A pyridinium salt compound connected with phenothiazine and triphenylamine and its preparation and application

Abstract

The present invention discloses a pyridinium salt compound connected with phenothiazine and triphenylamine, and its preparation and application. The compound has a structure as shown in formula (a), wherein R1 and R2 are independently selected from C1 to C 20 Alkyl. The present invention solves the problems of low selectivity and poor stability of viscosity fluorescent probes in the prior art, and provides a new highly selective viscosity fluorescent probe. The present invention combines phenothiazine with bending vibration characteristics with triphenylamine with freely rotating phenyl groups to enhance the viscosity response performance of the probe, and the phenothiazine and triphenylamine are distributed on both sides of the pyridinium salt, which can weaken the charge separation of excited state molecules and increase stability. The pyridinium salt compound fluorescent probe connected with phenothiazine and triphenylamine of the present invention has a stable structure, simple synthesis, large Stokes shift, good selectivity, and can effectively distinguish normal cells from cancer cells by utilizing the viscosity difference in cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional materials, and in particular relates to a pyridinium salt compound connected with phenothiazine and triphenylamine, and the preparation and application thereof. Background Art

[0002] Viscosity, as a key parameter of the microenvironment, is heterogeneously distributed in different regions of cells, tissues, and organs, and is involved in various biological processes, such as membrane fusion, material transport, and signal transduction. However, abnormal viscosity is often associated with major diseases such as cardiovascular disease, diabetes, and tumors. For example, the viscosity in the tumor environment is higher than that in normal tissues. Therefore, accurate detection of viscosity is of great significance for a better understanding of the pathology of related diseases. Common detection methods, such as viscometers, are not suitable for viscosity detection in cells and living tissues. Therefore, it is of great value to develop new and efficient viscosity detection methods for the diagnosis and pathological screening of related diseases.

[0003] Over the past decade, a large number of fluorescent probes have been designed and reported for detecting the viscosity microenvironment in cells. However, these probes generally involve an intramolecular charge transfer mechanism and possess a rotor structure that can rotate freely at low viscosities. Consequently, in low-viscosity environments, the probe's fluorescence is weak. In high-viscosity environments, rotor motion is restricted, and the probe's fluorescence is restored, thus achieving the desired detection. However, these probes are often affected by the polar microenvironment, and the large conjugated structure in the probe molecule is susceptible to reactions with intracellular active substances, resulting in false-positive signals. For example, a recently reported probe, LD-1, which utilizes an intramolecular charge transfer mechanism, responds to both viscosity and environmental polarity, but cannot be used to specifically image viscosity changes (Anal. Chem. 2022, 94, 4881-4888). Furthermore, the C=C double bond of the viscosity probe TP-1Bz undergoes oxidative cleavage in the presence of peroxynitrosyl ions, resulting in insufficient chemical stability (Sens. Actuators B, 2018, 276, 238-246). Therefore, there is an urgent need to develop a new class of viscosity fluorescent probes with high selectivity and good chemical stability. Summary of the Invention

[0004] The present invention aims to provide a pyridinium salt compound linked to phenothiazine and triphenylamine, and its preparation and application, which solves the problems of low selectivity and poor stability of viscosity fluorescent probes in the prior art. The probe is a new viscosity fluorescent probe with a stable structure, simple synthesis, good selectivity, and a large Stokes shift of 119 nm.

[0005] In order to achieve the above object, the present invention provides a pyridinium salt compound connected with phenothiazine and triphenylamine, wherein the compound has a structure as shown in formula (a);

[0006]

[0007] Wherein, R1 and R2 are each independently selected from C1 to C 20 of alkyl.

[0008] Preferably, R1 and R2 are each independently selected from C1 to C4 alkyl groups.

[0009] More preferably, the R1 is a C1 alkyl group; and the R2 is a C2 alkyl group.

[0010] The present invention provides a method for preparing the pyridinium salt compound connected with phenothiazine and triphenylamine, the method comprising:

[0011]

[0012] 4-Diphenylaminobenzaldehyde, compound I, and an organic base are dissolved in an anhydrous polar organic solvent and reacted at 70-90°C to prepare compound II; the prepared compound II, compound III, and an organic base are dissolved in an anhydrous polar organic solvent and reacted at 70-90°C to prepare a pyridinium salt compound connected with phenothiazine and triphenylamine, namely compound IV.

