A Multifunctional AIE Optical Diagnosis and Therapy Molecule, Its Azide Compound, Synthesis Method and Application

Abstract

The present invention discloses a multifunctional AIE optical diagnosis and treatment molecule and its azide compound, synthesis method and application. The multifunctional AIE optical diagnosis and treatment molecule uses triphenylamine as an electron donor and malononitrile fluorene as an electron acceptor. By adjusting the D-A structure of the molecule, a series of organic optical diagnosis and treatment molecules are synthesized, and a novel multifunctional optical diagnosis and treatment molecule with AIE properties is screened out. At the same time, further azide modification of the multifunctional optical diagnosis and treatment molecule can realize its covalent binding with functional groups such as polypeptides, DNA and small molecule compounds. The multifunctional AIE optical diagnosis and treatment molecule of the present invention has multifunctionality and can perform near-infrared fluorescence imaging, photodynamic therapy and photothermal therapy on tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of AIE molecules, and particularly relates to a multifunctional AIE optical diagnosis and treatment molecule, its azide compound, synthesis method and application. Background Art

[0002] Optical diagnosis and treatment has attracted great interest in recent years. It refers to the real-time diagnosis and treatment of diseases through light irradiation, opening up a new way for the treatment of cancer. Multifunctional optical diagnosis and treatment systems, including fluorescence imaging, photodynamic therapy and photothermal therapy, etc., can further improve the accuracy and efficacy of treatment. Designing a high-performance multifunctional optical diagnosis and treatment reagent is a difficult problem that urgently needs to be solved at present. Most traditional multifunctional optical diagnosis and treatment molecules have the ACQ effect. They emit strongly in good solutions, but their luminescence weakens or disappears in the aggregated state, which limits their application in the fields of biosensing and treatment. The emergence of aggregation-induced emission (AIE) solves this problem. It has no or only weak fluorescence in good solvents, but the fluorescence emission is enhanced in the aggregated state. At the same time, the AIE molecule can be endowed with photodynamic or photothermal therapy functions by adjusting the functional groups in the molecule. Since the AIE phenomenon was first reported in 2001, it has received extensive attention from researchers and has good application prospects in the biomedical field. However, at present, multifunctional AIE optical diagnosis and treatment molecules are still rare. How to construct high-performance multifunctional AIE optical diagnosis and treatment molecules to meet clinical needs is a difficult problem that urgently needs to be solved at present. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the above background art, and provide a multifunctional AIE optical diagnosis and treatment molecule, its azide compound, synthesis method and application. The optical diagnosis and treatment molecule uses triphenylamine as the electron donor (D) and malononitrile fluorene as the electron acceptor (A). By adjusting the connection mode between the donor and the acceptor, the conjugation situation of the molecule and the intramolecular charge transfer situation in the excited state are investigated, and the luminescence, photodynamic and photothermal properties of the AIE optical diagnosis and treatment molecule are regulated, so as to achieve the balance and optimization of the multifunction of diagnosis and treatment. Finally, the obtained optical diagnosis and treatment molecule has the comprehensive advantages of long emission wavelength, high brightness and good treatment effect.

[0004] To achieve the above purpose, the present invention provides a multifunctional AIE optical diagnosis and treatment molecule, which is obtained by screening through adjusting the connection mode between the electron donor and the electron acceptor. The multifunctional AIE optical diagnosis and treatment molecule uses triphenylamine as the electron donor (D) and malononitrile fluorene as the electron acceptor (A). The structural formula of the multifunctional AIE optical diagnosis and treatment molecule is as follows:

[0005]

[0006] The present invention also provides a method for synthesizing the multifunctional AIE optical diagnostic and therapeutic molecule. The synthesis route of the multifunctional AIE optical diagnostic and therapeutic molecule is as follows:

[0007]

[0008] Furthermore, the method for synthesizing the multifunctional AIE optical diagnostic and therapeutic molecule comprises the following steps:

[0009] S1: Add compound 1, diphenylamine, palladium acetate (Pd(OAc)2), tri-tert-butylphosphine tetrafluoroborate (P(t-Bu)3·HBF4), and potassium tert-butoxide (t-BuOK) into a container, then add anhydrous toluene, introduce inert gas into the container, heat and stir, and reflux at 100-110°C for 18-20h to obtain compound 2;

[0010] S2: Compound 2 and N-bromosuccinimide (NBS) were dissolved in anhydrous dichloromethane (DCM), respectively, the compound 2 / DCM solution was placed in an ice-water bath, and the NBS / DCM solution was added dropwise thereto. After the addition was completed, the mixture was stirred for 1-1.5 h in an ice-water bath, and then the mixture was stirred for 5-8 h at room temperature to obtain compound 3;

[0011] S3: Add compound 3 and 4-methoxyphenylboronic acid pinacol ester, potassium carbonate (K2CO3), tetrakistriphenylphosphine palladium (Pd(PPh3)4) into a container, then add deoxygenated tetrahydrofuran (THF) and deionized water, pass inert gas into the container and heat with stirring, reflux at 70-80°C for 20-24h to obtain compound 4;

[0012] S4: Compound 4 and N-bromosuccinimide (NBS) were dissolved in anhydrous DCM respectively, the compound 4 / DCM solution was placed in an ice-water bath, and the NBS / DCM solution was added dropwise thereto. After the addition was completed, stirring was continued in an ice-water bath for 1-1.5 h, and then the reaction was carried out at room temperature for 5-8 h to obtain compound 5;

[0013] S5: Add compound 5, bis(pinacol borate), potassium acetate (CH3COOK), and Pd(PPh3)2Cl2 into a container, then add dioxane, introduce inert gas into the container, heat and stir, and react at 100-120°C for 20-24h to obtain compound 6;

[0014] S6: Add compound 6, 3-bromofluorenone, K2CO3, and Pd(PPh3)4 into a container, then add deoxygenated THF and deionized water, introduce inert gas into the container, heat and stir, and reflux at 70-80°C for 20-24h to obtain compound 7;

