Near-infrared mitochondrial fluorescent tracer and application thereof

Abstract

The application designs a series of xanthene near-infrared mitochondrial fluorescent tracers, the self-assembly characteristics of the dyes are regulated by the length of the external hydrophobic carbon chain, and then the stability of the mitochondrial targeting and the signal-to-noise ratio of the imaging are optimized. The fluorescent tracers show strong NIR fluorescence, the emission wavelength can reach 700 nm, the molar extinction coefficient is 10.9 M ‑1 *cm ‑1 *10 4 , the fluorescence quantum yield is 0.64. In the aqueous phase, the ACQ effect is significant, but it can be quickly enriched and embedded into mitochondria, and then the strong fluorescence is restored, showing high signal-to-noise ratio; in living cells, it has stable mitochondrial targeting, is not affected by the membrane potential, and the imaging performance is better than that of the commercial probe TMRM. The fluorescent tracer can accurately and stably trace the microtubular morphological changes of mitochondria in ferroptosis.

Description

Technical Field

[0001] This invention relates to a class of near-infrared mitochondrial fluorescent tracers and their applications, belonging to the field of fine chemicals. Background Technology

[0002] Mitochondria are the respiratory center and energy hub of cells, playing a crucial role in cellular processes such as ATP production, oxidative respiration, and calcium-mediated signal transduction. Furthermore, mitochondrial function is directly related to metabolic disorders, cancer, and neurodegeneration. Long-wavelength fluorescent dyes possess excellent biocompatibility, including lower photodamage, longer penetration depth, and less background interference, making them highly sought after in live-cell fluorescence imaging. Simultaneously, as fluorescence imaging technology advances towards ultra-high resolution, multi-channel simultaneous acquisition, and long-term dynamic tracking, further demands are placed on the optical properties of fluorescent dyes, such as higher photostability, higher fluorescence quantum yield, and more precise organelle targeting. Therefore, developing near-infrared mitochondrial fluorescent tracers with precise and stable mitochondrial targeting and high imaging signal-to-noise ratio has significant application value. Summary of the Invention

[0003] This invention provides a class of near-infrared mitochondrial fluorescent tracers based on near-infrared xanthan dyes and their applications. By attaching an external hydrophobic carbon chain and adjusting the chain length, the self-assembly properties of the dye can be controlled, thereby optimizing its mitochondrial targeting stability and imaging signal-to-noise ratio. This fluorescent tracer has advantages such as high stability (unaffected by mitochondrial membrane potential) and high imaging signal-to-noise ratio (higher than commercial chromosome TMRM).

[0004] The technical solution of this invention: A class of near-infrared mitochondrial fluorescent tracers, having the following general structural formula:

[0005]

[0006] in, Structural fragments containing Y in the general formula for or

[0007] Y1 can be -CH3, -SO3H, -CF3, or -COOH. or

[0008] Y2 is Wherein, R is a straight-chain alkyl group with 1-30 carbon atoms, and Y2 replaces the hydrogen atom at the 4th or 5th position on the benzene ring;

[0009] R1 is or

[0010] R2 is or

[0011] Among them, X1 - The anion is an anion, and the total negative charge of the anion is equal to the total positive charge of the nitrogen-containing groups in the R2 structure.

[0012] Some specific fluorescent tracers, where R is a straight-chain alkyl group with 6-25 carbon atoms.

[0013] Some specific fluorescent tracers, where R is a straight-chain alkyl group with 6-20 carbon atoms.

[0014] Some specific fluorescent tracers, where R is a straight-chain alkyl group with 6-18 carbon atoms.

[0015] Some specific fluorescent tracers, where R is a straight-chain alkyl group with 6-16 carbon atoms.

[0016] Some specific fluorescent tracers, namely X1 - BF4 - Cl - ,Br - I - NO3 - SO4 2- ClO4 - CH3COO - CH3SO3 - or CF3SO3 - .