[0013] In the preparation of compound II, the reaction is carried out under reflux, and the reaction temperature is determined according to the boiling point of the selected solvent. If acetonitrile is used as the solvent, the reaction temperature is set to its boiling point of 80°C; if methanol is used, the reaction temperature is set to 65°C. Too high or too low a temperature will reduce the reaction yield.

[0014] Preferably, the compound I is prepared by dissolving 2,6-lutidine and a halogenated hydrocarbon in an anhydrous polar organic solvent and reacting at 50-90°C.

[0015]

[0016] In the preparation of compound I, the reaction is set to be carried out under reflux. Different halogenated hydrocarbons have different reaction activities. Specifically, iodine>bromine>chlorine. Therefore, for halogenated hydrocarbons with poor activity, the yield will be increased as much as possible by increasing the halogenated hydrocarbon equivalent and extending the reaction time. The reaction temperature will be determined according to the boiling point of the halogenated hydrocarbon and the boiling point of the solvent. For high-boiling-point halogenated hydrocarbons, a solvent with a higher boiling point can be selected. If acetonitrile is selected as the solvent, the reaction temperature is set to its boiling point of 80°C; if methanol is selected, it will be set to 65°C; too high or too low a temperature will reduce the reaction yield.

[0017] More preferably, the molar ratio of the halogenated hydrocarbon to 2,6-lutidine is (2.0-4.0):1; excessive addition of the halogenated hydrocarbon results in waste, while insufficient addition reduces yield. The molar volume ratio of the 2,6-lutidine to the anhydrous polar organic solvent is 1 mmol:(5-10) mL.

[0018] More preferably, in the preparation of compound I, the reaction time at 50-90°C is 4-12 hours.

[0019] More preferably, in the preparation of Compound I, the anhydrous polar organic solvent is selected from any one or more of anhydrous acetonitrile, anhydrous methanol, anhydrous ethanol, acetone, tetrahydrofuran, dichloromethane, dimethylformamide, and dimethyl sulfoxide. Excessive volume of anhydrous polar organic solvent results in waste, while insufficient volume leads to insufficient dissolution and reduced reaction yield.

[0020] Preferably, the molar ratio of 4-diphenylaminobenzaldehyde, compound I, and organic base is (0.8-1.0):1:(1.1-1.5). Properly reducing the molar ratio of 4-diphenylaminobenzaldehyde, compound I, and organic base to (0.6-0.8):1:(1.1-1.5) will maximize the production of the expected unilateral product (unilateral triphenylamine pyridinium salt, i.e., compound II). If the ratio is too large, the majority of the product will be the bilateral product (bilateral triphenylamine pyridinium salt).

[0021] Preferably, the molar volume ratio of the compound I to the anhydrous polar organic solvent is 1 mmol: (10-20) mL. Too much volume of the anhydrous polar organic solvent will cause waste, while too little will lead to insufficient dissolution and reduce the reaction yield.

[0022] Preferably, the molar ratio of Compound II, Compound III, and the anhydrous polar organic solvent is 1:(1.2-1.5):(1.2-1.5). Excessive addition of Compound III results in waste, while too little addition reduces the yield. The molar volume ratio of Compound II to the anhydrous polar organic solvent is 1 mmol:10 mL. Excessive volume of the anhydrous polar organic solvent results in waste, while too little leads to insufficient dissolution and reduces the reaction yield.

[0023] Preferably, in the preparation of compound II and compound IV, the reaction time at 70-90° C. is 4-12 hours. If the reaction time is too short, the reaction is not complete and the yield is reduced; if the time is too long, the yield is not significantly improved.

[0024] Preferably, in the preparation of Compound II and Compound IV, the anhydrous polar organic solvent is selected from any one or more of anhydrous acetonitrile, anhydrous methanol, anhydrous ethanol, acetone, tetrahydrofuran, dichloromethane, dimethylformamide, and dimethyl sulfoxide. Excessive volume of anhydrous polar organic solvent results in waste, while insufficient volume results in insufficient dissolution and reduces the reaction yield.

[0025] Preferably, in the preparation of compound II and compound IV, the organic base is any one or more of azacyclohexane (piperidine), 1,4-oxazacyclohexane (morpholine), potassium ethoxide, butyl lithium and lithium diisopropylamide.

[0026] The present invention provides an application of the pyridinium salt compound connected with phenothiazine and triphenylamine as a viscosity fluorescent probe.

