Viscosity-responsive TB-pyridine cyano vinyl derivatives and their synthesis and applications

Abstract

The present invention provides a viscosity-responsive TB-pyridine cyano vinyl derivative and its synthesis and application. The derivative is prepared by multi-step reaction using p-bromoaniline, paraformaldehyde, n-butyl lithium, 4-pyridine acetonitrile, etc. as raw materials. The structural formula is shown in the following derivative (6): #imgabs0# The product has a large Stokes shift (207 nm) in solution and has significant AIE properties. Compared with pure methanol, the fluorescence of 3 is enhanced by 5.3 times when methanol / glycerol = 1 / 9 (v / v), showing good viscosity response, easy entry into living A549 cells, and strong targeting ability to the endoplasmic reticulum (Pearson coefficient Pr = 0.75), with the potential to become an AIE-type endoplasmic reticulum viscosity-responsive fluorescent probe. The product has strong inhibitory ability against HpeG2 cells under both dark and light conditions (428 nm), indicating its potential for development as an anti-liver cancer drug.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis, and specifically relates to Base-pyridine cyano vinyl derivatives, their synthesis methods, and their applications in viscosity-responsive, endoplasmic reticulum-targeted anti-tumor activities. Background Art

[0002] Viscosity is a key parameter influencing numerous biological processes, determining the fluidity of substances and the rates of diffusion-controlled reactions. Within cells, viscosity significantly influences mass and signal transmission, as well as interactions between biomacromolecules. Abnormal intracellular viscosity is considered a key factor or indicator of numerous diseases and functional disorders, including diabetes, stroke, and hypertension. Therefore, real-time monitoring of intracellular viscosity is crucial for the investigation and diagnosis of these diseases.

[0003] Traditional viscometers cannot detect intracellular viscosity, but fluorescent small molecules have strong cell penetration ability and cause little damage to cells. Therefore, they can be used to monitor changes in intracellular viscosity and become the main choice for designing and synthesizing new intracellular viscosity-responsive fluorescent probes.

[0004] The dense polypeptide chains formed during protein misfolding and aggregation can cause changes in viscosity. The endoplasmic reticulum (ER), a crucial cellular organelle, organically connects the nucleus, cytoplasm, and cell membrane into a single entity. It is responsible for the transport of substances within the cell and serves as a hub for protein synthesis. ER-targeted viscosity probes can monitor viscosity changes caused by protein aggregation within the ER.

[0005] Pyridine has a variety of physiological activities, including antibacterial, antitumor, anti-inflammatory, and antihypertensive properties. At the same time, pyridine also has strong electrophilicity and can be used as an electron acceptor, and is widely used in new bipolar materials and organic devices. The cyano group has a high electron affinity and can therefore be used as a buffer layer for the introduction and extraction of electrons in organic matter. Some cyano-substituted compounds (R-CN) can exhibit unique enhanced emission behavior in the solid state. Cyanovinyl groups can be combined with fluorescent groups in the synthesis of organic molecules to act as electron donors or electron acceptors. Therefore, if cyanovinyl groups and pyridine are combined, their electron-withdrawing ability can be enhanced and AIE properties can be obtained.

[0006] Therefore, the present invention designs and synthesizes a D-π-A structure by combining electron-withdrawing fragments of cyanoethylene and pyridine on the TB skeleton. base-pyridine cyano vinyl derivatives. Summary of the Invention

[0007] Technical problem: The purpose of the present invention is to provide a TB-pyridine cyano vinyl derivative with viscosity response and its synthesis and application, using p-bromoaniline, paraformaldehyde, n-butyl lithium, 4-pyridine acetonitrile and the like as raw materials, and preparing the TB-pyridine cyano vinyl derivative through multi-step reaction. base-pyridine cyano vinyl derivatives, and applied them to viscosity recognition, endoplasmic reticulum localization and anti-tumor fields.

[0008] Technical solution: The structural formula of a viscosity-responsive TB-pyridine cyano vinyl derivative of the present invention is:

[0009]

[0010] The synthesis method of the viscosity-responsive TB-pyridine cyano vinyl derivative of the present invention comprises the following steps:

[0011] Step 1, 4-bromoaniline reacts with paraformaldehyde to obtain the first intermediate, and the reaction formula is as follows:

[0012]

[0013] Step 2: The first intermediate reacts with n-butyl lithium to obtain a second intermediate. The reaction formula is as follows:

[0014]

[0015] Step 3: The second intermediate is reacted with 4-pyridine acetonitrile through coupling reaction to obtain a derivative, and the reaction formula is as follows:

[0016]

[0017] The invention relates to the use of the viscosity-responsive TB-pyridine cyano vinyl derivative in the preparation of a viscosity probe.