[0017] The application of the fluorescent tracer in the preparation of mitochondrial-targeting drugs or materials.

[0018] The application of the fluorescent tracer in the preparation of drugs or materials for detecting and tracking mitochondria.

[0019] The application of the fluorescent tracer in the preparation of materials or reagents for intracellular mitochondrial confocal imaging.

[0020] The method for preparing the aforementioned near-infrared mitochondrial fluorescent tracer, including the reaction formula and reaction steps, is as follows:

[0021]

[0022] i) Compound A, Pd(OAc)2, BINAP, Cs2CO3 and HR1 were heated in an organic solvent under argon conditions to give compound B;

[0023] ii) Remove water using a water separator, and heat the mixture of compound B, TsOH and R4 to react and obtain compound C; iii) At room temperature, add compound C to an organic solvent, add NBS in batches, stir the reaction, and separate and purify to obtain compound D;

[0024] iv) Under argon protection, compound D was dissolved in an organic solvent, and tert-butyllithium was added and stirred for 0.5 h. The temperature was then raised to a certain level to dissolve the methyl benzoate compound. Dissolved in an organic solvent, the mixture was added dropwise to the above reaction solution, and the mixture was heated to room temperature to react. The reaction mixture was diluted with saturated NH4Cl and water, and extracted with EtOAc. The extracted organic phase was washed with brine, dried, and concentrated. Methanol and R5 were added to the concentrate, and the mixture was stirred at room temperature. The solvent was removed under reduced pressure to purify the product and obtain compound E.

[0025] R4 is or

[0026] R5 is HBF4, HCl, HBr, HI, HNO3, H2SO4, HClO4, CH3COOH, CH3SO3H, or CF3SO3H.

[0027] The definitions of R1, R2, Y, Y1, and Y2 are the same as those in the general structural formula.

[0028] The organic solvent in step i is one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, tetrahydrofuran, dioxane, N-methylpyrrolidone, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, hexamethylphosphoramide, sulfolane, acetonitrile, and benzonitrile, and the reaction temperature is 90-180℃; the reaction temperature in step ii is 130-170℃; and the organic solvent in step iii is one of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, tetrahydrofuran, dioxane, N-methylpyrrolidone, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, hexamethylphosphoramide, sulfolane, acetonitrile, and benzonitrile.

[0029] Methyl benzoate compound The following method was used for synthesis:

[0030]

[0031] 1) Under argon protection, compound I, trimethylethynylsilane, tetra(triphenylphosphine)palladium, triphenylphosphine, cuprous iodide, and triethylamine were dissolved in N,N-dimethylformamide after argon purging (to remove oxygen), and the mixture was stirred at 65°C for 2 h. After cooling in an ice bath, the mixture was washed with water, extracted with ethyl acetate, washed with saturated brine of organic phase, dried over anhydrous magnesium sulfate, and purified to obtain compound J.

[0032] 2) Compound J was dissolved in methanol, potassium hydroxide was added, and the mixture was stirred overnight at room temperature. The solvent was removed by rotary evaporation of the reaction solution, and compound K was obtained by separation and purification.

[0033] 3) Compound F and sodium azide were dissolved in dimethyl sulfoxide, stirred at room temperature for 30 h, washed with water, extracted with diethyl ether, dried over anhydrous magnesium sulfate in the organic phase, and the solvent was removed by rotary evaporation at low temperature to obtain compound G;

[0034] 4) Under argon protection, compound K, compound G, VcNa (sodium ascorbate) and copper sulfate were dissolved in a mixed solvent of tetrahydrofuran and water. The mixture was stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate in the organic phase, and the solvent was removed by rotary evaporation. The mixture was then separated and purified to obtain compound H.

[0035] Wherein, R is a straight-chain alkyl group with 1-30 carbon atoms.

