A Fluorescent Probe Based on Methoxybenzothiophene Derivatives, Its Preparation Method and Application

Abstract

The present invention belongs to the technical field of organic small molecule fluorescent probes, and particularly relates to a fluorescent probe based on methoxybenzothiophene derivatives, a preparation method thereof, and an application thereof. The fluorescent probe is composed of three parts: an energy donor naphthalimide, an energy acceptor methoxybenzothiophene derivative, and a linker (piperazine), and the fluorescent probe can be used for detecting SO2 derivatives and viscosity.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic small molecule fluorescent probes, and particularly relates to a fluorescent probe based on methoxybenzothiophene derivatives, a preparation method thereof, and applications thereof. Background Art

[0002] Sulfur dioxide (SO2) is a common environmental pollutant, which widely exists in the environment, food, and organisms in the form of bisulfite / sulfite (HSO3 - / SO3 2- ). When the concentration of sulfur dioxide in the environment is too high, acid rain is easily formed, posing a serious threat to the ecosystem. Excessive SO2 content in living organisms can lead to metabolic disorders and even diseases, such as cardiovascular diseases, endocrine disorders, cancer, etc.

[0003] The viscosity of the cellular microenvironment not only reflects the physical properties of the microenvironment but is also closely related to the physiological and pathological states of cells. For example, a highly viscous microenvironment may limit cell movement, affecting the migration path and speed of cells in tissues, which is of great significance in embryonic development, tissue repair, and cancer metastasis research. Additionally, the tumor microenvironment usually has abnormal viscosity, which is related to the growth, invasion, and metastasis of cancer cells. By detecting the viscosity in the tumor microenvironment, it is possible to help identify the invasiveness of tumors and predict the likelihood of their metastasis, thereby providing a basis for clinical diagnosis and treatment. Therefore, by detecting the viscosity in the cellular microenvironment, researchers can gain a deeper understanding of cell behavior, the impact of the microenvironment on cells, and how to utilize this information for applications and innovations in the fields of medicine and bioengineering.

[0004] The small molecule fluorescent probe method has many advantages such as good selectivity, strong anti-interference ability, high sensitivity, and low detection limit, and has thus become a current research hotspot; Ratio fluorescent probes based on the fluorescence resonance energy transfer (FRET) / intramolecular twisted charge transfer (TICT) mechanism generally consist of an energy donor, a linker, and an energy acceptor. Ratio fluorescent probes based on the FRET mechanism absorb energy by the energy donor and transfer the energy to the acceptor through an intramolecular resonance process, causing the probe to exhibit enhanced fluorescence of the acceptor. The TICT process mainly occurs in the acceptor part. As the concentration of the analyte changes, the FRET process is gradually blocked, the acceptor cannot receive the energy from the donor, and the fluorescence emission intensities of the donor and the acceptor gradually change.

[0005] Compared with traditional single-response fluorescent probes, dual-response fluorescent probes have the effect of "killing two birds with one stone". Currently, there are few reported mitochondrial-targeted fluorescent probes with dual responses to HSO3 - / SO3 2- and viscosity based on the FRET / TBET mechanism. Summary of the Invention

[0006] The first object of the present invention is to provide a fluorescence probe based on a methoxybenzothiophene derivative, which is composed of three parts: an energy donor naphthalimide, an energy acceptor methoxybenzothiophene derivative, and a linker piperazine; the chemical structural formula is as shown in formula (I):

[0007]

[0008] Formula (I).

[0009] The second object of the present invention is to provide a preparation method of the fluorescence probe based on the methoxybenzothiophene derivative, comprising the following steps:

[0010] S1. Synthesis of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid:

[0011] 4-(6-Bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid (1 mmol) and pyrrolidine (1.5 mol) are dissolved in DMF, and triethylamine is added as a catalyst. The reaction is carried out at 60 °C for 12 h, then the reaction is stopped, and the organic solvent is removed by rotary evaporation under reduced pressure. The target product 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid is separated by column chromatography using silica gel powder of 200 - 300 mesh.

[0012] S2. Synthesis of (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)styryl)benzothiazol-3-ium salt:

[0013] 6-Methoxy-2,3-dimethylbenzothiazol-3-ium salt (1.0 mmol) and 4-(piperazin-1-yl)benzaldehyde (1.2 mmol) are dissolved in absolute ethanol, and the mixture is heated under reflux for 12 hours. After the reaction is stopped, the solvent is removed by rotary evaporation under reduced pressure, and then purified by column chromatography. Using silica gel powder of 200 - 300 mesh and a mixed solution of dichloromethane and methanol with a volume ratio of 1:25 as the eluent, the target product (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)styryl)benzothiazol-3-ium salt is obtained.