[0018] The invention provides an application of the viscosity-responsive TB-pyridine cyanovinyl derivative in the preparation of an endoplasmic reticulum-targeting probe.

[0019] The application of the endoplasmic reticulum targeting probe is aimed at the localization of the endoplasmic reticulum of human lung cancer A549 cells.

[0020] The invention relates to the use of the viscosity-responsive TB-pyridine cyano vinyl derivative in the preparation of anticancer drugs.

[0021] The application of the invention in preparing anticancer drugs is aimed at inhibiting human liver cancer HpeG2 cells and human lung cancer A549 cells.

[0022] Beneficial effects:

[0023] 1. The synthesis method is simple and the post-processing is convenient;

[0024] 2. The product has excellent luminescence properties: large Stokes shift, excellent solid-state luminescence and significant AIE properties.

[0025] 3. It has a wide pH range and can be used in human physiological environment;

[0026] 4. It has a good response to viscosity and may become a viscosity-responsive fluorescent probe;

[0027] 5. It can easily enter living A549 cells and has a strong targeting ability to the endoplasmic reticulum (Pearson coefficient Pr is 0.75).

[0028] 6. It has strong inhibitory ability on HpeG2 cells under both dark and light conditions (428nm), indicating that it has the potential to be developed into an anti-liver cancer drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the product derivative 6 in the embodiment 1 HNMR spectrum;

[0030] Figure 2 is the product derivative 6 in the embodiment 13 C NMR spectrum;

[0031] Figure 3a is the UV absorption spectrum of derivative 6 in different solvents;

[0032] Figure 3b is the fluorescence emission spectrum of derivative 6 in different solvents;

[0033] FIG4 is (a) fluorescence emission spectra and (b) line graphs of derivative 6 at different pH values;

[0034] FIG5 is (a) fluorescence emission spectra and (b) line graph of derivative 6 in different ratios of THF / H2O (v / v);

[0035] Figure 6 is the SEM images of derivative 6 at different ratios of DMSO / H2O (v / v): (a) DMSO / H2O = 1 / 9 (v / v) and (b) DMSO / H2O = 1 / 99 (v / v);

[0036] FIG7 is (a) fluorescence emission spectra and (b) line graphs of derivative 6 at different viscosities;

[0037] Figure 8 is the fluorescence intensity of derivative 6 under the interaction with different ions and molecules;

[0038] FIG9 is (a) the fluorescence emission spectrum and (b) the broken line graph of compound 6 at different temperatures; (c) the fluorescence emission spectrum and (d) the broken line graph of compound 6 after binding to egg white;

[0039] Figure 10 It is the co-localization fluorescence imaging of HeLa cells treated with derivative 6 and ER-Tracker Green;

[0040] FIG11 shows the phototoxicity and dark toxicity tests of derivative 6 on (a) A549 cells and (b) HpEG2 cells. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the embodiments.

[0042] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. It will be understood by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0043] The following are: 4-bromoaniline 1, paraformaldehyde 2, first intermediate 3, second intermediate 4, 4-pyridine acetonitrile 5, derivative 6.

[0044] The structural formula of base-pyridine cyano vinyl derivatives is shown in the following table:

[0045]

[0046] Table 1. Structural formula of derivative 6

[0047]

[0048] The present invention also provides the above-mentioned novel The preparation method of base-pyridine cyano vinyl derivatives comprises:

[0049] In this embodiment, the reaction is prepared by coupling reaction using p-bromoaniline, paraformaldehyde, n-butyl lithium, 4-pyridine acetonitrile, etc. as raw materials. The steps include:

[0050] 4-Bromoaniline 1 reacts with paraformaldehyde 2 to obtain a first intermediate 3, the first intermediate 3 reacts with n-butyllithium to obtain a second intermediate 4, and the second intermediate 4 reacts with 4-pyridine acetonitrile 5 through a coupling reaction to obtain a derivative 6.