[0036] The beneficial effects of this invention are as follows: The series of xanthannaphthalene-based near-infrared mitochondrial fluorescent tracers designed in this invention link straight-chain alkyl groups at the 9-position of rhodamine. These straight-chain alkyl groups, acting as hydrophobic chains, control the stability of the entire molecular structure in targeting mitochondria, enabling the fluorescent tracers to stably and effectively track mitochondrial morphological changes over a long period. Furthermore, by linking carbon chains of different lengths, the self-assembly characteristics of the entire molecule in water can be adjusted, thereby regulating and improving the signal-to-noise ratio of fluorescence imaging. This series of molecules can be applied to confocal imaging, enabling real-time observation of the movement and trajectory of mitochondria after drug stimulation. It offers advantages such as high image quality, long imaging time, stable targeting (unaffected by membrane potential), and no washing required, making it valuable for research and application.

[0037] These mitochondrial fluorescent tracers exhibited strong NIR fluorescence, with emission wavelengths reaching 700 nm and a molar extinction coefficient of 10.9 M. -1 *cm -1 *10 4 The fluorescence quantum yield reaches 0.64. It exhibits a significant ACQ effect in aqueous phase, yet rapidly accumulates and embeds in mitochondria, thus restoring strong fluorescence and demonstrating a high signal-to-noise ratio. In living cells, it exhibits stable mitochondrial targeting, unaffected by membrane potential, and its imaging performance surpasses that of commercial probes such as TMRM. This type of mitochondrial fluorescent tracer can precisely and stably track microtubule morphological changes in mitochondria during ferroptosis with excellent imaging properties. Attached Figure Description

[0038] Figure 1 These are the fluorescence emission spectra of KRh-C0 and KRh-C16 in acetonitrile and water.

[0039] Where a is the fluorescence emission spectrum of KRh-C0 and b is the fluorescence emission spectrum of KRh-C16.

[0040] Figure 2 These are the fluorescence emission spectra of three probes, KRh-C0, KRh-C16, and TMRM, in saturated CTAB solution and water.

[0041] Where a is the fluorescence emission spectrum of KRh-C0, b is the fluorescence emission spectrum of KRh-C16, and c is the fluorescence emission spectrum of TMRM.

[0042] Figure 3 This is a colocalization imaging map of KRh-C0, KRh-C6, and KRh-C16 with TMRM in HeLa cells.

[0043] Figure 4 This is a comparison image of HeLa cells stained with KRh-C0, KRh-C6, and KRh-C16 before and after loss of membrane potential due to drug stimulation.

[0044] Figure 5 This is a confocal imaging and fluorescence signal map of mitochondria in KRh-C0.

[0045] Figure 6 This is a confocal imaging and fluorescence signal map of mitochondria in KRh-C16.

[0046] Figure 7 This is a mitochondrial confocal imaging and fluorescence signal map of TMRM.

[0047] Figure 8 This is a confocal image of Rh-C16 stained HeLa cells after erastin stimulation. Detailed Implementation

[0048] Synthesis of methyl benzoate compounds

[0049]

[0050] 1) Under argon protection, compound I1 (9.43 mmol), trimethylethynylsilane (37.72 mmol), tetrakis(triphenylphosphine)palladium (0.47 mmol), triphenylphosphine (1.89 mmol), cuprous iodide (2.83 mmol), and triethylamine (10 ml) were dissolved in N,N-dimethylformamide after purging with argon gas (to remove oxygen). The mixture was stirred at 65 °C for 2 h. After cooling in an ice bath, the mixture was washed with water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous magnesium sulfate to obtain compound J1.

[0051] 2) Dissolve compound J1 (0.06 mmol) in 4 ml of methanol, add potassium hydroxide (20 mg), stir overnight at room temperature, remove the solvent by rotary evaporation, and obtain compound K1 by separation and purification.

[0052] 3) Compound F1 (6.06 mmol) and sodium azide (6.67 mmol) were dissolved in dimethyl sulfoxide, stirred at room temperature for 30 h, washed with water, extracted with diethyl ether, dried the organic phase with anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation at low temperature to obtain compound G1.