[0027] The present invention discloses a pyridinium salt compound linked to phenothiazine and triphenylamine, and its preparation and application, which solve the problems of low selectivity and poor stability of viscosity fluorescent probes in the prior art and have the following advantages:

[0028] 1. In the present invention, phenothiazine and triphenylamine are connected to the 2- and 6-positions of the pyridinium salt, respectively. Since phenothiazine has bending vibration characteristics and triphenylamine has a freely rotating phenyl group, the combination of the two can enhance the viscosity response performance of the probe. In addition, the distribution of phenothiazine and triphenylamine on both sides of the pyridinium salt can weaken the charge separation of the excited state molecules and increase stability.

[0029] 2. The present invention provides a new viscosity fluorescent probe with stable structure, simple synthesis, good selectivity, large Stokes shift of 119 nm, and can effectively distinguish normal cells from cancer cells by utilizing the viscosity difference in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the hydrogen spectrum of compound IV prepared in Example 1 of the present invention.

[0031] Figure 2 This is the carbon spectrum of compound IV prepared in Example 1 of the present invention.

[0032] Figure 3 This is a fluorescence spectrum diagram of compound IV prepared in Example 1 of the present invention in the presence of different active substances, wherein the abscissa is wavelength and the ordinate is relative intensity.

[0033] Figure 4 This is an absorption spectrum of compound IV prepared in Example 1 of the present invention in different solvents, wherein the abscissa is wavelength and the ordinate is absorbance.

[0034] Figure 5This is a fluorescence spectrum of compound IV prepared in Example 1 of the present invention in different solvents, wherein the abscissa is wavelength and the ordinate is relative intensity.

[0035] Figure 6 This is a comparison chart of the fluorescence intensity of compound IV prepared in Example 1 of the present invention in different solvents, wherein the abscissa is the solvent and the ordinate is the relative intensity.

[0036] Figure 7 This is a fluorescence spectrum of compound IV prepared in Example 1 of the present invention in a methanol-glycerol system with different volume ratios, wherein the abscissa is the wavelength and the ordinate is the relative intensity.

[0037] Figure 8 These are probe imaging images of compound IV prepared in Example 1 of the present invention in different cells.

[0038] Figure 9 This is a fluorescence imaging image of compound IV prepared in Example 1 of the present invention after HeLa cells were stimulated with nystatin. DETAILED DESCRIPTION

[0039] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0040] The reagents and their preparation or models used in the following examples and experimental examples are as follows:

[0041] 1. Compound III was prepared according to the method of Observation of peroxynitrite overproduction in cells during 5-fluorouracil treatment via a ratiometric fluorescent probe. Scheme S1 (Chem. Commun., 2020, 56(18), 2759-2762).

[0042] 2. Model, specifications and manufacturer of culture medium:

[0043] Dulbecco's modified Eagle's medium (DMEM, 500mL, Gibco);

[0044] Fetal bovine serum (FBS, 500mL, Gibco).

[0045] Example 1

[0046] A method for preparing a pyridinium salt compound connected with phenothiazine and triphenylamine, the method comprising:

[0047]

[0048] (1) Methyl iodide and 2,6-lutidine at a molar ratio of 2.0:1 were dissolved in anhydrous acetonitrile (the molar volume ratio of 2,6-lutidine to anhydrous acetonitrile was 1 mmol:5 mL), and the mixture was reacted at 50° C. for 4 hours to prepare Compound I;

[0049] (2) dissolving 4-diphenylaminobenzaldehyde, compound I prepared in step (1) and azocyclohexane (piperidine) in anhydrous acetonitrile (the molar volume ratio of compound I to anhydrous acetonitrile is 1 mmol:10 mL) at a molar ratio of 0.8:1:1.1, and reacting at 70° C. for 4 hours to prepare compound II;

[0050] (3) Compound III was synthesized according to the literature method (Chem. Commun., 2020, 56(18), 2759-2762). Compound II, compound III and azocyclohexane prepared in step (2) at a molar ratio of 1:1.2:1.2 were dissolved in anhydrous acetonitrile (the molar volume ratio of compound II to anhydrous acetonitrile was 1 mmol:10 mL), and the mixture was reacted at 70°C for 4 hours to obtain compound IV (a pyridinium salt compound connected with phenothiazine and triphenylamine) with a yield of 38%.