[0015] S7: Compound 7 and malononitrile are added to a container, and then anhydrous pyridine is added, an inert gas is introduced into the container, and the container is heated and stirred, and refluxed at 110-120° C. for 20-24 h to obtain compound 8, which is a multifunctional AIE optical diagnostic and therapeutic molecule;

[0016] Wherein, the structural formula of the compound 1 is:

[0017]

[0018] The structural formula of the compound 2 is:

[0019]

[0020] The structural formula of the compound 3 is:

[0021]

[0022] The structural formula of the compound 4 is:

[0023]

[0024] The structural formula of the compound 5 is:

[0025]

[0026] The structural formula of the compound 6 is:

[0027]

[0028] The structural formula of the compound 7 is:

[0029]

[0030] Furthermore, in step S1, the molar ratio of compound 1 to diphenylamine, palladium acetate (Pd(OAc)2), tri-tert-butylphosphine tetrafluoroborate (P(t-Bu)3·HBF4), and potassium tert-butoxide (t-BuOK) is 1:(1.5-2.0):(0.01-0.10):(0.01-0.20):(3-10);

[0031] In the step S2, the molar ratio of compound 2 to N-bromosuccinimide is 1:1.0-1.1;

[0032] In the step S3, the molar ratio of compound 3 to 4-methoxyphenylboronic acid pinacol ester, potassium carbonate (K2CO3), and tetrakistriphenylphosphine palladium (Pd(PPh3)4) is 1:(1.5-2.0):(8.0-10.0):(0.02-0.10);

[0033] In the step S4, the molar ratio of compound 4 to N-bromosuccinimide is 1:1.0 - 1.1;

[0034] In the step S5, the molar ratio of compound 5 to bis(pinacolato)diboron, potassium acetate (CH3COOK), and Pd(PPh3)2Cl2 is 1:(2.0 - 3.0):(3.0 - 10.0):(0.01 - 0.10);

[0035] In the step S6, the molar ratio of compound 6 to 3-bromofluorone, K2CO3, and Pd(PPh3)4 is 1:(1.0 - 1.5):(5.0 - 10.0):(0.05 - 0.10);

[0036] In the step S7, the molar ratio of compound 7 to malononitrile is 1:5.0 - 10.0.

[0037] The present invention also provides an azide compound, which is obtained by azidation modification of the above-mentioned multifunctional AIE optical diagnosis and treatment molecule; the structural formula of the azide compound is as follows:

[0038]

[0039] The present invention also provides a synthesis method of the above-mentioned azide compound, and the synthesis route of the azide compound is as follows:

[0040]

[0041] Furthermore, the synthesis method of the azide compound includes the following steps:

[0042] 1) Dissolve the multifunctional AIE optical diagnosis and treatment molecule and boron tribromide (BBr3) in anhydrous DCM respectively. Place the multifunctional AIE optical diagnosis and treatment molecule / DCM solution in an ice-water bath, and dropwise add the BBr3 / DCM solution thereto. After the addition is completed, continue to stir for 1 - 1.5 h under the ice-water bath condition, and then react at room temperature for 4 - 6 h to obtain compound 9;

[0043] 2) Add compound 9, 1,6-diiodohexane, and potassium carbonate to a container, then add anhydrous DMF and ethyl acetate (EA). Pass an inert gas into the flask and heat with stirring, and reflux and react at 30 - 50 °C for 18 - 22 h to obtain compound 10;

[0044] 3) Add compound 10 and sodium azide to a container, then add anhydrous DMF. Pass an inert gas into the flask and stir, and react at 20 - 30 °C for 20 - 24 h to obtain azide compound 11;

[0045] Among them, the structural formula of the multifunctional AIE optical diagnosis and treatment molecule is as follows:

[0046]

[0047] The structural formula of the said Compound 9 is as follows:

[0048]

[0049] The structural formula of the said Compound 10 is as follows:

[0050]

[0051] The structural formula of the said Compound 11 is as follows:

[0052]

[0053] Further, in the said step 1), the molar ratio of the multifunctional AIE optical diagnosis and treatment molecule to BBr3 is 1:5.0 - 6.0;

[0054] In the said step 2), the molar ratio of Compound 9 to 1,6 - diiodohexane and potassium carbonate is 1:10.0 - 12.0:5.0 - 8.0;

[0055] In the said step 3), the molar ratio of Compound 10 to sodium azide is 1:3.0 - 5.0.

[0056] The present invention also provides the use of the above - mentioned multifunctional AIE optical diagnosis and treatment molecule for preparing a tumor therapeutic agent.

[0057] The present invention also provides the use of the above - mentioned multifunctional AIE optical diagnosis and treatment molecule for near - infrared fluorescence imaging, photodynamic therapy and photothermal therapy of tumors.

[0058] Compared with the prior art, the present invention has the following advantages:

[0059] Firstly, the present invention designs and synthesizes a novel multifunctional optical diagnosis and treatment molecule (TAF) with triphenylamine as the electron donor (D) and malononitrile fluorene as the electron acceptor (A). By adjusting the D - A structure of the molecule, optimizing the conjugation between groups, the charge transfer within the excited state molecule and the intermolecular interaction, this molecule is screened and obtained.

[0060] Secondly, the present invention further performs azide modification on the multifunctional AIE optical diagnosis and treatment molecule (TAF), which can achieve its covalent binding with functional groups (such as polypeptides, DNA, and small molecule compounds, etc.).

[0061] Thirdly, the multifunctional optical diagnosis and treatment molecule (TAF) of the present invention has multifunctionality, has good fluorescence intensity and photo - activation treatment ability, and can perform near - infrared fluorescence imaging, photodynamic therapy and photothermal therapy on tumors.

[0062] Fourthly, the raw materials for synthesizing the multifunctional optical diagnosis and treatment molecule (TAF) of the present invention are widely sourced, the preparation process is simple, each step of the chemical synthesis method is simple, and the cost is advantageous.

