An aie molecule responsive to unfolded protein and synthesis method and application thereof

Abstract

The application discloses an AIE molecule responding to unfolded proteins and a synthesis method and application thereof. The AIE molecule responding to unfolded proteins can target the endoplasmic reticulum of cells, and can react with sulfydryl in unfolded proteins to form a'sandwich' structure, so that RIM is enhanced, then fluorescence is lightened, and fluorescent surgical navigation of various cancers is realized.

Description

Technical Field

[0001] This invention relates to the technical field of AIE molecules, specifically to an AIE molecule that responds to unfolded proteins, its synthesis method, and its applications. Background Technology

[0002] Aggregation-induced emission (AIE) molecules exhibit no or weak fluorescence in well-dispersible solvents, but emit strong fluorescence in their aggregated state, offering new opportunities for fluorescence imaging and related biomedical applications. Restriction of movement within molecules (RIM) is currently the most widely accepted mechanism of AIE luminescence; the stronger the RIM, the stronger the luminescence, thus it can also be used to design fluorescent probes for activation imaging. Protein synthesis is an essential process for cell proliferation and replication, and it is even more vigorous in tumor cells. Tumors with higher proliferative activity often exhibit more vigorous protein synthesis, demonstrating its universality. Unfolded proteins are processed into folded proteins in the endoplasmic reticulum; the content of the former is a better indicator of the level of protein synthesis, making its detection significant.

[0003] With the advent of the precision surgery concept, intraoperative navigation technology has been gradually applied in clinical practice, providing surgical procedures with a "map" for guidance and eliminating "blind cutting." Fluorescence-guided surgery using fluorescence intraoperative navigation technology allows fluorescent molecular probes to illuminate cancer cells in real time during surgery, breaking through the precision limits of traditional surgical treatments. However, most current fluorescent probes respond to specific proteins, enzymes, or other conditions for imaging. While they are highly specific, they lack versatility, thus increasing probe development costs and reducing utilization. Tumors typically exhibit high proliferative activity, vigorous protein synthesis, and a high content of unfolded proteins. Designing a detection probe targeting unfolded proteins could solve the problem of poor versatility in surgical probes. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the aforementioned background technology and provide an AIE molecule that responds to unfolded proteins, its synthesis method, and its applications. The AIE molecule of this invention, which responds to unfolded proteins, can target the endoplasmic reticulum (ER) of cells and react with the sulfhydryl groups in unfolded proteins to form a "sandwich" structure, thereby enhancing RIM and illuminating fluorescence, enabling fluorescent surgical navigation for various cancers.

[0005] To achieve the above objectives, the present invention provides an AIE molecule that responds to unfolded proteins, the structural formula of which is as follows:

[0006]

[0007] This invention also provides a method for synthesizing the above-mentioned AIE molecule that responds to unfolded proteins, the synthetic route of which is as follows:

[0008]

[0009] Furthermore, the method for synthesizing the AIE molecule that responds to unfolded proteins includes the following steps:

[0010] 1) Synthesis of intermediate product 1:

[0011] Weigh Zn powder, 4-aminobenzophenone, and 4-bromobenzophenone into a double-necked flask, replace with nitrogen, and add ultra-dry tetrahydrofuran. Slowly add TiCl4 dropwise at 0°C, then restore to room temperature. The mixture is then refluxed overnight at 90-100°C. After the reaction is complete, quench the reaction with saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, and dry with anhydrous magnesium sulfate. Remove the solvent under reduced pressure using a rotary evaporator to obtain the crude product. Purify the crude product by column chromatography using petroleum ether and ethyl acetate as eluents to obtain a deep yellow oily substance, which is intermediate 1.

[0012] 2) Synthesis of intermediate product 2:

[0013] Intermediate 1 and THF were added to a two-necked flask, followed by Boc anhydride and Et3N. The mixture was stirred under reflux at 90-100°C for 3-4 hours. After the reaction was complete, hydrochloric acid was added. The aqueous phase was extracted with dichloromethane, and the organic phases were combined and extracted with dichloromethane again. The extract was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents to obtain a yellow solid, which was intermediate 2.

[0014] 3) Synthesis of intermediate product 3:

[0015] Intermediate 2, bis(pinacol)diboron, Pd(dppf)Cl2, and KOAc were added to a two-necked flask. After injecting dioxane, the system was degassed using a freeze-pump-thaw cycle, and the mixture was stirred overnight under reflux at 105-110°C. After the reaction was complete, dioxane was removed by rotary evaporation under reduced pressure, and the mixture was redissolved in dichloromethane. Hydrochloric acid was then added, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and extracted with dichloromethane again. The extract was washed with saturated brine and dried with anhydrous magnesium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents to obtain a yellow powder, which was intermediate 3.

[0016] 4) Synthesis of intermediate product 4:

[0017] Intermediate 3,4,7-dibromobenzo[c]-1,2,5-thiadiazole, Pd(PPh3)4, and K2CO3 were added to a two-necked flask. A reflux condenser was installed and sealed in the system, and then a THF / H2O mixture was injected. The system was degassed using a freeze-pump-thaw cycle, and the mixture was stirred overnight under reflux at 90-100°C. After the reaction was complete, THF was removed by rotary evaporation under reduced pressure, and the mixture was redissolved in dichloromethane. Hydrochloric acid was then added, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and extracted with dichloromethane again. The extract was washed with saturated brine and dried with anhydrous magnesium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents to obtain an orange solid, which was intermediate 4.

[0018] 5) Synthesis of intermediate product 5:

[0019] Intermediate 4, 4-(methoxycarbonyl)phenylboronic acid, Pd(PPh3)4, and K2CO3 were added to a two-necked flask, followed by the injection of a THF / H2O mixture. The system was degassed using a freeze-evacuation-thawing cycle, and the mixture was stirred overnight under reflux at 90-100°C. After the reaction was complete, THF was removed under reduced pressure using a rotary evaporator, and the mixture was redissolved in dichloromethane. Hydrochloric acid was then added, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and extracted again with dichloromethane. The extract was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure using a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents, yielding an orange solid, which was intermediate 5.