[0053] 4) Under argon protection, compound K1 (0.02 mmol), compound G1 (0.08 mmol), sodium ascorbate (5 mg) and copper sulfate (5 mg) were dissolved in a mixed solvent of tetrahydrofuran and water. The mixture was stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate in the organic phase, and the solvent was removed by rotary evaporation. The mixture was then separated and purified to obtain compound H1.

[0054] The methyl benzoate compound used in the embodiments of the present invention They were synthesized using the same method.

[0055] Example 1

[0056]

[0057] i) Under argon protection, a mixture of compound A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbled with argon (to remove oxygen) for 20 min. The reaction was carried out at 110 °C, and TLC was monitored until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B1.

[0058] ii) A mixture of B1 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110 °C for 4 h, then at 150 °C for 12 h. Water was removed. When TLC showed complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate on the organic phase, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to give a pale yellow solid, C1.

[0059] iii) At room temperature, C1 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The reaction solution was diluted with dichloromethane, washed with water, and the organic phase was dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to give a yellow solid D1.

[0060] iv) Under argon protection and at -78°C, D1 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and 0.95 mmol of tetrahydrofuran solution was added dropwise. The reaction mixture was heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol, and glacial acetic acid (100 μL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0061] Example 2

[0062]

[0063] i) Under argon protection, a mixture of A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol), and cyclohexylamine (1.4 mL) was dissolved in toluene bubbled with argon gas (to remove oxygen) for 20 min. The reaction was carried out at 110 °C, and TLC was monitored until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B₂.

[0064] ii) A mixture of B2 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110 °C for 4 h, then at 150 °C for 12 h. Water was removed. When TLC showed complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to give a pale yellow solid, C2.

[0065] iii) At room temperature, C2 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to give a yellow solid, D2.

[0066] iv) Under argon protection, at -78°C, D2 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and 0.95 mmol of tetrahydrofuran dissolved in it was added dropwise. The reaction mixture was then heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0067] Example

[0068]

[0069] i) Under argon protection, A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling (deoxygenation) under argon gas for 20 min, and reacted at 110 °C. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the solid was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B3.

[0070] ii) A mixture of B3 (0.94 mmol), 2-hydroxyethanethiol (2.1 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110 °C for 4 h, then at 150 °C for 12 h. Water was removed. When TLC showed complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to give a pale yellow solid, C3.

[0071] iii) At room temperature, C3 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to obtain a yellow solid, D3.

[0072] iv) Under argon protection and at -78°C, D3 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and 0.95 mmol of tetrahydrofuran dissolved in it was added dropwise. The reaction mixture was heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0073] Example 4

[0074]

[0075] i) Under argon protection, A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol) and A mixture of (1.5 mL) was dissolved in toluene purged with argon gas (to remove oxygen) for 20 min, and reacted at 110 °C, monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B4. ii) A mixture of B4 (0.94 mmol), ethylenedithiol (2.2 mL), and TsOH (0.29 mmol) was dissolved in toluene, reacted at 110 °C for 4 h, then at 150 °C for 12 h, and the water was removed. When the TLC showed that the starting material had completely disappeared, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to give a pale yellow solid C4.

[0076] iii) At room temperature, C4 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to give a yellow solid, D4.

[0077] iv) Under argon protection and at -78°C, D4 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction mixture was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and the tetrahydrofuran solution was added dropwise. The reaction mixture was then heated to room temperature and stirred for 12 h. The reaction was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid. Example 5

[0078]

[0079] i) Under argon protection, A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol) and (1.5 mL) of the mixture was dissolved in toluene purged with argon gas (to remove oxygen) for 20 min, and reacted at 110 °C, monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B5. ii) A mixture of B1 (0.94 mmol), 3-hydroxypropanethiol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene, reacted at 110 °C for 4 h, then at 150 °C for 12 h, and the water was removed. When TLC showed that the starting material had completely disappeared, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to give a pale yellow solid C5.