[0063] Fifthly, the preparation process of the multifunctional optical diagnosis and treatment molecule (TAF) of the present invention does not involve dangerous process equipment and harsh process conditions, and the chemical process safety is guaranteed. Description of the Drawings

[0064] Figure 1 is the 1H NMR characterization of Compound 2 in Example 1 of the present invention;

[0065] Figure 2 is the 1H NMR characterization of Compound 3 in Example 1 of the present invention;

[0066] Figure 3 is the 1H NMR characterization of Compound 4 in Example 1 of the present invention;

[0067] Figure 4 is the mass spectrometry characterization of Compound 4 in Example 1 of the present invention;

[0068] Figure 5 is the 1H NMR characterization of Compound 6 in Example 1 of the present invention;

[0069] Figure 6 is the mass spectrometry characterization of Compound 6 in Example 1 of the present invention;

[0070] Figure 7 is the 1H NMR characterization of Compound 7 in Example 1 of the present invention;

[0071] Figure 8 is the mass spectrometry characterization of Compound 7 in Example 1 of the present invention;

[0072] Figure 9 is the 1H NMR characterization of Compound 8 in Example 1 of the present invention;

[0073] Figure 10 is the mass spectrometry characterization of Compound 8 in Example 1 of the present invention;

[0074] Figure 11 is the 1H NMR characterization of Compound 9 in Example 1 of the present invention;

[0075] Figure 12 is the mass spectrometry characterization of Compound 9 in Example 1 of the present invention;

[0076] Figure 13 is the 1H NMR characterization of Compound 10 in Example 1 of the present invention;

[0077] Figure 14It is the mass spectrometry characterization of Compound 10 in Example 1 of the present invention;

[0078] Figure 15 It is the 1H NMR characterization of Compound 11 in Example 1 of the present invention;

[0079] Figure 16 It is the mass spectrometry characterization of Compound 11 in Example 1 of the present invention;

[0080] Figure 17 It is the ultraviolet absorption spectrum of the TAF molecule of Compound 1 in Example 1 of the present invention;

[0081] Figure 18 It is the fluorescence emission spectrum of the TAF molecule of Compound 1 in Example 1 of the present invention under different excitations; among them, Figure 18 Figure a in it is the fluorescence emission spectrum of the TAF molecule under 366 nm excitation; Figure 18 Figure b in it is the fluorescence emission spectrum of the TAF molecule under 600 nm excitation;

[0082] Figure 19 It is the determination of the AIE property of the TAF molecule of Compound 1 in Example 1 of the present invention. The relative fluorescence intensity (I / I0) of the TAF molecule in the tetrahydrofuran-water mixture and f 水 Relationship, excitation wavelength: 366 nm;

[0083] Figure 20 It is the ability diagram of the TAF molecule of Compound 1 in Example 1 of the present invention to generate ROS ( 1 O2) with Ce6;

[0084] Figure 21 It is the photothermal property of the TAF molecule of Compound 1 in Example 1 of the present invention; among them, Figure 21 Figure a in it is the photothermal heating curve of the TAF molecule; Figure 21 Figure b in it is the photothermal stability diagram of the TAF molecule;

[0085] Figure 22 It is the confocal image of detecting intracellular reactive oxygen species under 660 nm laser irradiation of HeLa cells incubated with TAF in Example 1 of the present invention;

[0086] Figure 23 Photothermal property diagram of HeLa cells incubated with TAF in Example 1 of the present invention; among them, Figure 23 Figure a in it is the curve of the temperature of HeLa cells incubated with TAF changing with irradiation time; Figure 23 Figure b in it is the infrared thermal image of HeLa cells incubated with TAF under 660 nm laser irradiation (800 mW cm -2 );

[0087] Figure 24Cytotoxicity experiment graphs of concentration gradient and time gradient of TAF incubating HeLa cells in Example 1 of the present invention; among them, Figure 24 Figure a in the middle is the cytotoxicity experiment graph of the concentration gradient of TAF; Figure 24 Figure b in the middle is the cytotoxicity experiment graph of the time gradient of TAF;

[0088] Figure 25 Confocal image of PI / CMFDA double staining of TAF incubating HeLa cells in Example 1 of the present invention. Detailed implementation manners

[0089] The implementation of the present invention will be described in detail below in combination with implementation cases, but they do not constitute a limitation to the present invention and are only for illustration purposes. At the same time, the advantages of the present invention will become clearer and easier to understand by description.

[0090] Example 1:

[0091] Step S1: Add compound 1 (2.50 g, 6.08 mmol), diphenylamine (1.54 g, 9.12 mmol), Pd(OAc)2 (136 mg, 0.607 mmol), P(t-Bu)3·HBF4 (176 mg, 0.607 mmol) and t-BuOK (2.05 g, 18.23 mmol) into a 100 mL two-necked round-bottom flask, inject 30 mL of anhydrous toluene under argon protection, and then heat to 100 °C and stir for 18 h. After the reaction is completed, first extract three times with DCM / H2O (1 / 3, v / v), then remove the organic solvent by rotary evaporation under reduced pressure. The obtained crude product is further recrystallized with anhydrous ethanol (1 g of powder is added with 50 mL of anhydrous ethanol), filtered by suction, and the white powder obtained is compound 2 with strong blue fluorescence. Yield: 2.86 g, yield rate: 94%.

[0092] Step S2: Add compound 2 (2.00 g, 4.00 mmol) into a 100 mL two-necked round-bottom flask and inject 10 mL of anhydrous DCM. Dissolve NBS (0.748 g, 4.20 mmol) in 20 mL of anhydrous DCM and add it dropwise to the above round-bottom flask under an ice-water bath condition. After the addition is completed, continue to stir for 60 min under an ice-water bath condition, and then transfer the round-bottom flask to an environment at 25 °C and continue to stir and react for 5 h. After the reaction is completed, first extract three times with DCM / H2O (1 / 3, v / v), then remove the organic solvent by rotary evaporation under reduced pressure. The obtained crude product is further recrystallized with anhydrous ethanol (1 g of powder is added with 50 mL of anhydrous ethanol), filtered by suction, and the light green powder obtained is compound 3 with strong blue fluorescence. Yield: 2.12 g, yield rate: 91.55%.