[0014] S3. Synthesis of the target probe:

[0015] 4-(1,3-Dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)styryl)benzothiazol-3-ium salt were dissolved in dry dichloromethane. 4-Dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added as catalysts and stirred at room temperature for 24 hours to stop the reaction. The solvent was removed by rotary evaporation under reduced pressure. The crude product was purified and separated by column chromatography using silica gel powder of 200 - 300 mesh, and the eluent was a mixed solution of dichloromethane and methanol with a volume ratio of 1:40 to obtain the target product;

[0016] Synthesis reaction formula of the probe:

[0017] 。

[0018] Further, in step S3, the molar ratio of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid to (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)styryl)benzothiazol-3-ium salt is 1:1; the molar ratio of 4-dimethylaminopyridine to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:10.

[0019] Further, the addition amount of the dry dichloromethane is limited to 40 mL of dry dichloromethane corresponding to 0.5 mmol of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid.

[0020] The present invention also provides the application of the fluorescent probe based on methoxybenzothiophene derivatives in the detection of HSO3 - / SO3 2- detection.

[0021] The present invention also provides the application of the fluorescent probe based on methoxybenzothiophene derivatives in viscosity detection.

[0022] Advantageous technical effects of the present invention:

[0023] (1)When the fluorescence probe of the methoxybenzothiophene derivative of the present invention is in the absence of SO2 derivatives, the probe is excited, and the energy donor transfers energy to the energy acceptor, and the probe emits enhanced fluorescence of the acceptor. In the presence of SO2 derivatives, the double-bond recognition site in the energy acceptor reacts with the SO2 derivative, the structure of the energy acceptor changes, and the intramolecular FRET process is blocked. At this time, the probe emits the fluorescence of the energy donor when excited by light. Different concentrations of SO2 derivatives result in different degrees of reaction between the probe and the SO2 derivative, manifested as changes in the fluorescence intensities at the two emission wavelengths of the probe.

[0024] (2)In addition, the energy acceptor part of the probe has an intramolecular TICT process. When the viscosity in the detection system increases, the TICT effect in the energy acceptor part weakens, and the probe emits enhanced fluorescence of the energy acceptor when excited. Based on the above changes, the purpose of detecting SO2 derivatives and viscosity using a ratio probe is achieved.

[0025] (3)The SO2 derivative and viscosity dual-responsive ratio probe based on the FRET / TICT mechanism of the present invention can not only quantitatively detect the concentration and viscosity level of SO2 derivatives, but also be used for real-time imaging of SO2 derivatives and viscosity in living cells, and has excellent mitochondrial targeting properties. In addition, the probe has been successfully used to detect the content of SO2 derivatives in food and water samples, and has a high spike recovery rate. Therefore, this probe is a good mitochondrial-targeted SO2 derivative and viscosity dual-responsive fluorescence probe based on the fluorescence resonance energy transfer and twisted intramolecular charge transfer mechanisms. It is expected to play an important role in environmental protection, food safety, and clinical medicine, and has broad application prospects. Description of the Drawings

[0026] Figure 1 1H NMR spectrum of the fluorescence probe prepared in Example 1 1 1H NMR

[0027] Figure 2 Fluorescence spectrum of the fluorescence probe prepared in Example 1 for selectively detecting SO2 derivatives

[0028] Figure 3 Fluorescence spectrum for characterizing the anti-interference ability of the fluorescence probe prepared in Example 1 to detect SO2 derivatives

[0029] Figure 4 Changes in the fluorescence intensities of the fluorescence spectrum of the fluorescence probe prepared in Example 1 at 530 nm and 582 nm, and the linear relationship diagram between the ratio (I 530 / I 582 ) and the concentration of SO2 derivatives

[0030] Figure 5Fluorescence intensity change at 582 nm of the fluorescence spectrum of the fluorescent probe prepared in Example 1, and the linear relationship diagram between Ln(I 582 ) and viscosity.