[0080] iii) At room temperature, C5 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to obtain a yellow solid, D5.

[0081] iv) Under argon protection and at -78°C, D5 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction mixture was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and the tetrahydrofuran solution was added dropwise. The reaction mixture was then heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0082] Example 6

[0083]

[0084] Under argon protection, a mixture of A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol), and thiomorpholine (1.5 mL) was dissolved in toluene bubbled with argon gas (to remove oxygen) for 20 min. The reaction was carried out at 110 °C, and TLC was monitored until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B6.

[0085] ii) A mixture of B6 (0.94 mmol), propylene glycol (2.3 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110 °C for 4 h, then at 150 °C for 12 h. Water was removed. When TLC showed complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to obtain a pale yellow solid, C6.

[0086] iii) At room temperature, C6 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to give a yellow solid, D6.

[0087] iv) Under argon protection and at -78°C, D6 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction mixture was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and the tetrahydrofuran solution was added dropwise. The reaction mixture was then heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0088] Example 7

[0089]

[0090] i) Under argon protection, A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling (deoxygenation) under argon gas for 20 min, and reacted at 110 °C. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the solid was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B7.

[0091] ii) A mixture of B7 (0.94 mmol), ethylenedithiol (2.2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110 °C for 4 h, then at 150 °C for 12 h. Water was removed. When TLC showed complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to give a pale yellow solid, C7.

[0092] iii) At room temperature, C7 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to give a yellow solid, D7.

[0093] iv) Under argon protection and at -78°C, D7 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction mixture was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and the tetrahydrofuran solution was added dropwise. The reaction mixture was then heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0094] Example 8

[0095]

[0096] Under argon protection, a mixture of A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol), and methylamine (1.5 mL) was dissolved in toluene bubbled with argon gas (to remove oxygen) for 20 min. The reaction was carried out at 110 °C, and TLC was monitored until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B8.

[0097] ii) A mixture of B8 (0.94 mmol), ethylene glycol (2.3 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110 °C for 4 h, then at 150 °C for 12 h. Water was removed. When TLC showed complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to obtain a pale yellow solid, C8.

[0098] iii) At room temperature, C8 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to obtain a yellow solid, D8.

[0099] iv) Under argon protection and at -78°C, D8 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction mixture was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and the tetrahydrofuran solution was added dropwise. The reaction mixture was then heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0100] Example 9

[0101]

[0102] i) Under argon protection, a mixture of A (2.97 mmol), Pd(OAc)₂ (0.45 mmol), BINAP (0.32 mmol), Cs₂CO₃ (8.62 mmol), and thiomorpholine dioxide (1.5 mL) was dissolved in toluene bubbled with argon gas (to remove oxygen) for 20 min. The reaction was carried out at 110 °C, and TLC was monitored until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the solid was purified by column chromatography (ethyl acetate: petroleum ether = 1:25) to give a pale yellow solid B9.

[0103] ii) A mixture of B9 (0.94 mmol), heptanediol (2.3 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110 °C for 4 h, then at 150 °C for 12 h. Water was removed. When TLC showed complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to obtain a pale yellow solid, C9.

[0104] iii) At room temperature, C9 (0.91 mmol) was dissolved in N,N-dimethylformamide and stirred until completely dissolved. NBS (1.82 mmol) was added to the reactants in five portions, 5 min apart, and reacted for 60 min after all portions were added. The mixture was then diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The solution was purified by column chromatography (dichloromethane:petroleum ether = 2:1) to obtain a yellow solid, D9.

[0105] iv) Under argon protection and at -78°C, D9 (0.38 mmol) was dissolved in ultra-dry tetrahydrofuran, and tert-butyllithium (1.3 M, 1.6 mL, 1.71 mmol) was added dropwise. The reaction mixture was stirred at -78°C for 30 min. The reaction mixture was then cooled to -20°C, and the tetrahydrofuran solution was added dropwise. The reaction mixture was then heated to room temperature and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol and added to the mixture. After stirring at room temperature for 10 min, the reaction mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain a blue-green solid.