[0051] Example 2

[0052] The preparation method of a pyridinium salt compound containing phenothiazine and triphenylamine is basically the same as that in Example 5, except that:

[0053] When anhydrous acetonitrile in steps (1) to (3) was replaced with anhydrous methanol (MeOH) or anhydrous ethanol (EtOH), the yield of compound IV decreased.

[0054] Example 3

[0055] The preparation method of a pyridinium salt compound containing phenothiazine and triphenylamine is basically the same as that in Example 5, except that:

[0056] The azacyclohexane (piperidine) in steps (2) and (3) was replaced by 1,4-oxazacyclohexane (morpholine); the yield of compound IV remained almost unchanged.

[0057] Experimental Example 1 Microscopic Characterization of the Compounds Prepared in Examples 1 to 4 of the Present Invention

[0058] like Figure 1As shown in FIG. 1 , the hydrogen spectrum of compound IV prepared in Example 1 of the present invention is shown in FIG. Figure 2 As shown, the carbon spectrum of compound IV prepared in Example 1 of the present invention. The nuclear magnetic resonance characterization of compound IV prepared in Examples 2 to 4 of the present invention is the same as that of compound IV prepared in Example 1. The nuclear magnetic resonance characterization data of compound IV prepared in Example 1 are as follows:

[0059] 1 HNMR(400MHz,DMSO-d6)δ8.34(t,J=7.8Hz,1H),8.18(dt,J=8.3,4.1Hz,2H),7.76–7.71(m ,3H),7.69(d,J=4.2Hz,1H),7.65(s,1H),7.61(dd,J=8.6,2.0Hz,1H),7.55(s,1H),7.51(d ,1H),7.40-7.36(m,3H),7.24-7.20(m,2H),7.18-7.14(m,3H),7.12-7.05(m,4H),6.99-6 .95(dm,2H),6.97(s,3H),4.27–4.22(m,3H),3.97(q,J=6.6Hz,2H),1.31(t,J=6.8Hz,3H).

[0060] 13 C NMR(100MHz,DMSO-d6)δ146.78,146.54,143.67,142.88,141.60,130.48,130.33,129.92,128.41,12 7.60,126.50,125.70,124.89,123.52,122.34,121.43,117.32,116.28,115.75,41.97,29.53,13.03.

[0061] Experimental Example 2 Fluorescent probe of compound IV prepared in Example 1 of the present invention in the presence of different active substances

[0062] Compound IV (a pyridinium salt compound containing phenothiazine and triphenylamine) prepared in Example 1 was dissolved in dimethyl sulfoxide to prepare a 5 mmol / L stock solution. Ten portions of 6 μL of the stock solution were added to ten portions of 3 ml of water to prepare 10 portions of test solutions with a probe concentration of 10 μmol / L. 100 μmol / L of different active substances were added to each of the test solutions, which were recorded as experimental groups. The blank group did not add any different active substances, and the other components were the same as the experimental group. The different active substances included: hypochlorite (ClO-), hydrogen peroxide (H2O2), peroxynitrosyl ion (ONOO - ), superoxide radicals (O2 - ), tert-butyl peroxide (t BuOOH), sulfite (SO3 2- ), sulfide ion (S 2- ), thiosulfate (S2O3 2- The excitation wavelength was 470 nm, and the fluorescence spectrum was measured by a fluorescence instrument.

[0063] like Figure 3 As shown in FIG. 1 , the fluorescence spectra of compound IV prepared in Example 1 of the present invention in the presence of different active substances, wherein the horizontal axis is the wavelength and the vertical axis is the relative intensity. Figure 3 As can be seen, in the blank group, where no active substances were added, Compound IV exhibited two weak fluorescence emission peaks at approximately 700 nm and 560 nm under an excitation wavelength of 470 nm. The fluorescence spectra of Compound IV did not change significantly after the addition of different active substances to the experimental groups, demonstrating its good chemical stability.

[0064] Experimental Example 3 Fluorescent probe of compound IV prepared in Example 1 of the present invention in different solvents

[0065] 6 μL of the mother solution prepared in Experimental Example 1 was added to 3 mL of water, dimethyl sulfoxide, acetonitrile, dimethylformamide, methanol, ethanol, 2,4-dioxane (1,4-Dioxane), tetrahydrofuran (THF), dichloromethane (DCM), or glycerol to prepare a test solution with a probe concentration of 10 μmol / L. The absorption spectrum was measured by a spectrophotometer; the excitation wavelength was 470 nm, and the fluorescence spectrum was measured by a fluorimeter.