[0031] Figure 6 Confocal fluorescence imaging diagrams of endogenous SO2 derivatives in AGS cells incubated under different conditions with the fluorescent probe prepared in Example 1, green fluorescence channel (490 - 550 nm) and red fluorescence channel (550 - 700 nm); where: (a) is the cell imaging diagram under the indicated conditions; (b) is the bar chart of the ratio of the fluorescence intensity of the green channel to the fluorescence intensity of the red channel corresponding to (a).

[0032] Figure 7 Confocal fluorescence imaging diagram of the viscosity change in AGS cells with the probe of the present invention, excitation wavelength 405 nm, red channel: 550 - 700 nm, where: (a) is the cell imaging diagram under the indicated conditions; (b) is the relative fluorescence intensity ratio diagram of the red channel corresponding to (a).

[0033] Figure 8 Schematic diagram of the organelle localization imaging of the probe of the present invention in live AGS cells, where: (a) is the imaging diagram of the probe of the present invention, λ ex = 405 nm, green fluorescence channel: 490 - 550 nm; (b) is the imaging diagram of the commercial dye Mito-Tracker™ Red targeting mitochondria, λ ex = 647 nm, red fluorescence channel: 647 - 700 nm; (c) is the overlapping diagram of (a) and (b); (d) is the schematic diagram of the co-localization coefficient. Detailed implementation manners

[0034] The content of the present invention will be described in detail below in conjunction with specific embodiments. The following examples are only the optimal implementation manners of the present invention, and do not impose any form of limitation on the present invention. Any simple modification, equivalent change and modification made to the implementation manner according to the technical essence of the present invention all fall within the scope of the technical solution of the present invention.

[0035] The drugs, reagents, consumables and cells used in the present invention are all commercially available products.

[0036] Example 1

[0037] S1. Synthesis of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid:

[0038] 4-(6-Bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid (1 mmol) and pyrrolidine (1.5 mol) were dissolved in DMF. Triethylamine was added as a catalyst, and the reaction was carried out at 60 °C for 12 h. The reaction was stopped, and the organic solvent was removed by rotary evaporation under reduced pressure. Silica gel powder with a mesh size of 200-300 was used, and the target product 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid was obtained by column chromatography;

[0039] S2. Synthesis of (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)styryl)benzothiazol-3-ium salt:

[0040] 6-Methoxy-2,3-dimethylbenzothiazol-3-ium salt (1.0 mmol) and 4-(piperazin-1-yl)benzaldehyde (1.2 mmol) were dissolved in absolute ethanol, and the mixture was refluxed for 12 hours. The reaction was stopped, and the solvent was removed by rotary evaporation under reduced pressure. Then, it was separated and purified by column chromatography. Silica gel powder with a mesh size of 200-300 was used, and the developing agent was a mixed solution of dichloromethane and methanol with a volume ratio of 1:25, to obtain the target product (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)styryl)benzothiazol-3-ium salt;

[0041] S3. 4-(1,3-Dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid II (0.5 mmol, 193.1 mg) and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)styryl)benzothiazol-3-ium salt III (0.5 mmol, 246.5 mg) were dissolved in dry dichloromethane (40 mL). 4-Dimethylaminopyridine (DMAP, 0.1 mmol, 12.5 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 1 mmol, 191.0 mg) were added as catalysts, and the mixture was stirred at room temperature for 24 hours. The reaction was stopped. The solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by column chromatography. Silica gel powder with a mesh size of 200-300 was used, and the developing agent was a mixed solution of dichloromethane and methanol (MeOH / DCM, 1 / 40, V / V). The yield of the target product was 36%.

[0042] The synthesis reaction formula of step 3 is as follows:

[0043] 。

[0044] Spectral data for confirming the structure of the probe molecule prepared in Example 1:

[0045] HRMS (ESI): m / z, calculated for [C 44 H 40 O4N5S] + is 734.2796, found 734.2805.