[0020] 6) Synthesis of intermediate product 6:

[0021] Weigh intermediate product 5 and add it to a THF / MeOH mixture. Add NaOH aqueous solution while continuously stirring. Reflux the mixture at 80-90℃ for 1-2 hours. After the reaction is complete, remove the organic solvent using a rotary evaporator under reduced pressure. Then add hydrochloric acid and extract the aqueous phase with dichloromethane. Combine the organic phases and continue extraction with dichloromethane. Wash the extract with saturated brine and dry with anhydrous magnesium sulfate. Remove the solvent using a rotary evaporator under reduced pressure to obtain the crude product. Purify the crude product by column chromatography using methanol, acetic acid, and dichloromethane as eluents. The final orange solid is intermediate product 6.

[0022] 7) Synthesis of intermediate product 7:

[0023] Add intermediate product 6 and 10 mL of TFA / DCM mixture to a two-necked flask, stir the solution for 1-2 hours, and then concentrate it under vacuum under nitrogen protection. The resulting yellow solid can be used directly without separation or purification. Add pure acetic acid and maleic anhydride to the solid, and stir the mixture overnight at 100-110°C. After cooling to room temperature, add toluene, and remove the solvent under reduced pressure using a rotary evaporator to obtain the crude product. Purify the crude product by column chromatography using acetic acid and dichloromethane as eluents to obtain the yellow solid product, which is intermediate product 7.

[0024] 8) Synthesis of MI-AIE-TsG:

[0025] The synthesized intermediate 7 was dissolved in dry DMF, and then HBTU was added directly to the solution. After standing, N-(2-aminoethyl)-4-methylbenzenesulfonamide was added to the mixture, and DIEA was added dropwise to adjust the pH of the solution to 8-8.5. Then, stirring was continued for 8-10 hours. After the reaction was completed, the solvent was air-dried, the remaining oily product was dissolved in methanol, and purified by high performance liquid chromatography. The product was lyophilized to obtain a yellow powder, which is MI-AIE-TsG.

[0026] The structural formula of intermediate product 1 is as follows:

[0027]

[0028] The structural formula of intermediate product 2 is:

[0029]

[0030] The structural formula of the intermediate product 3 is:

[0031]

[0032] The structural formula of intermediate product 4 is:

[0033]

[0034] The structural formula of the intermediate product 5 is as follows:

[0035]

[0036] The structural formula of the intermediate product 6 is:

[0037]

[0038] The structural formula of the intermediate product 7 is:

[0039]

[0040] The structural formula of the compound MI-AIE-TsG is:

[0041]

[0042] Further, in step 1), the molar ratio of Zn powder, 4-aminobenzophenone, 4-bromobenzophenone and TiCl4 is (4-6):1:1:(4-6); the eluent is petroleum ether:ethyl acetate = (5-8):1;

[0043] In step 2), the molar volume ratio of intermediate product 1 to THF is 1 mmol / (3.8-4) mL; the molar ratio of intermediate product 1, Boc anhydride and Et3N is 1:(3-5):(5-8); the eluent is petroleum ether:ethyl acetate = (20-22):1.

[0044] Further, in step 3), the molar ratio of intermediate product 2, bis(pinacol)diboron, Pd(dppf)Cl2 and KOAc is (2.1-3):(2.5-3):0.1:(8.4-9); the eluent is petroleum ether:ethyl acetate = (20-22):1;

[0045] In step 4), the molar ratio of intermediate products 3,4,7-dibromobenzo[c]-1,2,5-thiadiazole, Pd(PPh3)4, and K2CO3 is (1-1.1):(3.4-4):(0.11-0.2):(11.3-12).

[0046] The volume ratio of THF to H2O in the THF / H2O mixture is (8-10):1;

[0047] The eluent was petroleum ether:ethyl acetate = (20-22):1.

[0048] Further, in step 5), the molar ratio of intermediate product 4, 4-(methoxycarbonyl)phenylboronic acid, Pd(PPh3)4 to K2CO3 is (0.93-1):(1.9-2):(0.09-0.1):(9.3-10);

[0049] The volume ratio of THF to H2O in the THF / H2O mixture is (4-6):1;

[0050] The eluent was initially petroleum ether:ethyl acetate = (20-22):1, and then changed to petroleum ether:ethyl acetate = (10-12):1.

[0051] Further, in step 6), the molar volume ratio of intermediate product 5 to the THF / MeOH mixture is (0.4-0.6) mmol / 20mL;

[0052] The volume ratio of the THF / MeOH mixture is 1:(1-2);

[0053] The eluent is methanol:acetic acid:dichloromethane = 1:1:(100-110).

[0054] Further, in step 7), the molar volume ratio of intermediate product 6 and the TFA / DCM mixture is (0.32-0.4) mmol / 10mL;

[0055] The molar ratio of intermediate product 6 to maleic anhydride is 1:(5-8);

[0056] The volume ratio of TFA to DCM in the TFA / DCM mixture is 1:(1-2);

[0057] The eluent is acetic acid: dichloromethane = 1:(100-120).

[0058] Further, in step 8), the molar ratio of intermediate product 7, HBTU, and N-(2-aminoethyl)-4-methylbenzenesulfonamide is 0.1:(0.12-0.2):(0.12-0.2).

[0059] The present invention also provides an application of the above-mentioned AIE molecule that responds to unfolded proteins in a tumor surgical navigation tool.

[0060] Compared with the prior art, the present invention has the following advantages:

[0061] The AIE molecule designed in this invention, which responds to unfolded proteins, has two targeting ends: one end targets and binds to the endoplasmic reticulum (SUR1 protein), while the other end reacts with the thiol group of the unfolded protein, thus forming a "sandwich" structure. The more significantly the intramolecular movement of the AIE molecule responding to unfolded proteins is restricted, the stronger its luminescence becomes. This allows it to be used to distinguish between tumor cells and normal cells, regardless of tumor type, providing a tool for surgical navigation of various tumors. Attached Figure Description

[0062] Figure 1 High-resolution mass spectrum of intermediate product 2 in Example 1;

[0063] Figure 2 High-resolution mass spectrum of intermediate product 3 in Example 1;

[0064] Figure 3 High-resolution mass spectrum of intermediate product 4 in Example 1;

[0065] Figure 4 High-resolution mass spectrum of intermediate product 5 in Example 1;

[0066] Figure 5High-resolution mass spectrum of intermediate product 6 in Example 1;

[0067] Figure 6 High-resolution mass spectrum of intermediate product 7 in Example 1;

[0068] Figure 7 Example 1: High-resolution mass spectrum of small molecule MI-AIE-TsG;

[0069] Figure 8 Example 1: Small molecule MI-AIE-TsG in DMSO-d6 1 H NMR spectrum;

[0070] Figure 9 Example 1: Small molecule MI-AIE-TsG in DMSO-d6 13 C NMR spectrum;

[0071] Figure 10 The image shows the UV absorption spectrum of the small molecule MAT in a 1% DMSO aqueous solution, as shown in Example 1.