[0093] Step S3: Add compound 3 (2.5 g, 4.32 mmol), 4-methoxyphenylboronic acid pinacol ester (1.52 g, 6.48 mmol), potassium carbonate (4.78 g, 34.57 mmol) and tetrakis(triphenylphosphine)palladium(0) (138.67 mg, 0.12 mmol) into a 100 mL two-necked round-bottom flask. Under argon protection, inject 8 mL of deoxygenated deionized water and 30 mL of deoxygenated THF. Heat the resulting mixture to 80 °C and react for 20 h. After the reaction is completed, first extract with DCM / H2O (1 / 3, v / v) three times, then rotary evaporate under reduced pressure to remove the organic solvent. The obtained crude product is further purified by column chromatography (petroleum ether / dichloromethane = 3 / 1, v / v) to obtain a light yellow powder, which is compound 4 with yellow fluorescence. Yield: 1.88 g, yield rate: 71.82%.

[0094] Step S4: Add compound 4 (1.80 g, 2.97 mmol) into a 100 mL two-necked round-bottom flask and inject 10 mL of anhydrous DCM. Dissolve NBS (0.555 g, 3.12 mmol) in 15 mL of anhydrous DCM and add it dropwise to the above round-bottom flask under an ice-water bath. After the addition is completed, continue to stir for 60 min under an ice-water bath, and then transfer the round-bottom flask to an environment at 25 °C and continue to stir and react for 5 h. After the reaction is completed, first extract with DCM / H2O (1 / 3, v / v) three times, then rotary evaporate under reduced pressure to remove the organic solvent. The obtained crude product is further recrystallized with anhydrous ethanol (1 g of powder is added with 50 mL of anhydrous ethanol), filtered by suction to obtain a light yellow powder, which is compound 5 and is directly used for the next reaction without further purification.

[0095] Step S5: Add compound 5 (2.10 g, 3.07 mmol), bis(pinacolato)diboron (1.56 g, 6.13 mmol), potassium acetate (0.903 g, 9.20 mmol) and Pd(PPh3)2Cl2 (200 mg, 0.306 mmol) into a 100 mL two-necked round-bottom flask. Under argon protection, inject 30 mL of anhydrous dioxane. Heat the resulting mixture to 120 °C and react for 20 h. After the reaction is completed, first extract with DCM / H2O (1 / 3, v / v) three times, then rotary evaporate under reduced pressure to remove the organic solvent. The obtained crude product is further purified by column chromatography (petroleum ether / dichloromethane / ethyl acetate = 40 / 20 / 1, v / v / v) to obtain a yellow powder, which is compound 6 with yellow fluorescence. Yield: 1.50 g, yield rate: 66.83%.

[0096] Step S6: Add compound 6 (1.5 g, 2.05 mmol), 3-bromofluorone (0.796 g, 3.07 mmol), potassium carbonate (1.42 g, 10.25 mmol) and tetrakis(triphenylphosphine)palladium(0) (138.67 mg, 0.12 mmol) into a 100 mL two-necked round-bottom flask. Under argon protection, inject 5 mL of deoxygenated deionized water and 30 mL of deoxygenated THF. Heat the resulting mixture to 80 °C and react for 20 h. After the reaction is completed, first extract three times with DCM / H2O (1 / 3, v / v), then rotary evaporate under reduced pressure to remove the organic solvent. The obtained crude product is further purified by column chromatography (petroleum ether / dichloromethane = 3 / 1, v / v) to obtain an orange powder, which is compound 7 with orange-red fluorescence. Yield: 1.15 g, yield rate: 68.45%.

[0097] Step S7: Add compound 7 (200 mg, 0.255 mmol) and malononitrile (0.168 mg, 2.55 mmol) into a 25 mL two-necked round-bottom flask. Under argon protection, inject 5 mL of anhydrous pyridine. Heat the resulting mixture to 120 °C and react for 20 h. After the reaction is completed, first adjust the pH to weakly acidic with dilute hydrochloric acid, then extract three times with DCM / H2O (1 / 3, v / v), then rotary evaporate under reduced pressure to remove the organic solvent. The obtained crude product is further purified by column chromatography (petroleum ether / dichloromethane = 1 / 1.2, v / v) to obtain a black-red powder, which is compound 8. Compound 8 is the multifunctional AIE optical diagnosis and treatment molecule (TAF). Yield: 110 mg, yield rate: 51.16%.

[0098] Step S8: Add compound 8 (300 mg, 0.360 mmol) to a 50 mL two-necked round-bottom flask containing 5 mL of anhydrous DCM. Dissolve BBr3 (0.451 g, 1.80 mmol) in 10 mL of anhydrous DCM and add it dropwise to the above round-bottom flask under an ice-water bath. After the addition is completed, continue to stir for 60 min under an ice-water bath, then transfer the round-bottom flask to an environment at 25 °C and continue to stir and react for 4 h. After the reaction is completed, first quench the excess BBr3 with saturated sodium carbonate solution, then extract three times with DCM / H2O (1 / 3, v / v), then rotary evaporate under reduced pressure to remove the organic solvent. The obtained crude product is further purified by column chromatography (pure DCM) to obtain a purple powder, which is compound 9. Yield: 190 mg, yield rate: 64.42%.

[0099] Step S9: Compound 9 (250 mg, 0.305 mmol), 1,6-diiodohexane (1.03 g, 3.06 mmol) and potassium carbonate (211 mg, 1.53 mmol) were added to a 25 mL two-necked round-bottom flask. Under argon protection, 5 mL of anhydrous DMF and 5 mL of anhydrous ethyl acetate were injected. The resulting mixture was heated to 50 °C and reacted for 18 h. After the reaction was completed, it was extracted three times with DCM / H2O (1 / 3, v / v). Then, the organic solvents were removed by rotary evaporation under reduced pressure. The obtained crude product was further purified by column chromatography (petroleum ether / dichloromethane = 1 / 1, v / v) to obtain a dark purple powder, which was compound 10. Yield: 180 mg, yield rate: 57.29%.