[0106] Example 10

[0107]

[0108] KRh-C0 and KRh-C16 were dried at 50°C for at least 6 hours using an oil pump. DMSO was used as the solvent to prepare 1 mmol / L stock solutions for KRh-C0 and KRh-C16 probes, respectively. Using a 1000 μL pipette, 1 mL of the organic solvent or water to be tested was added to a 1 mL centrifuge tube. Using a 10 μL pipette, an appropriate amount of the KRh probe stock solution was added to the centrifuge tube. After thorough mixing, the solution was transferred to a 1 mL cuvette. The absorption spectrum of the corresponding probe was measured using a UV-Vis spectrophotometer. The excitation wavelength for fluorescence spectroscopy was determined based on the peak value displayed in the absorption spectrum. After adjusting the parameters, the emission spectrum of the KRh probe was measured.

[0109] The study analyzed the photophysical properties of two structures, KRh-C0 and KRh-C16, in organic solvents and water, and further analyzed the role of long carbon chains in the structure. Figure 1 These are the fluorescence emission spectra of the KRh-C0 and KRh-C16 structures in acetonitrile and water. The KRh-C16 structure is quenched in pure water, and its fluorescence almost disappears.

[0110] Example 11

[0111] The photophysical properties of KRh-C0, KRh-C16, and TMRM structures in surfactants and water were studied and analyzed. First, a saturated ddH2O solution of hexadecyltrimethylammonium bromide (CTAB) was prepared. Using a 1000 μL pipette, 1 mL of the saturated CTAB solution or ddH2O was added to a 1 mL centrifuge tube. Then, using a 10 μL pipette, an appropriate amount of the KRh probe stock solution was added to the centrifuge tube, preparing a saturated surfactant solution or water solution of a specific concentration. After thorough mixing, the solution was transferred to a 1 mL cuvette. The absorption spectrum of the corresponding probe was measured using a UV-Vis spectrophotometer. The excitation wavelength for fluorescence spectroscopy was determined based on the peak values ​​displayed in the absorption spectrum. After adjusting the parameters, the emission spectrum of the KRh probe was measured.

[0112] Depend on Figure 2 It is observed that the fluorescence emission intensities of KRh-C0 and TMRM are not significantly different in surfactants and water. However, the spectrum of the KRh-C16 structure shows that KRh-C16 almost loses fluorescence in water but recovers fluorescence in a surfactant (CTAB) solution. Combining the spectra of the probe in organic solvents, surfactants, and water, we infer that the KRh-C16 structure, due to its long carbon chain structure, undergoes self-assembly in water, forming micelles and producing a fluorescence quenching effect, resulting in almost complete loss of fluorescence. Therefore, the KRh-C16 structure can reduce noise through self-assembly, improving imaging accuracy.

[0113] Example 12

[0114] Table 1. Molar extinction coefficients and fluorescence quantum yields of KRh-C0 and KRh-C16 probes.

[0115]

[0116] KRh-C0 and KRh-C16 were dried at 50°C for at least 6 hours using an oil pump. DMSO was used as the solvent to prepare 1 mmol / L stock solutions for KRh-C0 and KRh-C16 probes, respectively. Using a 1000 μL pipette, 1 mL of the organic solvent or PBS to be tested was added to a 1 mL centrifuge tube. Using a 10 μL pipette, an appropriate amount of the KRh probe stock solution was added to the centrifuge tube. After thorough mixing, the solution was transferred to a 1 mL cuvette. The absorption spectrum of the corresponding probe was measured using a UV-Vis spectrophotometer. The excitation wavelength for fluorescence spectroscopy was determined based on the peak value displayed in the absorption spectrum. After adjusting the parameters, the emission spectrum of the KRh probe was measured.