[0072] Figure 11 The fluorescence emission spectrum of small molecule MAT in PBS in Example 1 is shown.

[0073] Figure 12 The fluorescence emission spectra of small molecule MAT in Example 1, co-incubated in folded BSA, unfolded BSA, and reduced unfolded BSA, respectively.

[0074] Figure 13 Fluorescence emission spectra of reduced unfolded BSA with different incubation sequences for MAT or NEM.

[0075] Figure 14 The image shows the binding of small molecule MAT to reduced unfolded BSA protein in Example 1, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0076] Figure 15 The fluorescence emission spectra of small molecule MAT in Example 1 after incubation with cysteine ​​and reduced unfolded BSA, respectively;

[0077] Figure 16 Here are the fluorescence emission spectra of the small molecule MAT reacting with different proteins in Example 1;

[0078] Figure 17 This is a graph showing the anisotropy of small molecule MAT as a function of SUR1 protein concentration in Example 1.

[0079] Figure 18Confocal images and quantitative analysis diagrams of A549, HeLa, and HESC cells after incubation with the small molecule MAT (10 μM) from Example 1 for 4 hours; among them Figure 18 A is a confocal image of A549, HeLa and HESC cells after incubation with MAT (10 μM) for 4 hours; Figure 18 B is a quantitative analysis diagram of A549, HeLa and HESC cells after incubation with MAT (10 μM) for 4 hours;

[0080] Figure 19 Confocal images of HeLa and HLF cells co-cultured with the small molecule MAT (10 μM) from Example 1 incubated for 4 hours;

[0081] Figure 20 Confocal images of HeLa and HESC cells co-cultured with small molecule MAT (10 μM) from Example 1 incubated for 4 hours;

[0082] Figure 21 This is a comparison of the photostability of small molecule MAT and a commercial endoplasmic reticulum fluorescent probe in living cells, as shown in Example 1. Figure 21 A is a confocal image of MAT and a commercial endoplasmic reticulum fluorescent probe in living cells after being irradiated with strong light; Figure 21 B is a quantitative analysis diagram of the fluorescence intensity of MAT and commercial endoplasmic reticulum fluorescent probes in live cells after irradiation with strong light;

[0083] Figure 22 This is a fluorescence imaging image of small molecule MAT used for surgical navigation in animals, as shown in Example 1; where... Figure 22 A represents the HeLa cervical cancer lymph node metastasis model; Figure 22 B is the SKOV3 model of ovarian cancer peritoneal metastasis; Figure 22 C represents a 4T1 breast cancer in situ model. Figure 22 D represents the intraoperative and postoperative in situ model of 4T1 breast cancer.

[0084] Figure 23 HE staining image of SKOV3 tumors in mice with SKOV3 ovarian cancer peritoneal metastasis model.

[0085] Figure 24 HE staining images of the lungs, liver, heart, kidneys, and spleen after subcutaneous injection of PBS and the small molecule MAT solution from Example 1 in SKOV3 mice with peritoneal metastasis of ovarian cancer.

[0086] Figure 25 Analysis of liver function indices ALT and AST, and kidney function indices ALT and AST in SKOV3 mice with peritoneal metastasis of ovarian cancer after different treatments. Detailed Implementation

[0087] The following examples illustrate the implementation of the present invention in detail, but they do not constitute a limitation on the invention and are merely illustrative. Furthermore, the advantages of the present invention will become clearer and easier to understand by explaining them.

[0088] Example 1:

[0089] The present invention provides a method for synthesizing an AIE fluorescent probe, comprising the following steps:

[0090] 1) Synthesis of intermediate product 1:

[0091] Weigh 5.3 g (81.1 mmol) of Zn powder, 4-aminobenzophenone (4.0 g, 20.3 mmol) and 4-bromobenzophenone (5.3 g, 20.3 mmol) into a 250 mL double-necked flask, purge with nitrogen three times, and add 100 mL of ultra-dry tetrahydrofuran. Slowly add 15.4 g (81.1 mmol) of TiCl4 dropwise at 0 °C, then allow to return to room temperature, followed by reflux at 90 °C overnight. After the reaction is complete, quench the reaction with 50 mL of saturated sodium bicarbonate aqueous solution, extract three times with 150 mL of ethyl acetate, and dry with anhydrous magnesium sulfate. Remove the solvent under reduced pressure using a rotary evaporator to obtain the crude product. Purify the crude product by column chromatography with petroleum ether:ethyl acetate = 5:1 as the eluent, yielding a deep yellow oily substance, which is intermediate 1.

[0092] 2) Synthesis of intermediate product 2:

[0093] In a 100 mL two-necked flask, intermediate 1 (2.2 g, 5.2 mmol) and THF (20 mL) were added, followed by Boc anhydride (3.4 g, 15.6 mmol) and Et3N (2.6 g, 26.0 mmol). The mixture was stirred under reflux at 90 °C for 3 hours. After the reaction was complete, an appropriate amount of hydrochloric acid (1 M) was added. The aqueous phase was extracted three times with dichloromethane, and the organic phases were combined and extracted three more times with dichloromethane. The extracts were washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography with petroleum ether:ethyl acetate as the eluent in a ratio of 20:1, yielding a yellow solid, which was intermediate 2.