[0051] The derivative 6 of Example was prepared by the above-mentioned synthesis method:

[0052] 4-Bromoaniline (50.0 mmol) and paraformaldehyde (100.0 mmol) were added sequentially to a 200.0 mL round-bottom flask, which was placed in a low-temperature tank and adjusted to -15°C. Trifluoroacetic acid (100.0 mL, approximately 30 minutes) was slowly added dropwise to the flask with stirring. The mixture was allowed to react at room temperature for 7 days. After the reaction was complete (TLC tracking), the mixture was poured into ice water, adjusted to pH 9-10 with aqueous ammonia, cooled to room temperature, extracted with dichloromethane (50.0 mL x 3), and dried to obtain a crude product. Acetone was added and heated until the crude product was completely dissolved. The product was recrystallized at room temperature, filtered, and washed with acetone to obtain the first intermediate 3.

[0053]

[0054] Synthesis of intermediate 3 of formula 1

[0055] (3) 3 (5.0 mmol) was added to a 100 mL round-bottom flask. After three evacuations, the flask was placed in a low-temperature tank and the temperature was adjusted to -78 ° C. 20.0 mL of anhydrous tetrahydrofuran was added to the flask with stirring, and 2.5 mL of n-butyl lithium was added dropwise. After the reaction was carried out under argon protection for 1 hour, 0.6 mL of DMF was added dropwise, and the flask was allowed to react at room temperature for 4 hours. After TLC tracking until the reaction was complete, the flask was extracted with dichloromethane (30.0 mL × 3) and dried to obtain a crude product. The crude product was purified by column chromatography (V 石油醚 :V 乙酸 Ethyl ester = 5:1) to give the second intermediate 4 (33%).

[0056]

[0057] Synthesis of intermediate 4 from formula 2

[0058] (4) The second intermediate 4 (1.0 mmol), 4-pyridineacetonitrile (1.2 mmol), and 30 mL of anhydrous methanol were added to a 100 mL round-bottom flask in sequence. The reaction mixture was heated to 80°C under argon protection. After TLC was followed until the reaction was complete, water was added to quench the reaction. The organic phase was extracted with dichloromethane (20.0 mL × 3) and dried over anhydrous Na2SO4 and then dried to obtain a crude product. The crude product was purified by column chromatography (V 石油醚 :V 乙酸乙酯 =5:1) to give derivative 6 (77%).

[0059]

[0060] Synthesis of derivative 6 of formula 3

[0061] (Z)-3-(8-Bromo-6H,12H-5,11-methyldibenzo[b,f][1,5]diazocin)-2-(pyridin-4-yl)acrylonitrile(6)

[0062] 1 H NMR (400MHz, DMSO-d6) δ8.84(d,J=5.0Hz,2H),8.43(s,1H),8.04(d,J=5.9Hz,2H),7.93(d,J=8.5Hz,1H,Ar-H),7.68(s,1 H,Ar-H),7.41-7.29(m,2H,Ar-H),7.25-7.11(m,2H),4.71(t,J=15.8Hz,2H,-CH2-bridge),4.42-4.16(m,4H,TB-CH2*2). 13 C NMR(100MHz,DMSO-d6)δ152.8,148.8,147.3,146.5,131.2,130.5,130.3,130 .0,129.4,129.1,128.4,127.5,125.9,121.6,117.4,105.0,66.2,58.4,58.3.

[0063] Optical performance

[0064] The solvation effect of the compounds of the present invention was tested, and the specific test protocol is as follows:

[0065] Derivative 6 was prepared with methanol, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), ethyl acetate, chloroform (CHCl3), toluene, and n-hexane to a concentration of 1×10 -5 mol·L -1 The working solution was prepared and its UV absorption spectrum and fluorescence emission spectrum were tested. Figure 3a-3b shown.

[0066] from Figure 3a-3b As can be seen, as the polarity of the solution increases, the energy difference between the π-π* orbitals decreases, and the UV absorption band red-shifts, due to the greater stabilization of derivative 6 on the π* orbital than on the π orbital. The fluorescence emission wavelength of derivative 6 also red-shifts, and the fluorescence intensity decreases. This is because when derivative 6 is in a highly polar solution, the electrostatic interaction with the solvent increases, stabilizing the excited state of derivative 6 and causing the fluorescence emission wavelength to red-shift, indicating that derivative 6 exhibits an ICT effect.