[0100] Step S10: Compound 10 (70 mg, 0.068 mmol) was added to a 50 mL reaction tube. Under argon protection, 5 mL of anhydrous DMF was injected. Sodium azide (15 mg, 0.204 mmol) was added with stirring. The resulting mixture was heated to 30 °C and reacted overnight. After the reaction was completed, a small amount of sodium hypochlorite was added to consume the excess raw materials. Then, it was extracted three times with DCM / H2O (1 / 3, v / v). Next, the organic solvents were removed by rotary evaporation under reduced pressure. The obtained crude product was further purified by column chromatography (petroleum ether / dichloromethane = 1 / 1, v / v) to obtain a dark purple powder, which was the target compound 11. Yield: 30 mg, yield rate: 46.71%. The molecular structural formulas of the above compounds are shown in Table 1:

[0101] Table 1

[0102]

[0103]

[0104]

[0105]

[0106] To prove the progress of the reaction, the following characterizations were specifically made:

[0107] As Figure 1 shown, the 1H NMR characterization of compound 2 in Example 1 is as follows:

[0108] 1 H NMR (400 MHz, CDCl3)): δ [ppm]: 7.21 (t, J = 7.8 Hz, 4H), 7.16–7.00 (m, 19H), 6.97 (t, J = 7.2 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H).

[0109] As Figure 2As shown, the 1H NMR characterization of Compound 3 in Example 1 is as follows:

[0110] 1 H NMR(400MHz,DMSO-d6)):δ[ppm]:7.41(d,J=8.8Hz,2H),7.30(t,J=7.8Hz,2H),7.22–7.07(m,10H),7.04(t,J=7.6Hz,2H),6.96(t,J=6.4Hz,6H),6.88(d,J=8.6Hz,2H),6.83(d,J=8.8Hz,2H),6.75(d,J=8.6Hz,2H).

[0111] As Figure 3 shown, the 1H NMR characterization of Compound 4 in Example 1 is as follows:

[0112] 1 H NMR(400MHz,CDCl3)):δ[ppm]:7.49(d,J=8.6Hz,2H),7.40(d,J=8.6Hz,2H),7.23(t,J=7.8Hz,2H),7.13–6.97(m,20H),6.96(d,J=8.6Hz,2H),6.88–6.82(dd,J=10.2,4.8Hz,4H),3.84(s,3H).

[0113] As Figure 4 shown, the mass spectrometry characterization of Compound 4 in Example 1 is as follows:

[0114] HRMS(APCI):C 45 H 35 NO:[M+H] + =606.2797;Found:606.2771。

[0115] As Figure 5 shown, the 1H NMR characterization of Compound 6 in Example 1 is as follows:

[0116] 1 H NMR(400MHz,CDCl3)):δ[ppm]:7.65(d,J=8.4Hz,2H),7.49(d,J=8.6Hz,2H),7.41(d,J=8.4Hz,2H),7.18–6.98(m,20H),6.95(d,J=8.8Hz,2H),6.89–6.84(dd,J=11.2,4.4Hz,4H),3.84(s,3H),1.33(s,12H).

[0117] As Figure 6As shown, the mass spectrometry characterization of Compound 6 in Example 1 is as follows:

[0118] HRMS(APCI): C 51 H 47 BNO3: [M + H] + = 732.3649; Found: 732.3655.

[0119] As Figure 7 shown, the 1H NMR characterization of Compound 7 in Example 1 is as follows:

[0120] 1 1H NMR(400 MHz, CDCl3)): δ [ppm]: 8.39 (d, J = 8.0 Hz, 2H), 7.74 (s, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.58–7.43 (m, 8H), 7.33 (t, J = 7.4 Hz, 1H), 7.20–7.01 (m, 19H), 6.95 (t, J = 7.8 Hz, 4H), 6.90 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H).

[0121] As Figure 8 shown, the mass spectrometry characterization of Compound 7 in Example 1 is as follows:

[0122] HRMS(APCI): C 58 H 41 NO2: [M + H] + = 784.3216; Found: 784.3213.

[0123] As Figure 9 shown, the 1H NMR characterization of Compound 8 in Example 1 is as follows:

[0124] 1 1H NMR(400 MHz, CDCl3)): δ [ppm]: 8.40 (d, J = 5.0 Hz, 1H), 8.39 (d, J = 4.6 Hz, 1H), 7.74 (s, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.57–7.43 (m, 8H), 7.33 (t, J = 7.4 Hz, 1H), 7.20–7.01 (m, 19H), 6.96 (t, J = 9.2 Hz, 4H), 6.90 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H).

[0125] As Figure 10 shown, the mass spectrometry characterization of Compound 8 in Example 1 is as follows:

[0126] HRMS(APCI): C 61 H 41N3O: [M+H] + = 832.3328; Found: 832.3305.

[0127] As Figure 11 shown, the 1H NMR characterization of Compound 9 in Example 1 is as follows:

[0128] 1 H NMR (400 MHz, DMSO-d6): δ [ppm]: 9.48 (s, 1H), 8.19 (d, J = 8.0 Hz, 2H), 8.15 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 3H), 7.19–7.11 (m, 10H), 7.01–6.98 (m, 10H), 6.92 (d, J = 8.0 Hz, 2H), 6.86 (s, 1H), 6.83 (d, J = 8.0 Hz, 2H).

[0129] As Figure 12 shown, the mass spectrometry characterization of Compound 9 in Example 1 is as follows:

[0130] HRMS (APCI): C 60 H 40 N3O: [M+H] + = 818.3171; Found: 818.3194.

[0131] As Figure 13 shown, the 1H NMR characterization of Compound 10 in Example 1 is as follows:

[0132] 1 H NMR (400 MHz, CDCl3): δ [ppm]: 8.43–8.36 (m, 2H), 7.74 (s, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.58–7.43 (m, 8H), 7.34 (t, J = 7.8 Hz, 1H), 7.21–7.01 (m, 22H), 6.94 (t, J = 12.8 Hz, 3H), 4.01 (t, J = 6.4 Hz, 2H), 3.22 (t, J = 7.0 Hz, 2H), 1.94–1.77 (m, 4H), 1.53–1.50 (m, 4H).