[0117] The fluorescence quantum yield and molar extinction coefficient of KRh-C0 and KRh-C16 were calculated. KRh-C0 exhibited a higher molar extinction coefficient and fluorescence quantum yield, with an extinction coefficient as high as 10.9 M.-1 *cm -1 *10 4 The fluorescence quantum yield in chloroform reached 0.64. Of noteworthy significance is that the fluorescence quantum yield of the KRh-C16 structure in pure water was as low as 0.06, with fluorescence almost completely absent.

[0118] Combination Figure 1 , Figure 2 As shown in Table 1, the KRh-C16 structure, due to its long carbon chain, undergoes self-assembly in water, forming micelles and producing a fluorescence quenching effect, resulting in almost complete loss of fluorescence. In cells, when the probe binds to mitochondria, it is equivalent to binding to surfactant molecules in a surfactant solution; the structure is in an extended state and emits strong fluorescence. Probe molecules that do not bind to mitochondria and remain free in the cytoplasm are analogous to those in water, exhibiting an aggregated state, resulting in fluorescence quenching and almost no fluorescence. Therefore, the KRh-C16 structure can improve imaging accuracy through noise reduction via self-assembly.

[0119] Example 13

[0120] Normally passaged HeLa cells were seeded into three clean cell culture dishes and allowed to adhere for approximately 4 hours before imaging experiments. HeLa cells were incubated for 15 minutes with 1 mL of 1 μM KRh-C probe culture medium, washed three times with PBS, and then incubated for 1 minute with 1 mL of 1 μM tetramethylrhodamine methyl ester (TMRM) culture medium, washed three times with PBS. Confocal microscopy was used to image the HeLa cells stained with the three dyes (excitation wavelengths of 545 nm and 655 nm, respectively).

[0121] like Figure 3 As shown, the first row, from left to right, displays confocal imaging images of KRh-C0, TMRM, and KRh-C0 plus TMRM; the second row, from left to right, displays confocal imaging images of KRh-C6, TMRM, and KRh-C6 plus TMRM; and the third row, from left to right, displays confocal imaging images of KRh-C16, TMRM, and KRh-C16 plus TMRM. These confocal imaging images clearly demonstrate that KRh-C0, KRh-C6, and KRh-C16 specifically target mitochondria. Therefore, KRhC probes are reliable tracers for mitochondrial imaging.

[0122] Example 14

[0123] Normally passaged HeLa cells were seeded in clean cell culture dishes and allowed to adhere for approximately 4 hours before imaging experiments. Cells were stained with 1 μM KRh-C0, KRh-C6, KRh-C16, and TMRM (tetramethylrhodamine methyl ester) in 1 mL of culture medium for 15 min, respectively, followed by washing three times with PBS. Confocal microscopy was used to perform confocal imaging of the KRh-C0, KRh-C6, KRh-C16, and TMRM-stained HeLa cells. KRh-C0, KRh-C6, KRh-C16, and TMRM-stained HeLa cells were incubated with 1 μM cccp in 1 mL of culture medium for 30 min, followed by washing three times with PBS. Confocal microscopy was then used to perform confocal imaging of the four dye-stained HeLa cells, observing changes in brightness and cell morphology before and after cccp stimulation.

[0124] Depend on Figure 4 It can be seen that, without changing the imaging parameters, the brightness of mitochondria stained with KRhC probes is hardly reduced, and the outline of mitochondria can still be clearly distinguished; however, mitochondria stained with membrane potential-dependent TMRM probes lose most of their fluorescence. Therefore, it can be concluded that the staining effect of KRhC probes is almost unaffected by cell membrane potential. When cells lose membrane potential, KRhC probes can stain better than TMRM, which also proves that KRhC probes can be used as reliable tracers for mitochondrial imaging when cells lose membrane potential.