[0094] 3) Synthesis of intermediate product 3:

[0095] In a 100 mL two-necked flask, intermediate 2 (1.1 g, 2.1 mmol), bis(pinacol)diboron (637.4 mg, 2.5 mmol), Pd(dppf)Cl2 (73.2 mg, 0.1 mmol), and KOAc (820.5 mg, 8.4 mmol) were added. Dioxane (20 mL) was then injected, and the system was degassed using a three-cycle freeze-pump-thaw cycle. The mixture was stirred overnight under reflux at 105 °C. After the reaction was complete, dioxane was removed under reduced pressure using a rotary evaporator, and the mixture was redissolved in dichloromethane. A suitable amount of hydrochloric acid (1 M) was then added, and the aqueous phase was extracted three times with dichloromethane. The organic phases were combined and extracted three more times with dichloromethane. The extracts were washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure using a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography with petroleum ether:ethyl acetate = 20:1 as the eluent, and the resulting yellow powder was intermediate product 3.

[0096] 4) Synthesis of intermediate product 4:

[0097] In a 500 mL two-necked flask, intermediate 3 (650 mg, 1.1 mmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (1.0 g, 3.4 mmol), Pd(PPh3)4 (130.6 mg, 0.11 mmol), and K2CO3 (1.6 g, 11.3 mmol) were added. A reflux condenser was installed and sealed in the system, and then 180 mL of a THF / H2O mixture (8:1) was added. The system was degassed using three freeze-pump-thaw cycles, and the mixture was stirred overnight under reflux at 90 °C. After the reaction was complete, THF was removed under reduced pressure using a rotary evaporator, and the mixture was redissolved in dichloromethane. A suitable amount of hydrochloric acid (1 M) was then added, and the aqueous phase was extracted three times with dichloromethane. The organic phases were combined and extracted three more times with dichloromethane. The extracts were washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure using a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography with petroleum ether:ethyl acetate = 20:1 as the eluent, and the orange solid obtained was intermediate product 4.

[0098] 5) Synthesis of intermediate product 5:

[0099] In a 100 mL two-necked flask, intermediate 4 (612 mg, 0.93 mmol), 4-(methoxycarbonyl)phenylboronic acid (333 mg, 1.9 mmol), Pd(PPh3)4 (107.5 mg, 0.09 mmol), and K2CO3 (1.3 g, 9.3 mmol) were added, followed by the addition of 40 mL of a THF / H2O mixture (4:1, V:V). The system was degassed using a three-cycle freeze-evaporation-thawing process, and the mixture was stirred overnight under reflux at 90 °C. After the reaction was complete, THF was removed under reduced pressure using a rotary evaporator, and the mixture was redissolved in dichloromethane. A suitable amount of hydrochloric acid (1 M) was then added, and the aqueous phase was extracted three times with dichloromethane. The organic phases were combined and extracted three more times with dichloromethane. The extracts were washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure using a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography. The eluent was initially petroleum ether:ethyl acetate = 20:1, and then changed to petroleum ether:ethyl acetate = 10:1. The final orange solid was intermediate product 5.

[0100] 6) Synthesis of intermediate product 6:

[0101] 290 mg (0.4 mmol) of intermediate product 5 was weighed and injected into 20 mL of THF / MeOH (1:1, V / V). 3 mL of NaOH aqueous solution (0.67 M) was added while stirring continuously. The mixture was refluxed and stirred at 80 °C for 1 hour. After the reaction was complete, the organic solvent was removed under reduced pressure using a rotary evaporator. Then, an appropriate amount of hydrochloric acid (1 M) was added, and the aqueous phase was extracted three times with dichloromethane. The organic phases were combined and extracted three more times with dichloromethane. The extract was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure using a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography using methanol:acetic acid:dichloromethane = 1:1:100 as the eluent, finally yielding an orange solid, which was intermediate product 6.

[0102] 7) Synthesis of intermediate product 7:

[0103] Intermediate product 6 (224 mg, 0.32 mmol) and 10 mL of a TFA / DCM mixture (1:1, v / v) were added to a 100 mL two-necked flask. The solution was stirred for 1 hour, and then concentrated under vacuum under nitrogen protection. The resulting yellow solid was used directly without separation or purification. 20 mL of pure acetic acid and maleic anhydride (156 mg, 1.6 mmol) were added to the solid, and the mixture was stirred overnight at 100 °C. After cooling to room temperature, toluene was added, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography with acetic acid:dichloromethane as the eluent at a ratio of 1:100, yielding a yellow solid product, which was intermediate product 7 (373 mg, yield = 54.9%).

[0104] 8) Synthesis of small molecule MI-AIE-TsG:

[0105] The synthesized intermediate 7 (0.68 g, 0.1 mmol) was dissolved in 4 mL of dry DMF, and then 0.12 mmol of HBTU was added directly to the solution. After standing for 30 minutes, 0.12 mmol of N-(2-aminoethyl)-4-methylbenzenesulfonamide (0.26 g) was added to the mixture, and DIEA was added dropwise to adjust the pH of the solution to approximately 8. The mixture was then stirred for 8 hours. After the reaction was complete, the solvent was air-dried, and the remaining oily product was dissolved in methanol and purified by high performance liquid chromatography. The product was then lyophilized to obtain 0.67 g of yellow powder, which was MI-AIE-TsG (yield = 76.3%).

[0106] The molecular structural formulas of the above compounds are shown in Table 1:

[0107] Table 1

[0108]

[0109]

[0110] To demonstrate the progress of the reaction, the following characterization was specifically performed:

[0111] like Figure 1 As shown, the high-resolution mass spectrometry characterization of intermediate product 2 in Example 1 is as follows: HRMS(ESI) m / z [C 31 H 28 BrNO2] + The calculated value is 525.1303, and the found value is 525.1294.

[0112] like Figure 2 As shown, the high-resolution mass spectrometry characterization of intermediate product 3 in Example 1 is as follows: HRMS(ESI) m / z [C 37 H 40 BNO4] + The calculated value is 573.3050, and the found value is 573.3040.

[0113] like Figure 3 As shown, the high-resolution mass spectrometry characterization of intermediate product 4 in Example 1 is as follows: HRMS(ESI) m / z [C 37 H 30 BrN3O2S] + The calculated value is 659.1242, and the found value is 659.1235.

[0114] like Figure 4 As shown, the high-resolution mass spectrometry characterization of intermediate product 5 in Example 1 is as follows: HRMS(ESI) m / z [C45 H 37 N3O4S] + The calculated value is 715.2505, and the found value is 716.2557.