[0133] As Figure 14 shown, the mass spectrometry characterization of Compound 10 in Example 1 is as follows:

[0134] HRMS (APCI): C66 H 51 N3OI: [M+H] + = 1028.3077; Found:

[0135] 1028.3042。

[0136] As Figure 15 shown, the 1H NMR characterization of Compound 11 in Example 1 is as follows:

[0137] 1 H NMR(400MHz, CDCl3): δ [ppm]: 8.40(m, J = 8.0Hz, 2H), 7.74(s, 1H), 7.62(d, J = 7.4Hz, 1H), 7.62–7.45(m, 8H), 7.34(t, J = 7.2Hz, 1H), 7.20–7.01(m, 19H), 6.99–6.87(m, 6H), 4.01(t, J = 6.4Hz, 2H), 3.30(t, J = 7.0Hz, 2H), 1.86–1.80(m, 2H), 1.69–1.60(m, 2H), 1.52–1.45(m, 4H).

[0138] As Figure 16 shown, the mass spectrometry characterization of Compound 11 in Example 1 is as follows:

[0139] HRMS(APCI): C 66 H 51 N6O: [M+H] + = 943.4124; Found: 943.4085。

[0140] Test Example:

[0141] (I) Photophysical Properties of TAF Molecules

[0142] Figure 17 is the ultraviolet absorption spectrum of the TAF molecule;

[0143] The photophysical properties of Compound 8 in Example 1 were studied by ultraviolet-visible absorption spectroscopy. We first evaluated the absorption properties of the TAF molecule in a 10 μM, 1% DMSO solution. It can be seen that TAF shows two characteristic ultraviolet absorption peaks at 366 nm and 600 nm ( Figure 17 ).

[0144] Figure 18 Figure a in the middle is the fluorescence emission spectrum of the TAF molecule excited at 366 nm;

[0145] Figure 18 Figure b in the middle is the fluorescence emission spectrum of the TAF molecule excited at 600 nm;

[0146] The fluorescence emission of TAF molecules (10 μM, 1% DMSO solvent) under different excitations was evaluated by fluorescence emission spectroscopy. It can be seen that under 366 nm excitation, obvious emission fluorescence of TAF molecules can be detected ( Figure 18 as shown in Figure a in the middle), under 600 nm excitation, TAF molecules produce fluorescence emission, and its highest fluorescence emission peak is at 820 nm, indicating that TAF has good NIR fluorescence imaging ability ( Figure 18 as shown in Figure b in the middle).

[0147] Figure 19 For the relationship between the relative fluorescence intensity (I / I0) of TAF molecules in a tetrahydrofuran-water mixture and f 水 ; excitation wavelength: 366 nm;

[0148] The fluorescence intensities of TAF molecules (10 μM) in different tetrahydrofuran-water solvent ratios were detected by a fluorescence spectrometer respectively. It can be seen from Figure 19 that as the water ratio (f 水 ) in the mixture increases, the emission of TAF molecules increases sharply, showing typical AIE characteristics.

[0149] (II) Photodynamic properties of TAF molecules

[0150] Figure 20 For the ability diagram of TAF molecules and Ce6 to generate ROS ( 1 O2)

[0151] The synthesized TAF molecules in the examples were tested for photodynamic properties; the active oxygen indicator used in the test was 9,10-anthracene-bis(methylene)dimalonic acid (ABDA). Test conditions: ABDA concentration was 10 μM; laser wavelength was 660 nm, laser intensity was 800 mW / cm 2 , illumination time was 10 minutes; the concentrations of both TAF molecules and Ce6 were 10 μM (the solvent was H2O containing 1% DMSO). The ultraviolet absorption spectrum of ABDA was tested every 1 minute, indicating that a large amount of reactive oxygen was generated in the solution. As Figure 20 shown, under the same conditions, compared with the photosensitizer Ce6 and the solvent H2O, the absorption peak of ABDA in TAF molecules gradually decreased with the extension of the illumination time, indicating that TAF molecules have better ability to generate reactive oxygen.

[0152] (III) Photothermal properties of TAF molecules

[0153] Figure 21 Figure a in the middle is the photothermal heating curve of TAF molecules;

[0154] Figure 21Figure b in the middle is the photothermal stability diagram of the TAF molecule;

[0155] The photothermal performance of the synthesized TAF molecule in the examples was tested. Test conditions: The concentration of the TAF molecule was 100 μM; the wavelength of the laser was 660 nm, and the intensity was 800 mW / cm 2 , and the irradiation time was 5 minutes. The temperature change of the TAF molecule solution was observed through a photothermal imager. As Figure 21 shown in Figure a in the middle, the temperature increase of the TAF molecule could reach 20 °C, indicating that TAF has good photothermal performance.

[0156] Then, the photothermal stability of the TAF molecule was tested. Test conditions: The concentration of the TAF molecule was 100 μM; the wavelength of the laser was 660 nm, and the intensity was 800 mW / cm 2 , and the irradiation time was 5 minutes. As Figure 21 shown in Figure b in the middle, after 4 photothermal cycle tests, it was found that the TAF molecule solution had good photothermal stability.

[0157] (IV) Photodynamic therapy effect of TAF in cells

[0158] Photodynamic therapy was carried out on the TAF prepared in Example 1 in vitro cells.

[0159] Figure 22 Figure is the confocal image of intracellular reactive oxygen species detected under 660 nm laser irradiation of HeLa cells incubated with TAF.

[0160] Photodynamic therapy was carried out on the synthesized TAF in the examples in vitro cells. DCFH was used as an intracellular reactive oxygen species indicator, which could be transformed from non-fluorescent DCFH-DA into green fluorescent DCF under the action of reactive oxygen species. After incubating HeLa cells with TAF for 12 hours, DCFH (10 μM) was added and incubated for 30 minutes. Subsequently, laser irradiation treatment was carried out (laser wavelength 660 nm, irradiation intensity 800 mW / cm 2 , irradiation time 5 minutes), and bright green fluorescence was observed in the cells by CLSM. As Figure 22 shown, it was proved that a large amount of reactive oxygen species were generated in HeLa cells, indicating that the TAF molecule has good photodynamic properties.