[0125] Example 15

[0126] Images of HeLa cells stained with KRh-C0, KRh-C6, KRh-C16, and tetramethylrhodamine methyl ester (TMRM) were obtained respectively. The FV10-ASW Viewer and Fiji software were used to generate images based on the signal strength, calculate the signal and background noise, and perform lateral comparisons. Figure 5-7 The images shown are confocal images and signal strength plots of KRh-C0, KRh-C16, and TMRM. Calculations showed that KRh-C6 and KRh-C16 significantly improved the signal-to-noise ratio (SNR) compared to KRh-C0, with KRh-C16 exhibiting the lowest SNR, even lower than the commercial chromosome TMRM (as shown in Table 1). This demonstrates that the introduction of carbon chains can significantly improve the imaging SNR. These compounds, through their quenching effect, offer convenient staining, strong versatility, weak background, and good stability, making them suitable for high-precision research.

[0127] Table 2. Signal-to-noise ratio (S / N) of confocal images from KRh-C0, KRh-C6, KRh-C16, and TMRM.

[0128]

[0129] Example 16

[0130] After incubating HeLa cells at 37°C and 5% CO2 for 24 hours, the cells were stimulated with 1 ml of culture medium containing 25 μM erastin. At 0 h, 24 h, and 36 h post-stimulation, the cells were stained with 1 ml of culture medium containing 1 μM KRh-C16 and incubated for 15 minutes. After washing three times with PBS, confocal imaging was performed to obtain confocal images before drug stimulation and at 24 h and 36 h post-stimulation.

[0131] like Figure 8 As shown in the first image, the filamentous mitochondrial structure of the cell is clearly visible. The image after 24 hours of stimulation reveals localized breakage of the mitochondria, with the filaments becoming discontinuous and the mitochondria appearing disordered. The image after 36 hours shows significant breakage and contraction of the mitochondria, generating numerous small circles, and their morphology has almost completely changed. Through observation of these images, we can conclude that the KRh-C16 probe can provide long-term dynamic monitoring of mitochondria during ferroptosis, allowing for real-time monitoring of morphological and structural changes and detailed observation of mitochondria. This makes it a powerful tool for studying mitochondrial changes during ferroptosis.

[0132] The same erastin drug stimulation imaging experiment was performed on compounds KRh-C0, KRh-C6, E9 and E18. KRh-C6, E9 and E18 also showed clear changes in mitochondrial morphology 24 h after stimulation, while the fluorescence of KRh-C0 disappeared 30 min after stimulation.

Claims

1. A class of near-infrared mitochondrial fluorescent tracers characterized in that, having the general structure: ； wherein Y = O or S; or ; structural fragment with Y in the general formula is , , , , , or ; Y1= -CH3, -SO3H, -CF3, -COOH, or ; Y2is wherein Y2substitutes a hydrogen atom at 4-position or 5-position of the benzene ring; R1is or ; R2 is or ; wherein X1 - is an anion, the total negative charge carried by said anion being equal to the total positive charge carried by the nitrogen-containing group in R2; R is a linear alkyl group having 6 to 25 carbon atoms.

2. The fluorescent tracer of claim 1, wherein, R is a linear alkyl group having 6 to 20 carbon atoms.

3. The fluorescent tracer of claim 1, wherein, R is a linear alkyl group having 6 to 18 carbon atoms.

4. The fluorescent tracer of claim 1, wherein, R is a linear alkyl group having 6 to 16 carbon atoms.

5. The fluorescent tracer of any one of claims 1-4, wherein, X1 - BF4 - Cl - Br - I - NO3 - 0.5 SO4 2- ClO4 - CH3COO - CH3SO3 - or CF3SO3 - .

6. Use of the fluorescent tracer according to any one of claims 1-5 in the preparation of a drug or material targeting mitochondria.

7. Use of the fluorescent tracer according to any one of claims 1-5 in the preparation of a drug or material for detecting and tracking mitochondria.

8. Use of the fluorescent tracer according to any one of claims 1-5 in the preparation of a material or reagent for co-localization imaging of mitochondria in living cells.

Images