[0046] 1 HNMR (400 MHz, DMSO- d 6 ): δ = 2.041 (s, 4H), 3.590 (s, 6H), 3.810 (s, 6H), 3.906 (s, 3H), 4.242 (s, 3H), 6.948 (d, J = 8.8 Hz, 1H), 7.101 (d, J = 8.8 Hz), 7.400 (dd, J = 9.2 and 2.4 Hz, 1H), 7.445 (d, J = 8.8 Hz, 2H), 7.601 (d, J = 8.0 Hz, 2H), 7.652 - 7.718 (m, 2H), 7.935 (d, J = 8.0 Hz, 3H), 8.013 (d, J = 15.6 Hz, 1H), 8.0557 (d, J = 9.6 Hz, 1H), 8.270 (d, J = 8.4 Hz, 1H), 8.463 (d, J = 7.2 Hz, 1H), 8.820 (d, J = 8.8 Hz, 1H), see Figure 1 。

[0047] 13 CNMR (100 MHz, DMSO- d 6 ): δ = 26.06, 36.37, 53.43, 56.00, 56.70, 107.06, 109.02, 109.36, 114.45, 115.82, 117.68, 122.37, 123.77, 124.09, 128.09, 129.44, 129.93, 131.31, 131.63, 132.58, 133.58, 136.60, 153.03, 153.52, 159.52, 163.04, 163.49, 164.52, 169.31, 169.80.

[0048] Example 2

[0049] To a 10 ml volumetric flask containing 5 μM of the probe prepared in Example 1, make up the volume (EtOH / PBS, V / V, 5 / 5, pH = 7.4), and then use a microsyringe to add 50 μl each of: Hcy, C2O4 2- , CH3COO - , SO4 2- , HCO3 - , CO32- , F - , Br - , Cys, I, Gly, NO3 - , S2O3 2- , NH4 + , Cl - , S 2- , K + , Zn 2+ , Mg 2+ , Cu 2+ , Al 3+ Plasma, followed by fluorescence testing.

[0050] The test results are as Figure 2 shown. As can be seen from the figure, the orange curve represents HSO3⁻ / SO3²⁻, which is significantly different from the fluorescence intensity curves of other ions, indicating that HSO3⁻ / SO3²⁻ has a significant inhibitory effect on fluorescence intensity. For other anions and cations such as Hcy, C2O4²⁻, CH3COO⁻, SO4²⁻, HCO3⁻, CO3²⁻, F⁻, Br⁻, Cys, I⁻, Gly, NO3⁻, S2O3²⁻, NH4⁺, Cl⁻, S²⁻, K⁺, Zn²⁺, Mg²⁺, Cu²⁺, Al³⁺, etc., there is no significant change in the peak position or intensity of the fluorescence intensity, which indicates that the above ions have little interference on the intensity and peak of the fluorescence probe prepared in Example 1. In summary, the fluorescence probe prepared in Example 1 has high selectivity in detecting HSO3⁻ / SO3²⁻.

[0051] Example 3

[0052] To a 10 ml volumetric flask containing 5 μM of the probe prepared in Example 1, make up the volume (EtOH / PBS, V / V, 5 / 5, pH = 7.4), and then use a microsyringe to add 50 μl each of: Hcy, C2O4 2- , CH3COO - , SO4 2- , HCO3 - , CO3 2- , F - , Br - , Cys, I, Gly, NO3 - , S2O3 2- , NH4 + , Cl - , S 2- , K + , Zn 2+ , Mg 2+ , Cu 2+ , Al 3+ plasma, and then add 50 μl each of HSO3 - / SO3 2- After the action is complete, fluorescence testing is carried out, and the results obtained are as Figure 3 shown. It can be seen from the figure that even in the presence of other ions, the fluorescent probe prepared in Example 1 can still detect HSO3⁻ / SO3²⁻, indicating that the fluorescent probe detects HSO3 - / SO3 2- has good anti-interference ability.

[0053] Example 4

[0054] To a 10 ml volumetric flask containing 5 μM of the fluorescent probe prepared in Example 1, make up the volume (EtOH / PBS, V / V, 5 / 5, pH = 7.4), and then use a microsyringe to add different concentrations of HSO3 - / SO3 2- , and then carry out fluorescence testing. The results are as Figure 4 shown, indicating that with the increase in the concentration of the SO2 derivative of the fluorescent probe, the fluorescence emission intensity of the probe at 530 nm gradually increases, and the fluorescence emission intensity at 582 nm gradually decreases; the ratio of the fluorescence intensities at the two emission peaks (I 530 / I 582 ) shows a linear relationship with the concentration of the SO2 derivative within a certain range, that is, the fluorescent probe can quantitatively detect the concentration of HSO3 - / SO3 2- .