[0115] like Figure 5 As shown, the high-resolution mass spectrometry characterization of intermediate product 6 in Example 1 is as follows: HRMS(ESI) m / z [C 44 H 35 N3O4S] + The calculated value is 701.2348, and the found value is 701.2345.

[0116] like Figure 6 As shown, the high-resolution mass spectrometry characterization of intermediate product 7 in Example 1 is as follows: HRMS(ESI) m / z [C 43 H 27 N3O4SH] + The theoretical value is 681.1722, and the observed value is 682.1797.

[0117] like Figure 7 As shown, the high-resolution mass spectrometry characterization of the MI-AIE-TsG synthesized in Example 1 is as follows: HRMS(ESI) m / z [C 53 H 47 N5O5S2H] + The calculated value is 877.2393, and the found value is 878.2459.

[0118] like Figure 8 As shown, the 1H NMR characterization of the MI-AIE-TsG synthesized in Example 1 is as follows: 1 H NMR (400MHz, DMSO-d6) δ8.57 (t, J=5.7Hz, 1H), 8.10 (d, J=8.1Hz, 2H), 8.05-7.83 (m, 5H), 7.83 (s, 1H), 7.70 (t, J=6.3Hz, 3H), 7.3 8 (d, J=7.9Hz, 2H), 7.28-7.08 (m, 16H), 7.05 (dd, J=12.3, 7.4Hz, 2H), 3.35 (q, J=6.5Hz, 2H), 2.93 (q, J=6.6Hz, 2H), 2.36 (s, 3H).

[0119] like Figure 9 As shown, the carbon NMR characterization of the MI-AIE-TsG synthesized in Example 1 is as follows: 13C NMR (101MHz, DMSO-d6) δ170.23, 166.41, 162.74, 153.75, 153.66, 143.73, 143.49, 14 3.44, 143.40, 143.08, 142.79, 141.09, 140.74, 139.83, 138.04, 135.22, 135.09, 134. 26, 132.57, 131.65, 131.64, 131.50, 131.45, 131.35, 131.22, 131.18, 130.27, 130.10, 129.33, 129.18, 128.96, 128.55, 128.38, 127.87, 127.34, 126.97, 126.31, 126.18.

[0120] Test example:

[0121] 1. Photophysical properties of MAT molecules

[0122] The photophysical properties of the MAT molecule synthesized in Example 1 were characterized.

[0123] Figure 10 The image shows the UV absorption spectrum of MAT molecules in a 1% DMSO aqueous solution.

[0124] The photophysical properties of the MAT molecule were investigated using UV-Vis absorption spectroscopy. We first evaluated the absorption characteristics of this molecule in a 1% DMSO aqueous solution. Figure 11 As shown, MAT has two ultraviolet characteristic peaks at approximately 320 nm and 420 nm.

[0125] Figure 11 The image shows the fluorescence emission spectrum of MAT molecules in PBS.

[0126] The fluorescence spectrum of MAT in PBS was studied using fluorescence spectroscopy. Figure 12 As shown, the highest emission peak of MAT is at 560 nm.

[0127] 2. Properties of MAT binding to thiol groups

[0128] Experiments were conducted to investigate the thiol binding properties of the MAT molecule synthesized in Example 1.

[0129] Figure 12 The fluorescence emission spectra of MAT co-incubated in folded BSA, unfolded BSA, and reduced unfolded BSA, respectively.

[0130] MAT was incubated with folded BSA, unfolded BSA, and reduced unfolded BSA for 4 hours using a fluorescence spectrometer. The fluorescence intensity of the mixed solution was then measured. Figure 12 It can be seen that, in terms of the magnitude of fluorescence enhancement, MAT has a smaller response to folded BSA, a larger response to unfolded BSA, and the largest response to reduced unfolded BSA.

[0131] Figure 13 Fluorescence emission spectra of reduced unfolded BSA with different incubation sequences for MAT or NEM.

[0132] Fluorescence emission spectra of MAT / NEM (10 μM) and unfolded BSA (1 μM) with different incubation sequences were detected using a fluorescence spectrometer. To further elucidate the luminescence mechanism of MAT, N-acetylacetamide (NEM) was introduced to competitively bind to the thiol groups in the protein. Figure 13 As shown, when MAT is first added to reduced unfolded BSA and reacted for 4 hours, followed by the addition of NEM and a further 4 hours of reaction, the fluorescence of the solution is significantly enhanced. This indicates that most of the MAT reacts first with the thiol groups of reduced unfolded BSA, leaving almost no active thiol groups in the reduced unfolded BSA to react with NEM. However, when MAT and NEM are added to reduced unfolded BSA simultaneously and reacted for 4 hours, the fluorescence of the solution is somewhat enhanced, indicating that MAT and NEM competitively react with the thiol groups in reduced unfolded BSA, with only a portion of MAT reacting with these groups. When NEM is first added to reduced unfolded BSA and reacted for 4 hours, followed by the addition of MAT and a further 4 hours of reaction, the fluorescence intensity of the solution remains almost unchanged. This indicates that most of the NEM reacts first with the thiol groups of reduced unfolded BSA, leaving almost no active thiol groups in the reduced unfolded BSA to react with MAT, thus resulting in almost no change in the fluorescence intensity of MAT. In summary, MAT exhibits significant specificity for thiol groups in proteins.

[0133] Figure 14 Image of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showing the binding of MAT to reduced unfolded BSA protein.

[0134] To more directly observe the binding of MAT to reduced unfolded BSA protein, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used in the experiment. Figure 14 As shown, the molecular weight of the unfolded BSA decreased to between 53 and 70 kDa, which is consistent with the actual situation. When the reduced unfolded BSA binds to MAT, the molecular weight increases slightly. At the same time, fluorescence imaging can reveal a bright band between 53 and 70 kDa, which is the complex of MAT and reduced unfolded BSA. These results fully demonstrate the response of MAT to thiol groups.

[0135] Figure 15The fluorescence emission spectra are those of MAT incubated with cysteine ​​and reduced unfolded BSA, respectively.

[0136] The fluorescence emission spectra of MAT (10 μM) after incubation with cysteine ​​(35 μM) and folded-reduced BSA (1 μM) were detected using a fluorescence spectrometer. Figure 15 As shown, the binding of MAT to reduced unfolded BSA significantly improved the RIM of TPE molecules, thereby greatly increasing the fluorescence emission intensity, which fully demonstrates that MAT is responsive to thiol groups.

