1. Figure 3

[0086] The above examples describe the basic principles, main features and advantages of the present application, and those skilled in the art should understand that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application, and various changes and improvements can be made without departing from the scope of the present application, and these changes and improvements all fall within the scope of the present application.​

Claims

1. A mitochondrial-targeted fluorescent probe, characterized in that: It has the general structural formula shown in formula (I): In formula (I), R1 is One of the groups; Wherein R2 is an alkyl group having less than 6 carbon atoms.

2. The mitochondria-targeted fluorescent probe according to claim 1, characterized in that: R2 is methyl or ethyl.

3. The mitochondria-targeted fluorescent probe according to claim 2, characterized in that Including compounds with the following structures:

4. The method for preparing the mitochondria-targeting fluorescent probe according to claim 1, characterized in that: Specifically: In the presence of a basic catalyst, a nitrogen-containing heterocyclic onium iodide and 4-(di(2-chloroethyl)amino)-2-fluorobenzaldehyde undergo a Knoevenagel condensation reaction to obtain a compound of the general structural formula shown in formula (I), which is a mitochondrial-targeted fluorescent probe; The nitrogen-containing heterocyclic onium iodide is Any one of the following, wherein R2 is an alkyl group having less than 6 carbon atoms.

5. The method for preparing a mitochondrial-targeted fluorescent probe according to claim 4, wherein: The alkaline catalyst is piperidine, pyridine or triethylamine.

6. The method for preparing a mitochondrial-targeted fluorescent probe according to claim 4, wherein: The nitrogen-containing heterocyclic onium iodide is prepared by reacting a nitrogen-containing heterocyclic compound with an alkyl iodide; The nitrogen-containing heterocyclic compound is One of the following; The alkyl iodide is an alkyl iodide having less than 6 carbon atoms.

7. The method for preparing a mitochondrial-targeted fluorescent probe according to claim 6, wherein: The alkyl iodide is methyl iodide or ethyl iodide.

8. The method for preparing a mitochondrial-targeted fluorescent probe according to claim 4, wherein: The 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde is prepared by the following process: Slowly add POCl3 dropwise into DMF at 0-5°C to form Vilsmeier reagent; add 2,2'-((3-fluorophenyl)azanediyl)diethanol dropwise, raise the temperature to 80-120°C, and react for 8-12 hours. After the reaction, cool to room temperature, pour into ice water, adjust the pH to neutral, filter, and purify to obtain 4-(bis(2-chloroethyl)amino)-2-fluorobenzaldehyde.

9. Use of the mitochondria-targeted fluorescent probe according to any one of claims 1 to 3 in the preparation of a tumor cell mitochondrial imaging reagent.

10. Use of the mitochondria-targeted fluorescent probe according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

Images