[0161] Figure 23 Figure a in the middle is the curve of the temperature change of HeLa cells incubated with TAF over the irradiation time;

[0162] Figure 23 Figure b in the middle is the infrared thermogram of HeLa cells incubated with TAF under 660 nm laser irradiation (800 mW / cm 2 );

[0163] After incubating HeLa cells with 100 mM TAF for 12 hours, wash them three times with PBS. Then use a cell scraper to scrape the HeLa cells on the culture dish wall and place them in an EP tube, and centrifuge at 1000 rpm for 4 minutes. Place the cell pellet under 660 nm laser irradiation (laser power is 800 mW / cm 2 , irradiate for 5 minutes), and then use an infrared thermal imager to collect the temperature change of the cell pellet in real time. As Figure 23 shown in Figure a in Figure 23 and Figure b in

[0164] Figure 24 , under laser irradiation, the temperature of the HeLa cell pellet rises rapidly, while the temperature of the HeLa cells treated with PBS hardly changes, indicating that TAF has good photothermal effect in cells.

[0165] Figure 24 Figure a in

[0166] is the cytotoxicity test chart of the concentration gradient of TAF incubated with HeLa cells; 2 , irradiate for 5 minutes) and use MTT to detect the viability of HeLa. From Figure 24 the results, it can be seen that with the increase of the nanoparticle concentration, the photothermal performance of the nanoparticles is activated under light irradiation conditions, thus significantly inhibiting cell viability and promoting cell apoptosis.

[0167] Figure 25 is the confocal image of PI / CMFDA double staining of TAF incubated with HeLa cells;

[0168] Incubate HeLa cells with 100 μM TAF for 24 hours. After laser irradiation of the cells, wash the cells three times with PBS, and then add 10 μL of PI (50 μg mL -1 ) and 5 μL of CMFDA (5 μM mL -1 ) to the new medium and stain for 30 minutes, and then use CLSM to observe the fluorescence staining in the cells. From Figure 25 it can be seen that the PI probe (red fluorescence) is used to stain dead cells, and the CMFDA probe (green fluorescence) is used to stain live cells. The results show that TAF can promote the apoptosis of HeLa cells under laser irradiation, indicating that the nanoparticles have good cell killing effect under laser action.

[0169] Example 2:

[0170] The specific steps of this embodiment are the same as those of Embodiment 1 except for the following differences:

[0171] In step S1, the molar ratio of compound 1 to diphenylamine, palladium acetate (Pd(OAc)2), tri-tert-butylphosphine tetrafluoroborate (P(t-Bu)3·HBF4), and potassium tert-butoxide (t-BuOK) is 1:2.0:0.10:0.20:10; the reaction is refluxed at 110° C. for 20 h;

[0172] In step S2, the molar ratio of compound 2 to N-bromosuccinimide is 1:1.1; after the dropwise addition is completed, stirring is continued for 1.5 hours in an ice-water bath, and then stirring is continued for reaction at room temperature for 8 hours;

[0173] In step S3, the molar ratio of compound 3 to 4-methoxyphenylboronic acid pinacol ester, potassium carbonate (K2CO3), and tetrakistriphenylphosphine palladium (Pd(PPh3)4) is 1:2.0:10.0:0.10; the reaction is refluxed at 70°C for 24h;

[0174] In step S4, the molar ratio of compound 4 to N-bromosuccinimide is 1:1.1; after the dropwise addition is completed, stirring is continued for 1.5 hours in an ice-water bath, and then the reaction is carried out at room temperature for 8 hours;

[0175] In step S5, the molar ratio of compound 5 to bis(pinacol borate), potassium acetate (CH3COOK), and Pd(PPh3)2Cl2 is 1:3.0:10.0:0.01; the reaction is carried out at 100°C for 24h;

[0176] In step S6, the molar ratio of compound 6 to 3-bromofluorenone, K2CO3, and Pd(PPh3)4 is 1:1.0:10.0:0.10; the reaction is refluxed at 70°C for 24h;

[0177] In step S7, the molar ratio of compound 7 to malononitrile is 1:5.0; the reaction is refluxed at 110° C. for 24 h;

[0178] In step S8, the molar ratio of compound 8 to BBr3 is 1:6.0; after the dropwise addition is completed, stirring is continued for 1.5 hours in an ice-water bath, and then the reaction is carried out at room temperature for 6 hours;

[0179] In step S9, the molar ratio of compound 9 to 1,6-diiodohexane and potassium carbonate is 1:12.0:8.0; the reaction is refluxed at 30° C. for 22 hours;

[0180] In step S10, the molar ratio of compound 10 to sodium azide is 1:5.0, and the reaction is carried out at 20°C for 20 hours.

[0181] The intermediate products and the target product obtained in this embodiment are basically consistent with those in Embodiment 1 in terms of yield and characterization results.

[0182] The above is only a specific embodiment of the present invention. It should be noted that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. The rest not described in detail is the prior art.