[0055] Example 5

[0056] To a 10 ml volumetric flask containing 5 μM of the fluorescent probe prepared in Example 1, add a PBS / glycerol mixed solution with different PBS contents to make up the volume. Adding glycerol to the PBS buffer can increase the viscosity of the solution, thereby affecting the diffusion rate of molecules or particles in the solution. In this example, PBS-glycerol mixed solutions with different ratios are used to simulate the in vivo environment, and the proportion of glycerol increases from 0% to 99.5%. After mixing evenly, fluorescence testing is carried out. The results are as Figure 5 shown. As the proportion of glycerol increases, the color of the fluorescence changes from purple to orange. The fluorescence intensity of the fluorescent probe at 582 nm increases with the increase in the proportion of glycerol, that is, with the increase in viscosity. In addition, the value of Ln(I 582 ) shows a linear relationship with the proportion of glycerol, that is, the viscosity.

[0057] Example 6

[0058] Control group (Ctr): AGS cells are incubated with the solution of the fluorescent probe (4 μM) prepared in Example 1 for 1 h, rinsed three times with PBS, and then confocal imaging is carried out;

[0059] GSH / Na2S2O3 group: AGS cells were first incubated with the fluorescent probe (4 μM) solution for another 1 h, then incubated with GSH (500 μM) and Na2S2O3 (250 μM) for 1 h, washed three times with PBS, and subjected to confocal imaging;

[0060] GSH group: AGS cells were first incubated with the probe (4 μM) solution for another 1 h, then incubated with GSH (500 μM) for 1 h, washed three times with PBS, and subjected to confocal imaging;

[0061] TNBS / GSH / Na2S2O3 group: AGS cells were first incubated with TNBS (500 μM) for 1 h, then incubated with the probe (4 μM) for 1 h, and finally incubated with GSH (500 μM) / Na2S2O3 (250 μM) for 1 h, washed three times with PBS, and subjected to confocal imaging.

[0062] The results are as Figure 6 shown: In the presence of both GSH and Na2S2O3, AGS cells can produce endogenous SO2 derivatives, the green fluorescence intensity of the probe in the cells increases, the red fluorescence intensity decreases, and the relative fluorescence intensity ratio I Green / I Red increases significantly; in the presence of TNBS, GSH, and Na2S2O3 simultaneously, TNBS can inhibit the production of endogenous SO2 derivatives, and there is no obvious change in the probe fluorescence signal at this time. The above experiments show that the probe can effectively detect endogenous SO2 derivatives in living cells.

[0063] Example 7

[0064] AGS cells were incubated with the fluorescent probe prepared in Example 1 (4 μM) for 1 h, and then incubated with different concentrations (0, 10, 30, 40 μM) of nystatin for 0.5 h. After washing three times with PBS, confocal imaging was performed. Red fluorescence channel: 550 - 700 nm. Culturing cells with nystatin will cause an increase in intracellular viscosity. The results are as Figure 7 shown. As the concentration of nystatin for culturing cells increases, the intracellular viscosity gradually increases, and the red fluorescence of the probe in the cells gradually increases, indicating that the fluorescent probe can effectively detect the intracellular viscosity.

[0065] Example 8

[0066] (a) AGS cells were incubated with the fluorescent probe prepared in Example 1 (4 μM) for 1 h, washed three times with PBS, and then subjected to confocal imaging, λ ex = 405 nm, green light channel: 490 - 550 nm; The results are shown in Figure 8 a. The fluorescent probe has strong green fluorescence in mitochondria;

[0067] (b) Incubate AGS cells with the commercial mitochondrion-targeting dye Mito-Tracker™ Red (0.2 μM) for 1 h, wash three times with PBS, and then perform confocal imaging, λ ex = 647 nm, red channel: 647 - 700 nm; Mito-Tracker™ Red is a fluorescent dye commonly used for mitochondrial staining, which can specifically target and label mitochondria in living cells. Mito-Tracker™ Red has strong red fluorescence in mitochondria, as shown in Figure 8 b;

[0068] Figure 8 c is Figure 8 a and Figure 8 the overlay of b, and it can be seen that the two achieve good coincidence. Figure 8 d is the schematic diagram of the colocalization coefficient of the two, and the coefficient is obtained as 0.93. The results of the colocalization experiment show that the fluorescent probe has high mitochondrial localization targeting.