[0066] like Figure 4 As shown in FIG. 1 , the absorption spectra of compound IV prepared in Example 1 of the present invention in different solvents, wherein the horizontal axis is the wavelength and the vertical axis is the absorbance. Figure 4 As can be seen, the maximum absorption wavelength of compound IV in different solvents ranges from 472 to 527 nm, with the maximum absorption wavelength in glycerol being 486 nm. It is important to note that, with the exception of dichloromethane, the maximum absorption wavelength of compound IV in solvents of varying polarity lies between 472 and 490 nm, indicating that the maximum absorption wavelength of this compound is minimally affected by polarity.

[0067] like Figure 5 As shown in FIG. 1 , the fluorescence spectra of compound IV prepared in Example 1 of the present invention in different solvents, wherein the horizontal axis is the wavelength and the vertical axis is the relative intensity. Figure 5As can be seen, compound IV exhibits the strongest fluorescence emission in glycerol, with an emission wavelength of 605 nm and a Stokes shift of 119 nm. In other low-viscosity solvents, the fluorescence intensity is weaker. Notably, compound IV also exhibits weak fluorescence in less polar solvents, such as dichloromethane and 1,4-dioxane, indicating that the fluorescence intensity of this compound is minimally affected by polarity.

[0068] like Figure 6 As shown in FIG. 1 , the fluorescence intensity comparison diagram of compound IV prepared in Example 1 of the present invention in different solvents, wherein the horizontal axis is the solvent and the vertical axis is the relative intensity. Figure 6 It can be seen that compound IV has good selectivity for glycerol, which is a commonly used water-soluble high-viscosity substance. Therefore, compound IV has a selective response to viscosity.

[0069] Experimental Example 4 Fluorescent probe of compound IV prepared in Example 1 of the present invention in methanol-glycerol system with different volume ratios

[0070] 6 μL of the mother solution prepared in Experimental Example 1 was added sequentially to 11 3 mL methanol-glycerol mixed systems with different volume ratios (the total volume of the methanol-glycerol mixed system was 3 mL, and the volume percentages of glycerol were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively). The excitation wavelength was 470 nm, the probe concentration was 10 μmol / L, and the changes in the fluorescence signal were detected by a fluorimeter.

[0071] like Figure 7 As shown in FIG. 1 , the fluorescence spectra of compound IV prepared in the present invention in a methanol-glycerol system with different volume ratios are shown, wherein the horizontal axis is the wavelength and the vertical axis is the relative intensity. Figure 7 It can be concluded that as the viscosity coefficient increases (the proportion of glycerol increases), the fluorescence intensity gradually increases, proving that the probe of the present invention has a high sensitivity in detecting viscosity changes.

[0072] Experimental Example 5 Fluorescent probe of compound IV prepared in Example 1 of the present invention in different cells

[0073] 1 μL of the mother solution prepared in Experimental Example 1 was diluted to 1 mL with culture medium and added to mouse macrophages (RAW264.7 cells), mouse fibroblasts (L929 cells), cervical cancer cells (HeLa cells), and human lung cancer cells (A549 cells) (the number of cells of each type was controlled between 200,000 and 250,000). The cells were incubated at 37°C for 40 minutes, and the fluorescence signal was observed at room temperature using a laser confocal fluorescence microscope.

[0074] like Figure 8As shown, the probe imaging images of compound IV prepared in Example 1 of the present invention in different cells, wherein RAW 264.7 is mouse macrophage (normal cell); L929 is mouse fibroblast (normal cell); HeLa is cervical cancer cell; A549 is human lung cancer cell; the first row (A, D, G, J) is fluorescence image; the second row (B, E, H, K) is bright field image, and the third row (C, F, I, L) is the superposition of the first two rows. Figure 8 Viscosity values ​​in cancer cells are higher than in normal cells, indicating that Compound IV exhibits a superior viscosity response to cancer cells in the fluorescence image. Therefore, the difference in the degree of fluorescence signal expressed by Compound IV in different cell types can be used to distinguish between normal and cancer cells.