[0067] The UV absorption and fluorescence emission spectra of derivative 6 in DMSO solution and its solid-state fluorescence emission spectrum were tested. The specific experimental scheme is as follows:

[0068] Weigh 10 -5mol of 4-bromoaniline 1, the second intermediate 4 and the derivative 6 were diluted with DMSO solution to a concentration of 1×10 -5 mol / L, and tested its UV absorption, fluorescence emission and solid-state fluorescence emission spectra.

[0069] The spectral data of 4-bromoaniline 1, the second intermediate 4, and the derivative 6 are shown in Table 2.

[0070] Table 2 Spectral data (DMSO) of 4-bromoaniline 1, second intermediate 4, and derivative 6

[0071]

[0072] a UV absorption wavelength in the solution (slit is 2.5 / 5 nm); b molar extinction coefficient ε = A / bC, unit is 1×10 5 L·mol -1 cm -1 ; c Fluorescence emission wavelength in solution; d Stokes shift in solution; e relative fluorescence quantum yield (reference: quinine sulfate); f fluorescence brightness, unit: L·mol -1 cm -1 ; g solid-state excitation wavelength (slit is 5 / 5 nm); h solid-state fluorescence emission wavelength; i solid-state Stokes shift.

[0073] (1) The solution and solid λem of derivative 6 showed a significant red shift, and the solution and solid Stokes shifts (155 and 102 nm, respectively) increased significantly;

[0074] (2) The relative fluorescence quantum yield of the solution of derivative 6 increased significantly.

[0075] This may be because the introduction of 4-pyridineacetonitrile group into derivative 6 promotes the flow of electrons, reduces the overall energy of the molecule, makes its fluorescence emission easier, has a longer maximum fluorescence emission wavelength, and increases the Stokes shift;

[0076] (3) C=C restricts intramolecular rotation, giving the molecule a highly twisted structure, thereby enhancing the solid-state luminescence intensity.

[0077] The above results show that combining the TB skeleton with the 4-pyridine cyanoethylene fragment can amplify the advantages of both in luminescence performance, and is a new way to obtain products with excellent luminescence performance.

[0078] pH response

[0079] Derivative 6 was prepared with DMSO as solvent at a concentration of 1×10 -4 mol·L -11.0 mL of derivative 6 working solution was measured and placed in nine 10.0 mL volumetric flasks, and then 1.0 mL of buffer solution with a pH value of 2.2-10.0 was added (citric acid / disodium hydrogen phosphate system was selected when the pH value was 2.2-8.0, and sodium bicarbonate / sodium carbonate system was selected when the pH value was 9.0-10.0), and DMSO was used to adjust the volume to a concentration of 1 × 10 -5 mol L -1 , and its fluorescence emission spectrum (λ ex =365nm, slit: 10 / 10nm, Figure 4a-4b ).

[0080] Depend on Figure 4a-4b It can be seen that the fluorescence intensity of derivative 6 remains almost constant in the pH range of 2.2-10.0, indicating that derivative 6 has a wide pH applicability range.

[0081] AIE characteristics

[0082] Since derivative 6 is easily soluble in DMSO but poorly soluble in water, 6 was prepared to a concentration of 1×10 -4 mol·L -1 1.0 mL of working solution was measured and placed in ten 10.0 mL volumetric flasks. 0.0-9.0 mL of double distilled water and DMSO were added to the ten 10.0 mL volumetric flasks to make the concentration 1 × 10 -5 mol·L -1 (DMSO / H2O (v / v) was 1 / 9-9 / 1, respectively), and the derivative 6 was prepared to a concentration of 1×10 -3 mol·L -1 Measure 100.0 μL of the working solution into a 10.0 mL volumetric flask, then add 9.9 mL of double-distilled water and DMSO to the 10.0 mL volumetric flask to make the concentration 1×10 -5 mol L -1 , so that DMSO / H2O=1 / 99 (v / v). The fluorescence emission spectrum was measured as follows Figure 5a-5b As shown (λ ex =365nm, slit: 5 / 10nm).