Claims

1. A multifunctional AIE optical diagnosis and treatment molecule, characterized in that: The multifunctional AIE optical diagnostic and therapeutic molecule uses triphenylamine as an electron donor (D) and malononitrile fluorene as an electron acceptor (A), and is screened by adjusting the connection mode between the electron donor and the electron acceptor; the structural formula of the multifunctional AIE optical diagnostic and therapeutic molecule is as follows:

2. The synthesis method of the multifunctional AIE optical diagnosis and treatment molecule according to claim 1, characterized in that: The synthesis route of the multifunctional AIE optical diagnostic and therapeutic molecule is as follows:

3. The synthesis method of the multifunctional AIE optical diagnosis and treatment molecule according to claim 2, characterized in that: The steps include: S1: Add compound 1, diphenylamine, palladium acetate (Pd(OAc)2), tri-tert-butylphosphine tetrafluoroborate (P(t-Bu)3·HBF4), and potassium tert-butoxide (t-BuOK) into a container, then add anhydrous toluene, introduce inert gas into the container, heat and stir, and reflux at 100-110°C for 18-20h to obtain compound 2; S2: Compound 2 and N-bromosuccinimide (NBS) were dissolved in anhydrous dichloromethane (DCM), respectively, the compound 2 / DCM solution was placed in an ice-water bath, and the NBS / DCM solution was added dropwise thereto. After the addition was completed, the mixture was stirred for 1-1.5 h in an ice-water bath, and then the mixture was stirred for 5-8 h at room temperature to obtain compound 3; S3: Add compound 3 and 4-methoxyphenylboronic acid pinacol ester, potassium carbonate (K2CO3), tetrakistriphenylphosphine palladium (Pd(PPh3)4) into a container, then add deoxygenated tetrahydrofuran (THF) and deionized water, pass inert gas into the container and heat with stirring, reflux at 70-80°C for 20-24h to obtain compound 4; S4: Compound 4 and N-bromosuccinimide (NBS) were dissolved in anhydrous DCM respectively, the compound 4 / DCM solution was placed in an ice-water bath, and the NBS / DCM solution was added dropwise thereto. After the addition was completed, stirring was continued in an ice-water bath for 1-1.5 h, and then the reaction was carried out at room temperature for 5-8 h to obtain compound 5; S5: Add compound 5, bis(pinacol borate), potassium acetate (CH3COOK), and Pd(PPh3)2Cl2 into a container, then add dioxane, introduce inert gas into the container, heat and stir, and react at 100-120°C for 20-24h to obtain compound 6; S6: Add compound 6, 3-bromofluorenone, K2CO3, and Pd(PPh3)4 into a container, then add deoxygenated THF and deionized water, introduce inert gas into the container, heat and stir, and reflux at 70-80°C for 20-24h to obtain compound 7; S7: Compound 7 and malononitrile are added to a container, and then anhydrous pyridine is added, an inert gas is introduced into the container, and the container is heated and stirred, and refluxed at 110-120° C. for 20-24 h to obtain a multifunctional AIE optical diagnostic and therapeutic molecule; Wherein, the structural formula of the compound 1 is: The structural formula of the compound 2 is: The structural formula of the compound 3 is: The structural formula of the compound 4 is: The structural formula of the compound 5 is: The structural formula of the compound 6 is: The structural formula of the compound 7 is:

4. The method for synthesizing the multifunctional AIE optical diagnostic and therapeutic molecule according to claim 3, characterized in that: In the step S1, the molar ratio of compound 1 to diphenylamine, palladium acetate (Pd(OAc)2), tri-tert-butylphosphine tetrafluoroborate (P(t-Bu)3·HBF4), and potassium tert-butoxide (t-BuOK) is 1:(1.5-2.0):(0.01-0.10):(0.01-0.20):(3-10); In the step S2, the molar ratio of compound 2 to N-bromosuccinimide is 1:1.0-1.1; In the step S3, the molar ratio of compound 3 to 4-methoxyphenylboronic acid pinacol ester, potassium carbonate (K2CO3), and tetrakistriphenylphosphine palladium (Pd(PPh3)4) is 1:(1.5-2.0):(8.0-10.0):(0.02-0.10); In the step S4, the molar ratio of compound 4 to N-bromosuccinimide is 1:1.0-1.1; In the step S5, the molar ratio of compound 5 to bis(pinacol) borate, potassium acetate (CH3COOK), and Pd(PPh3)2Cl2 is 1:(2.0-3.0):(3.0-10.0):(0.01-0.10); In the step S6, the molar ratio of compound 6 to 3-bromofluorenone, K2CO3, and Pd(PPh3)4 is 1:(1.0-1.5):(5.0-10.0):(0.05-0.10); In the step S7, the molar ratio of compound 7 to malononitrile is 1:5.0-10.

0.

5. An azide compound, characterized in that: The azide compound is obtained by modifying the multifunctional AIE optical diagnostic and therapeutic molecule according to claim 1 through azidation; the structural formula of the azide compound is as follows:

6. A method for synthesizing the azide compound according to claim 5, characterized in that: The synthetic route of the azide compound is as follows:

7. The method for synthesizing an azide compound according to claim 6, wherein The steps include: 1) The multifunctional AIE optical diagnostic and therapeutic molecule and boron tribromide (BBr3) were dissolved in anhydrous DCM respectively, the multifunctional AIE optical diagnostic and therapeutic molecule / DCM solution was placed in an ice-water bath, and the BBr3 / DCM solution was added dropwise thereto. After the addition was completed, stirring was continued in an ice-water bath for 1-1.5 hours, and then the reaction was carried out at room temperature for 4-6 hours to obtain compound 9; 2) Compound 9, 1,6-diiodohexane and potassium carbonate were added to a container, and then anhydrous DMF and ethyl acetate (EA) were added. Inert gas was introduced into the flask and heated with stirring. The mixture was refluxed at 30-50° C. for 18-22 h to obtain compound 10; 3) Compound 10 and sodium azide were added to a container, and then anhydrous DMF was added. Inert gas was introduced into the flask and stirred. The reaction was carried out at 20-30° C. for 20-24 h to obtain azide compound 11; The structural formula of the multifunctional AIE optical diagnostic and therapeutic molecule is as follows: The structural formula of the compound 9 is as follows: The structural formula of the compound 10 is as follows: The structural formula of the compound 11 is as follows:

8. The method for synthesizing an azide compound according to claim 7, characterized in that: In the step 1), the molar ratio of the multifunctional AIE optical diagnostic molecule to BBr3 is 1:5.0-6.0; In the said step 2), the molar ratio of compound 9, 1,6-diiodohexane and potassium carbonate is 1:10.0 - 12.0:5.0 - 8.0; In the said step 3), the molar ratio of compound 10 and sodium azide is 1:3.0 - 5.

0.

9. Use of the multifunctional AIE optical diagnosis and treatment molecule according to claim 1 for preparing a tumor therapeutic agent.

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