[0137] 3. Endoplasmic reticulum binding capacity of MAT molecules

[0138] An experiment was conducted to investigate the endoplasmic reticulum binding ability of the MAT molecule synthesized in Example 1.

[0139] Figure 16 The fluorescence emission spectra of MAT molecules reacting with different proteins are shown.

[0140] MAT molecules react with a variety of proteins, including thrombin, casein, trypsin, BSA, and SUR1 protein. For example... Figure 16 As shown, the MAT molecule exhibits a more significant increase in fluorescence intensity when interacting with the SUR1 protein compared to other proteins.

[0141] Figure 17 The graph shows the anisotropy of MAT molecules as a function of SUR1 protein concentration.

[0142] The affinity of MAT for the SUR1 protein was determined using fluorescence anisotropy assays. Figure 17 As shown, the dissociation constant (KD) of MAT is in the nanoscale range (8.64 nM) of the SUR1 protein, indicating that it has a high affinity for the SUR1 protein.

[0143] 4. MAT's responsiveness to intracellular unfolded proteins

[0144] An experiment was conducted to investigate the responsiveness of the MAT molecule synthesized in Example 1 to intracellular unfolded proteins.

[0145] Figure 18 Confocal images and quantitative analysis diagrams of A549, HeLa and HESC cells after incubation with MAT (10 μM) for 4 hours.

[0146] Figure 18 A is a confocal image of A549, HeLa and HESC cells after incubation with MAT (10 μM) for 4 hours;

[0147] Figure 18B is a quantitative analysis diagram of A549, HeLa and HESC cells after incubation with MAT (10 μM) for 4 hours.

[0148] A549, HeLa, and HESC cells were incubated with MAT (10 μM) for 4 hours, and then intracellular fluorescence staining was observed using CLSM. Figure 18 As shown, the fluorescence intensity of HESC is significantly weaker than that of HeLa and A549. Compared with normal cells (human endometrial stromal cells, HESC), tumor cells, including cervical cancer cells HeLa and lung cancer cells A549, contain more sulfhydryl groups. Therefore, MAT molecules can effectively distinguish cancer cells from normal cells based on unfolded proteins.

[0149] 5. MAT can target and induce apoptosis in cancer cells.

[0150] Experiments were conducted to investigate the ability of the MAT molecule synthesized in Example 1 to target and induce apoptosis in cancer cells.

[0151] Figure 19 Confocal images of co-cultured HeLa and HLF cells incubated with MAT (10 μM) for 4 hours.

[0152] After co-culturing HeLa and HLF cells and incubating them with MAT molecules (10 μM) for 4 hours, the intracellular fluorescence staining was observed using CLSM.

[0153] like Figure 19 As shown, there is a significant difference in MAT fluorescence between HLF cells and HeLa cells, with a fluorescence intensity difference of 2 times, and the fluorescence intensity is greater in HeLa cells.

[0154] Figure 20 Confocal images of co-cultured HeLa and HESC cells incubated with MAT (10 μM) for 4 hours.

[0155] After co-culturing HeLa and HESC cells and incubating them with MAT molecules (10 μM) for 4 hours, the intracellular fluorescence staining was observed using CLSM.

[0156] like Figure 20 As shown, there is a significant difference in MAT fluorescence between HESC cells and HeLa cells, with a fluorescence intensity difference of 2 times, and the fluorescence intensity is greater in HeLa cells.

[0157] Figure 21 A comparison of the photostability of MAT and commercial endoplasmic reticulum fluorescent probes in live cells.

[0158] Figure 21 A is a confocal image of MAT and a commercial endoplasmic reticulum fluorescent probe in living cells after being irradiated with strong light;

[0159] Figure 21 B is a quantitative analysis diagram of the fluorescence intensity of MAT and commercial endoplasmic reticulum fluorescent probes in living cells after irradiation with strong light.

[0160] After incubating HeLa cells with MAT and the commercial endoplasmic reticulum fluorescent probe ER-Tracker Green for 4 hours, the cells were then irradiated with laser. Figure 21 As shown, after 70 seconds, the fluorescence brightness of MAT remained above 70%, while ER-TrackerGreen decreased to below 10%, demonstrating MAT's excellent anti-photobleaching performance.

[0161] 6. MAT is used for precise surgical navigation and biosafety analysis.

[0162] The MAT molecule synthesized in Example 1 was used for surgical navigation in vivo.

[0163] Figure 22 Fluorescence imaging of MAT molecules used for surgical navigation in animals.

[0164] Figure 22 A represents the HeLa cervical cancer lymph node metastasis model;

[0165] Figure 22 B is the SKOV3 model of ovarian cancer peritoneal metastasis;

[0166] Figure 22 C represents a 4T1 breast cancer in situ model.

[0167] Figure 22 D represents the in situ model of 4T1 breast cancer during and after surgery.

[0168] A 100 μM, 100 μL solution of MAT was injected into tumor-bearing mice, and imaging was performed using an in vivo imaging system (IVIS) 4 hours after injection. Figure 22 As shown in A, 22B, and 22C, MAT can accurately image three types of tumors: HeLa (human cervical cancer), SKOV3 (human ovarian cancer), and 4T1 (mouse breast cancer), as... Figure 22 As shown in Figure D, the tumor's fluorescence imaging is clearly visible before surgery, while the fluorescence disappears after surgery, confirming the complete removal of the lesion and enabling surgical navigation. Therefore, MAT provides excellent imaging and guides surgical treatment, and its versatility is demonstrated by the various surgical models available.

[0169] Figure 23 HE staining image of SKOV3 tumors in mice with peritoneal metastasis of SKOV3 ovarian cancer.

[0170] like Figure 23As shown, hematoxylin-eosin (HE) staining confirmed that surgery under MAT guidance could precisely remove the tumor.

[0171] Figure 24 HE staining images of the lungs, liver, heart, kidneys, and spleen after subcutaneous injection of PBS and MAT solution in SKOV3 mice with peritoneal metastasis of ovarian cancer.

[0172] like Figure 24 As shown, HE staining of organ anatomical sections from the PBS-injected and MAT-injected groups revealed no obvious damage to normal organs, indicating that MAT has biocompatibility.