[0083] Depend on Figure 5a-5bIt can be seen that when the water content is less than 90%, the fluorescence emitted by the system is weak; when the water content reaches 99%, the fluorescence intensity is significantly enhanced. The possible reason is that when the water content is 0-90%, the energy of the excited state of derivative 6 is dissipated through non-radiative transitions, resulting in a weak fluorescence signal. When the volume fraction of the poor solvent water reaches 99%, derivative 6 rapidly aggregates in the poor solvent system, causing the aromatic rings and conjugated double bonds that were originally freely rotatable to become non-rotatable due to aggregation, resulting in strong fluorescence emission. This shows that derivative 6 exhibits typical AIE properties.

[0084] In order to explore the formation mechanism of the AIE performance of derivative 6, we used scanning electron microscopy (SEM) to observe the morphological characteristics of derivative 6 in DMSO / H2O=1 / 9 (v / v) and DMSO / H2O=1 / 99 (v / v). Figure 6a-6b shown.

[0085] Depend on Figure 6a-6b The results show that at a water content of 90%, derivative 6 appears as amorphous particles. However, at a water content of 99%, the first intermediate 3 exhibits regularly shaped, uniformly sized, rice-like aggregates with an average diameter of 3 μm. This indicates that 6 can self-assemble into nanoaggregates at a water content of 99%, with a dramatic increase in fluorescence, demonstrating AIE properties. The formation of stable nanoaggregates may be the reason for its AIE behavior.

[0086] Viscosity response

[0087] The viscosity responsiveness of 6 was tested: the derivative 6 was prepared with methanol as solvent to a concentration of 1×10 - 4 mol·L -1 Take 10 10.0mL volumetric flasks, add 0.0-9.0mL propylene glycol to each, measure 1.0mL of working solution into the volumetric flask, and adjust the volume with methanol to make the concentration of 1×10 -5 mol·L -1 , the fluorescence emission spectrum was measured (the volume ratio of methanol / glycerol was 1 / 9-10 / 0), as shown in Figures 7a-7b As shown (λex=365nm, slit: 5 / 10nm).

[0088] Depend on Figures 7a-7b As can be seen, the fluorescence intensity of derivative 6 increases with increasing viscosity. This is because at low viscosity, the molecular rotor can rotate freely, and this rotational motion consumes exciton energy and increases the nonradiative decay rate, resulting in a weaker fluorescence signal for derivative 6. As the viscosity increases, the free rotation of the molecular rotor is inhibited, weakening the nonradiative transition mode and enhancing its fluorescence. This result suggests that derivative 6 has the potential to be a viscosity-responsive fluorescent probe.

[0089] Interference experiment

[0090] The common cation Fe was examined 3+ 、Al 3+ 、Na + , Ca 2+ 、Cu 2+ Cr 3+ and K + ; anion CO3 2- 、HCO3 - 、CH3COO - PO4 2- 、SO4 2- 、SCN - and HS - Effects of biothiols Cys, Hcy and GSH and 90% glycerol (2-18 from left to right, 1 is a blank control) on the fluorescence emission spectrum of derivative 6, as shown Figure 8 As shown (λ ex =365nm, slit: 10 / 10nm).

[0091] The results show that the fluorescence intensity of derivative 6 remains basically unchanged after the addition of various anions, cations and biothiols, while the fluorescence intensity of derivative 6 in 90% propylene glycol is significantly enhanced, indicating that derivative 6 can achieve specific detection of viscosity in complex biological environments.

[0092] Protein aggregation experiments

[0093] The dense polypeptide chains formed during protein misfolding and aggregation can lead to changes in viscosity

[74] Denaturation of proteins changes their physical and chemical properties, exposing hydrophobic groups within the molecules, accelerating their aggregation and causing them to precipitate from aqueous solutions, resulting in increased viscosity. Therefore, we used the thermal denaturation process of egg white to simulate the viscosity changes during protein aggregation and used derivative 6 to monitor the viscosity changes during denaturation, observing its fluorescence changes.

[0094] Firstly, the fluorescence change of derivative 6 at 0-100℃ was tested ( Figures 9a-9b ), the results showed that the fluorescence intensity of derivative 6 did not change significantly at 0-100℃, indicating that it has good thermal stability (λ ex =365nm, slit: 10 / 10nm).

[0095] When derivative 6 was mixed with an appropriate amount of egg white, as the temperature increased, the protein gradually aggregated and the viscosity increased, and the fluorescence of derivative 6 gradually increased, indicating that it can be used to monitor the viscosity changes during protein aggregation and has the potential to become a viscosity-responsive probe for protein aggregation ( Figures 9c-9d ).