[0069] Example 9

[0070] Accurately weigh 20 g of granulated sugar and prepare it in 1000 ml of PBS buffer containing sucrose. Add 50 μl of the probe mother liquor to a 10 ml volumetric flask in sequence, and then make up the volume with EtOH / PBS (V / V, 5 / 5, pH = 7.4). Then measure the fluorescence to obtain the fluorescence intensity ratio I 530 / I 582 , substitute it into the working curve obtained in Example 4, and calculate the content of its SO2 derivative. Subsequently, add different concentration gradients of HSO3 - / SO3 2- (8, 10, 15 μM), and measure the fluorescence spectrum to calculate the spike recovery rate of the probe. The determination method of white wine is as shown above. The results are shown in Table 1, and the calculated spike recovery rates are all in the range of 86% - 108%, indicating that the probe has high accuracy, anti-interference ability, and reproducibility in detecting SO2 derivatives in actual samples.

[0071] Table 1 Determination of the concentration of SO2 derivatives in granulated sugar and white wine by the probe and determination of spike recovery rate

[0072] .

Claims

1. A fluorescent probe based on a methoxybenzothiazole derivative, characterized in that: The fluorescent probe is composed of three parts: energy donor naphthalimide, energy acceptor methoxybenzothiazole derivative, and linker piperazine; the chemical structure is shown in formula (I): Formula (I).

2. A method for preparing a fluorescent probe based on a methoxybenzothiazole derivative as claimed in claim 1, characterized in that: The steps include: S1. Synthesis of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)benzoic acid: 4-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and pyrrolidine were dissolved in DMF, and triethylamine was added as a catalyst. The reaction was carried out at 60° C. for 12 h, and the reaction was stopped. The organic solvent was removed by rotary evaporation under reduced pressure, and the target product 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid was separated by column chromatography using silica gel powder 200-300 mesh; S2. Synthesis of (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt: Dissolve 6-methoxy-2,3-dimethylbenzo[d]thiazol-3-ium salt and 4-(piperazin-1-yl)benzaldehyde in anhydrous ethanol, heat and reflux for 12 hours, stop the reaction, remove the solvent by vacuum rotary evaporation, and separate and purify by column chromatography, silica gel powder 200-300 mesh, and a mixed solution of dichloromethane and methanol in a volume ratio of 1:25 as a developing solvent to obtain the target product (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt; S3. Synthesis of target probe: Dissolve 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt in dry dichloromethane, add 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as catalysts, stir at room temperature for 24 hours, and stop the reaction; remove the solvent by rotary evaporation under reduced pressure, purify and separate the crude product by column chromatography, silica gel powder 200-300 mesh, and a mixed solution of dichloromethane and methanol in a volume ratio of 1:40 as a developing solvent to obtain the target product; The synthetic reaction formula of the target probe is: 。 3. The method for preparing a fluorescent probe based on a methoxybenzothiazole derivative according to claim 2, characterized in that: In step S1, the molar ratio of 4-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid to pyrrolidine is 1:1.5; In step S2, the molar ratio of 6-methoxy-2,3-dimethylbenzo[d]thiazol-3-ium salt to 4-(piperazin-1-yl)benzaldehyde is 1:1.2; In step S3, the molar ratio of the 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt is 1:1; the molar ratio of the 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:

10.

4. A fluorescent probe based on a methoxybenzothiazole derivative as claimed in claim 1 in HSO3 - / SO3 2- Applications in detection, including non-disease treatment and disease diagnosis.

5. The fluorescent probe based on methoxybenzothiazole derivatives according to claim 4 is prepared in HSO - / SO3 2- The application in detection is characterized by: The fluorescent probe is used to detect HSO3 - / SO3 2- Be selective.

6. The fluorescent probe based on methoxybenzothiazole derivatives according to claim 4 is prepared in HSO - / SO3 2- The application in detection is characterized by: The fluorescent probe is measured by the fluorescence intensity ratio I 530 / I 582 To achieve HSO3 - / SO3 2- Quantitative detection of concentration.

7. The fluorescent probe based on methoxybenzothiazole derivatives according to claim 4 is prepared in HSO - / SO3 2- The application in detection is characterized by: The fluorescent probe is used to detect endogenous HSO3 - / SO3 2- Detection.

8. Use of the fluorescent probe based on methoxybenzothiazole derivatives as claimed in claim 1 in viscosity detection, characterized in that: The fluorescent probe is used for detecting intracellular viscosity, and the application is non-disease treatment use and disease diagnosis use.

9. Use of the fluorescent probe based on methoxybenzothiazole derivatives as claimed in claim 1 in mitochondrial localization and targeting, wherein the application is for non-disease treatment and disease diagnosis.

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