[0173] Figure 25 Analysis of liver function indices ALT and AST, and kidney function indices ALT and AST in SKOV3 mice with peritoneal metastasis of ovarian cancer after different treatments.

[0174] like Figure 25 As shown, there were no significant differences in liver function-related biochemical indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and kidney function-related biochemical indicators such as blood urea nitrogen (BUN) and creatinine (CREA), indicating that MAT has no effect on liver and kidney function.

[0175] Example 2:

[0176] The specific steps in this embodiment are the same as those in Embodiment 1, except for the following differences:

[0177] In step 1), the molar ratio of Zn powder, 4-aminobenzophenone, 4-bromobenzophenone and TiCl4 is 6:1:1:6; the eluent is petroleum ether:ethyl acetate = 8:1.

[0178] In step 2), the molar volume ratio of intermediate product 1 to THF is 1 mmol / (3.8-4) mL; the molar ratio of intermediate product 1, Boc anhydride and Et3N is 1:5:(8); the eluent is petroleum ether:ethyl acetate = 22:1;

[0179] In step 3), the molar ratio of intermediate 2, bis(pinacol)diboron, Pd(dppf)Cl2 and KOAc is 3:-3:0.1:-9; the eluent is petroleum ether:ethyl acetate = 22:1;

[0180] In step 4), the intermediates 3,4,7-dibromobenzo[c]-1,2,5-thiadiazole and Pd(PPh3) are produced. 4、 The molar ratio of K2CO3 is 1:-4:0.2):12;

[0181] The volume ratio of THF to H2O in the THF / H2O mixture is 10:1;

[0182] The eluent was petroleum ether:ethyl acetate = 22:1.

[0183] In step 5), the molar ratio of intermediate product 4, 4-(methoxycarbonyl)phenylboronic acid, Pd(PPh3)4 and K2CO3 is 1:2:0.1:10;

[0184] The volume ratio of THF to H2O in the THF / H2O mixture is 6:1;

[0185] The eluent was initially petroleum ether:ethyl acetate = 22:1, and then changed to petroleum ether:ethyl acetate = 12:1.

[0186] In step 6), the molar volume ratio of intermediate product 5 to the THF / MeOH mixture is 0.6 mmol / 20 mL;

[0187] The volume ratio of the THF / MeOH mixture is 1:2;

[0188] The eluent is methanol:acetic acid:dichloromethane = 1:1:110.

[0189] In step 7), the molar volume ratio of intermediate product 6 and the TFA / DCM mixture is 0.4 mmol / 10 mL;

[0190] The molar ratio of intermediate product 6 to maleic anhydride is 1:-8;

[0191] The volume ratio of TFA to DCM in the TFA / DCM mixture is 1:2.

[0192] The eluent is acetic acid: dichloromethane = 1:120;

[0193] In step 8), the molar ratio of intermediate product 7, HBTU, and N-(2-aminoethyl)-4-methylbenzenesulfonamide is 0.1:0.2:0.2.

[0194] The intermediate products and target products obtained in this embodiment are basically consistent with those in Example 1 in terms of yield and characterization results.

[0195] Example 3:

[0196] The specific steps in this embodiment are the same as those in Embodiment 1, except for the following differences:

[0197] In step 1), the mixture is stirred overnight under reflux at 100°C;

[0198] In step 2), the mixture is stirred for 4 hours under reflux at 100°C;

[0199] In step 3), the mixture is stirred overnight under reflux at 110°C;

[0200] In step 4), the mixture is stirred overnight under reflux at 100°C;

[0201] In step 5), the mixture is stirred overnight under reflux at 100°C;

[0202] In step 6), the mixture is stirred under reflux at 90°C for 2 hours;

[0203] In step 7), the solution is stirred for 2 hours, and the mixture is stirred overnight at 110°C;

[0204] In step 8), DIEA is added dropwise to adjust the pH of the solution to 8.5, and then stirring is continued for 10 hours.

[0205] The intermediate products and target products obtained in this embodiment are basically consistent with those in Example 1 in terms of yield and characterization results.

[0206] The above are merely specific embodiments of the present invention. It should be noted that any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the protection scope of the present invention. Any other aspects not described in detail are prior art.