[0096] ER localization experiments

[0097] The ER, a crucial cellular organelle, organically connects the nucleus, cytoplasm, and cell membrane into a single entity. It is responsible for the transport of substances within the cell and serves as a base for the synthesis of proteins and lipids. Since cyano groups are highly lipid-soluble, they could theoretically be localized to the ER. Therefore, a rational co-localization experiment was designed to verify this.

[0098] First, the commercial ER probe ER-Tracker Green and derivative 6 were incubated with HeLa cells under appropriate conditions for 30 minutes, then washed twice with phosphate buffered saline (PBS), and then added with PBS for confocal imaging. The results are shown in Figure 2. Figure 10 shown.

[0099] The results showed that derivative 6 could easily penetrate living HeLa cells and co-localize with ER-Tracker Green with a Pr of 0.75, indicating that it could easily enter HeLa cells and accurately localize in the ER.

[0100] Extracorporeal photodynamic therapy

[0101] Human non-small cell lung cancer (A549) cells and human liver cancer (HepG2) cells were used as models to detect the cytotoxicity of derivative 6 on A549 cells and HepG2 cells using MTT assay. A549 cells and HepG2 cells were seeded in 96 microplates (1×10 -5 / mL), 100μL of culture medium was added to each well, and after culturing in a CO2 incubator at 37°C for 24h, different concentrations of compound 6 were added to the seeded cells and incubated for 24h. The microplate was then rinsed 3 times with PBS buffer solution, and 10μL of MTT solution was added to each well and cultured for another 4h. The culture medium in the wells was removed, and 150μL of DMSO was added to each well to dissolve the blue-purple formazam crystals in the cells. The wells were placed on a shaker and shaken at low speed for 5-7min to fully dissolve the crystalline material. Finally, the absorbance values ​​of each well at 560nm and 670nm were measured using an enzyme-linked immunosorbent assay. Cytotoxicity was calculated using the following formula:

[0102] %viability=[∑(A i / A0×100) / n]

[0103] Where A i are the absorbance values ​​of compounds at different concentrations; A0 is the average absorbance value of the control wells without added compounds; n (=3) means three parallel experiments.

[0104] The dark toxicity and phototoxicity of derivative 6 on HpeG2 cells and A549 cells were detected by MTT assay. The light source was 365 nm. The control group was not exposed to light. The absorbance of each well was measured at 560 nm and 670 nm using an enzyme-linked immunosorbent assay. Figures 11a-11b shown.

[0105] Table 3 Half inhibition rate (IC) of derivative 6 on two cell lines 50 )

[0106]

[0107] The half inhibition rates of derivative 6 on HpeG2 and A549 cells are shown in Table 3. The results showed that the IC 50 76.5 and 11.2 μmol·L, respectively -1 Under light conditions (428 nm), the inhibition rates on HpeG2 cells and A549 cells were 36.2 and 5.5 μmol·L -1 , indicating that it has excellent PDT effect. Moreover, its inhibitory ability against HpeG2 cells is strong under both dark and light conditions (428nm), indicating that it has the potential to be developed into an anti-liver cancer drug.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A TB-pyridine cyano vinyl derivative with viscosity response, characterized in that: The structural formula of the derivative is:

2. A method for synthesizing the viscosity-responsive TB-pyridine cyano vinyl derivative according to claim 1, characterized in that: The following steps are involved: Step 1: 4-bromoaniline (1) reacts with paraformaldehyde (2) to obtain a first intermediate (3). The reaction formula is as follows: Step 2: The first intermediate (3) reacts with n-butyl lithium to obtain the second intermediate (4). The reaction formula is as follows: Step 3: The second intermediate (4) and 4-pyridine acetonitrile (5) are coupled to obtain a derivative (6). The reaction formula is as follows:

3. Use of the TB-pyridine cyanovinyl derivative with viscosity response as claimed in claim 1 in the preparation of a viscosity probe.

4. Use of the viscosity-responsive TB-pyridine cyanovinyl derivative according to claim 1 in the preparation of an endoplasmic reticulum targeting probe.

5. The use according to claim 4, characterized in that The application of the endoplasmic reticulum targeting probe is aimed at the localization of the endoplasmic reticulum of human lung cancer A549 cells.

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