[0075] Experimental Example 6 Fluorescent probe of compound IV prepared in Example 1 of the present invention after HeLa cells were stimulated with nystatin

[0076] Nystatin (10 μg / mL) was added to cervical cancer cells (HeLa cells) and incubated at 37°C for 2 hours. The culture medium was removed and the cells were washed three times with PBS (phosphate buffered saline). Then, 1 μL of the mother solution prepared in Experimental Example 1 was diluted to 1 mL with culture medium and added to the cells. The cells were cultured for 40 minutes, which was designated as the +Nys group (stimulated by nystatin). 1 μL of the mother solution prepared in Experimental Example 1 was added to cervical cancer cells (HeLa cells) and incubated at 37°C for 40 minutes, which was designated as the control group. The fluorescence signal was observed at room temperature using a laser confocal fluorescence microscope.

[0077] like Figure 9 As shown, the fluorescence imaging of compound IV prepared in Example 1 of the present invention after HeLa cells were stimulated with nystatin, wherein the Control group was not stimulated by the drug; the +Nys group was stimulated by nystatin; the first column (A, D) is the fluorescence image, the second column (B, E) is the bright field image, and the third column (C, F) is the overlay image. Figure 9 It can be seen that after stimulation by nystatin, the intracellular microenvironment of the +Nys group changed, the viscosity increased, and the fluorescence signal was enhanced.

[0078] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A pyridinium salt compound connected to phenothiazine and triphenylamine, characterized in that: The compound has a structure as shown in formula (a); Wherein, R1 and R2 are each independently selected from C1 to C4 alkyl groups.

2. A method for preparing a pyridinium salt compound containing phenothiazine and triphenylamine as claimed in claim 1, characterized in that: The method includes: 4-Diphenylaminobenzaldehyde, compound I, and an organic base are dissolved in an anhydrous polar organic solvent and reacted at 70-90°C to prepare compound II; compound III, the prepared compound II, and an organic base are dissolved in an anhydrous polar organic solvent and reacted at 70-90°C to prepare a pyridinium salt compound connected to phenothiazine and triphenylamine, i.e., a compound of formula (a).

3. The method for preparing a pyridinium salt compound containing phenothiazine and triphenylamine according to claim 2, wherein: The compound I is prepared by dissolving 2,6-lutidine and a halogenated hydrocarbon in an anhydrous polar organic solvent and reacting at 50-90°C; 4. The method for preparing a pyridinium salt compound containing phenothiazine and triphenylamine according to claim 3, wherein: The molar ratio of the 2,6-lutidine and the halogenated hydrocarbon is (2.0-4.0):1; the molar volume ratio of the 2,6-lutidine to the anhydrous polar organic solvent is 1 mmol:(5-10) mL; in the preparation of compound I, the anhydrous polar organic solvent is selected from any one or more of anhydrous acetonitrile, anhydrous methanol, anhydrous ethanol, acetone, tetrahydrofuran, dichloromethane, dimethylformamide and dimethyl sulfoxide.

5. The method for preparing a pyridinium salt compound containing phenothiazine and triphenylamine according to claim 2, wherein: The molar ratio of the 4-diphenylaminobenzaldehyde, compound I, and organic base is (0.8-1.0):1:(1.1-1.5); the molar volume ratio of the compound I to the anhydrous polar organic solvent is 1 mmol:(10-20) mL.

6. The method for preparing a pyridinium salt compound containing phenothiazine and triphenylamine according to claim 2, wherein: The molar ratio of the compound II, compound III and the anhydrous polar organic solvent is 1:(1.2-1.5):(1.2-1.5); the molar volume ratio of the compound II and the anhydrous polar organic solvent is 1 mmol:10 mL.

7. The method for preparing a pyridinium salt compound containing phenothiazine and triphenylamine according to claim 2, wherein: In the preparation of compound II and the compound of formula (a), the anhydrous polar organic solvent is selected from any one or more of anhydrous acetonitrile, anhydrous methanol, anhydrous ethanol, acetone, tetrahydrofuran, dichloromethane, dimethylformamide and dimethyl sulfoxide.

8. The method for preparing a pyridinium salt compound containing phenothiazine and triphenylamine according to claim 2, wherein: In the preparation of compound II and the compound of formula (a), the organic base is any one or more of azacyclohexane, 1,4-oxazacyclohexane, potassium ethoxide, butyl lithium and lithium diisopropylamide.

9. Use of the pyridinium salt compound linked to phenothiazine and triphenylamine as claimed in claim 1 in the preparation of a viscosity fluorescent probe.

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