Claims

1. An AIE molecule that responds to unfolded proteins, characterized in that: The structural formula of the AIE molecule that responds to unfolded proteins is as follows: 。 2. A method for synthesizing an AIE molecule responsive to unfolded proteins as described in claim 1, characterized in that: Its synthetic route is as follows: 。 3. The synthesis method according to claim 2, characterized in that: The steps include the following: 1) Synthesis of intermediate product 1: Weigh Zn powder, 4-aminobenzophenone, and 4-bromobenzophenone into a double-necked flask, replace with nitrogen, and add ultra-dry tetrahydrofuran. Slowly add TiCl4 dropwise at 0°C, then restore to room temperature. The mixture is then refluxed overnight at 90-100°C. After the reaction is complete, quench the reaction with saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, and dry with anhydrous magnesium sulfate. Remove the solvent under reduced pressure using a rotary evaporator to obtain the crude product. Purify the crude product by column chromatography using petroleum ether and ethyl acetate as eluents to obtain a deep yellow oily substance, which is intermediate 1. 2) Synthesis of intermediate product 2: Intermediate 1 and THF were added to a two-necked flask, followed by Boc anhydride and Et3N. The mixture was stirred under reflux at 90-100°C for 3-4 hours. After the reaction was complete, hydrochloric acid was added. The aqueous phase was extracted with dichloromethane, and the organic phases were combined and extracted with dichloromethane again. The extract was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents to obtain a yellow solid, which was intermediate 2. 3) Synthesis of intermediate product 3: Intermediate 2, bis(pinacol)diboron, Pd(dppf)Cl2, and KOAc were added to a two-necked flask. After injecting dioxane, the system was degassed using a freeze-pump-thaw cycle, and the mixture was stirred overnight under reflux at 105-110°C. After the reaction was complete, dioxane was removed by rotary evaporation under reduced pressure, and the mixture was redissolved in dichloromethane. Hydrochloric acid was then added, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and extracted with dichloromethane again. The extract was washed with saturated brine and dried with anhydrous magnesium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents to obtain a yellow powder, which was intermediate 3. 4) Synthesis of intermediate product 4: Intermediate 3,4,7-dibromobenzo[c]-1,2,5-thiadiazole, Pd(PPh3)4, and K2CO3 were added to a two-necked flask. A reflux condenser was installed and sealed in the system, and then a THF / H2O mixture was injected. The system was degassed using a freeze-pump-thaw cycle, and the mixture was stirred overnight under reflux at 90-100°C. After the reaction was complete, THF was removed by rotary evaporation under reduced pressure, and the mixture was redissolved in dichloromethane. Hydrochloric acid was then added, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and extracted with dichloromethane again. The extract was washed with saturated brine and dried with anhydrous magnesium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents to obtain an orange solid, which was intermediate 4. 5) Synthesis of intermediate product 5: Intermediate 4, 4-(methoxycarbonyl)phenylboronic acid, Pd(PPh3)4, and K2CO3 were added to a two-necked flask, followed by the injection of a THF / H2O mixture. The system was degassed using a freeze-evacuation-thawing cycle, and the mixture was stirred overnight under reflux at 90-100°C. After the reaction was complete, THF was removed under reduced pressure using a rotary evaporator, and the mixture was redissolved in dichloromethane. Hydrochloric acid was then added, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and extracted again with dichloromethane. The extract was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure using a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography using petroleum ether and ethyl acetate as eluents, yielding an orange solid, which was intermediate 5. 6) Synthesis of intermediate product 6: Weigh intermediate product 5 and add it to a THF / MeOH mixture. Add NaOH aqueous solution while continuously stirring. Reflux the mixture at 80-90℃ for 1-2 hours. After the reaction is complete, remove the organic solvent using a rotary evaporator under reduced pressure. Then add hydrochloric acid and extract the aqueous phase with dichloromethane. Combine the organic phases and continue extraction with dichloromethane. Wash the extract with saturated brine and dry with anhydrous magnesium sulfate. Remove the solvent using a rotary evaporator under reduced pressure to obtain the crude product. Purify the crude product by column chromatography using methanol, acetic acid, and dichloromethane as eluents. The final orange solid is intermediate product 6. 7) Synthesis of intermediate product 7: Add intermediate product 6 and 10 mL of TFA / DCM mixture to a two-necked flask, stir the solution for 1-2 hours, and then concentrate it under vacuum under nitrogen protection. The resulting yellow solid can be used directly without separation or purification. Add pure acetic acid and maleic anhydride to the solid, and stir the mixture overnight at 100-110℃. After cooling to room temperature, add toluene, and remove the solvent under reduced pressure using a rotary evaporator to obtain the crude product. Purify the crude product by column chromatography with acetic acid and dichloromethane as eluents to obtain the yellow solid product, which is intermediate product 7. 8) Synthesis of MI-AIE-TsG: The synthesized intermediate product 7 was dissolved in dry DMF, and then HBTU was added directly to the solution. After standing, N-(2-aminoethyl)-4-methylbenzenesulfonamide was added to the mixture, and DIPEA was added dropwise to adjust the pH of the solution to 8-8.

5. Then, stirring was continued for 8-10 hours. After the reaction was completed, the solvent was air-dried, the remaining oily product was dissolved in methanol, and purified by high performance liquid chromatography. The product was lyophilized to obtain a yellow powder, which is MI-AIE-TsG.

4. The synthesis method according to claim 3, characterized in that: In step 1), the molar ratio of Zn powder, 4-aminobenzophenone, 4-bromobenzophenone and TiCl4 is (4-6):1:1:(4-6); the eluent is petroleum ether:ethyl acetate = (5-8):1; In step 2), the molar volume ratio of intermediate product 1 to THF is 1 mmol / (3.8-4) mL; the molar ratio of intermediate product 1, Boc anhydride and Et3N is 1:(3-5):(5-8); the eluent is petroleum ether:ethyl acetate = (20-22):

1.

5. The synthesis method according to claim 3, characterized in that: In step 3), the molar ratio of intermediate product 2, bis(pinacol)diboron, Pd(dppf)Cl2 and KOAc is (2.1-3):(2.5-3):0.1:(8.4-9); the eluent is petroleum ether:ethyl acetate = (20-22):1; In step 4), the intermediate products 3,4,7-dibromobenzo[c]-1,2,5-thiadiazole and Pd(PPh3) are involved. 4、 The molar ratio of K2CO3 is (1-1.1):(3.4-4):(0.11-0.2):(11.3-12). The volume ratio of THF to H2O in the THF / H2O mixture is (8-10):1; The eluent is petroleum ether:ethyl acetate = (20-22):

1.

6. The synthesis method according to claim 3, characterized in that: In step 5), the molar ratio of intermediate product 4, 4-(methoxycarbonyl)phenylboronic acid, Pd(PPh3)4 to K2CO3 is (0.93-1):(1.9-2):(0.09-0.1):(9.3-10). The volume ratio of THF to H2O in the THF / H2O mixture is (4-6):1; The eluent was initially petroleum ether:ethyl acetate = (20-22):1, and then changed to petroleum ether:ethyl acetate = (10-12):

1.

7. The synthesis method according to claim 3, characterized in that: In step 6), the molar volume ratio of intermediate product 5 to the THF / MeOH mixture is (0.4-0.6) mmol / 20mL; The volume ratio of the THF / MeOH mixture is 1:(1-2); The eluent is methanol:acetic acid:dichloromethane = 1:1:(100-110).

8. The synthesis method according to claim 3, characterized in that: In step 7), the molar volume ratio of intermediate product 6 and the TFA / DCM mixture is (0.32-0.4) mmol / 10 mL; The molar ratio of intermediate product 6 to maleic anhydride is 1:(5-8). The volume ratio of TFA to DCM in the TFA / DCM mixture is 1:(1-2). The eluent is acetic acid: dichloromethane = 1: (100-120).

9. The synthesis method according to claim 3, characterized in that: In step 8), the molar ratio of intermediate product 7, HBTU, and N-(2-aminoethyl)-4-methylbenzenesulfonamide is 0.1:(0.12-0.2):(0.12-0.2).

10. The use of the AIE molecule of claim 1, which is responsive to unfolded proteins, in the preparation of a tumor surgical navigation tool, wherein the tumor is cervical cancer, ovarian cancer, or breast cancer.

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