Free radical type photosensitizer with fluorescence lightening characteristic and preparation method and application thereof

Abstract

The invention discloses a free radical type photosensitizer with a fluorescent lightening characteristic as well as a preparation method and application of the free radical type photosensitizer. The preparation method comprises the following steps: taking 4, 7-dibromo-2, 1, 3-benzothiazole and a triphenylamine derivative as raw materials, and carrying out Suzuki coupling reaction to obtain an intermediate product 1; carrying out Suzuki coupling reaction on the obtained intermediate product 1 and 4-aminophenylboronic acid pinacol ester to obtain an intermediate product 2; and reacting the obtained intermediate product 2 with maleic anhydride to obtain a final product. The method has the advantages that the maleimide which can specifically recognize the Cys and is a strong electron acceptor is introduced into the molecular skeleton of the photosensitizer, so that the photosensitizer can specifically recognize the Cys to be lightened by fluorescence, the generation efficiency of free radical type ROS is improved, and a good photodynamic therapy effect on cancer cells is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of photosensitizer preparation, and particularly relates to the application of a photosensitizer in the preparation of anti-tumor drugs. Background Art

[0002] Cysteine is an important biological thiol, widely present in cells and having various functions. The level of cysteine is related to various diseases, and abnormal concentrations can lead to edema, weakness, lethargy, hair loss, slow growth, liver damage, skin damage, etc. Cancer is a disease with a very high mortality rate. It is reported that cysteine is crucial for promoting the proliferation and survival of cancer cells and is highly expressed in tumors.

[0003] Photodynamic therapy (PDT) has unique advantages such as non-invasive / minimally invasive, strong controllability, good curative effect, and low drug resistance, and has important application values in clinical aspects such as cancer treatment, wound healing promotion, and anti-skin infection. Reactive oxygen species (ROS) generated by irradiating a photosensitizer with appropriate excitation light can effectively kill cancer cells, thereby achieving the effect of treating diseases. ROS can be divided into two categories. One is singlet oxygen mainly based on energy transfer, which has the problem of high oxygen dependence. The photosensitizer, oxygen, and excitation light source are three important components of PDT for generating this type of ROS. The other is free radical type ROS mainly based on electron transfer, which can still efficiently generate free radical type ROS even at low oxygen concentrations.

[0004] Therefore, developing a class of photosensitizers that can specifically recognize intracellular Cys and generate free radical type ROS is expected to achieve precise diagnosis and efficient treatment of tumors. Summary of the Invention

[0005] The present invention provides a free radical type photosensitizer with fluorescence "turn-on" characteristics, its preparation method and application, to solve the problem of the lack of non-invasive / minimally invasive, strong controllability, good curative effect, and low drug resistance anti-tumor drugs in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A free radical type photosensitizer with fluorescence "turn-on" characteristics, the structural formula is:

[0008]

[0009] The present invention also provides a preparation method of the photosensitizer, including the following steps:

[0010] (1) Using 4,7-dibromo-2,1,3-benzothiadiazole and a triphenylamine derivative as raw materials, obtaining intermediate 1 through a Suzuki coupling reaction;

[0011] (2) The obtained intermediate 1 and 4-aminophenylboronic acid pinacol ester are subjected to a Suzuki coupling reaction to obtain intermediate 2;

[0012] (3) The obtained intermediate 2 and maleic anhydride are reacted to obtain the final product TBZMA.

[0013] Furthermore, in the step (1), when 4,7-dibromo-2,1,3-benzothiazole reacts with the triphenylamine derivative, a catalyst and a salt need to be added. The molar ratio of 4,7-dibromo-2,1,3-benzothiazole, the triphenylamine derivative, the catalyst and the salt is 1:(1 - 1.2):(0.05 - 0.1):12;

[0014] In the step (2), when intermediate 1 reacts with 4-aminophenylboronic acid pinacol ester, a catalyst and a salt need to be added. The molar ratio of intermediate 1, 4-aminophenylboronic acid pinacol ester, the catalyst and the salt is 1:(1 - 1.2):(0.05 - 0.1):18;

[0015] In the step (3), when intermediate 2 reacts with maleic anhydride, sodium acetate anhydrous and acetic anhydride need to be added. The molar ratio of intermediate 2, maleic anhydride, sodium acetate anhydrous and acetic anhydride is 1:(1.8 - 2.2):(7.6 - 7.8):(330 - 335).

[0016] Furthermore, the detailed process of the step (1) includes: adding 1.5 mmol of 4,7-dibromo-2,1,3-benzothiazole, 1.8 mmol of 4-borotriphenylamine, 0.06 mmol of Pd(PPh 3 ) 4 and 18 mmol of anhydrous potassium carbonate into a mixed solvent of tetrahydrofuran and water, with the volume ratio of tetrahydrofuran to water being 20 mL:8 mL, and reacting to obtain TBZ-Br;

[0017] The detailed process of the step (2) includes: adding 1 mmol of TBZ-Br, 1.2 mmol of 4-aminophenylboronic acid pinacol ester, 0.06 mmol of Pd(PPh 3 ) 4 and 18 mmol of anhydrous potassium carbonate into a mixed solvent of tetrahydrofuran and water, with the volume ratio of tetrahydrofuran to water being 20 mL:8 mL, and reacting to obtain TBZ-NH 2 ;

[0018] The detailed process of the step (3) includes: adding 0.64 mmol of TBZ-NH 2 and 1.28 mmol of maleic anhydride into tetrahydrofuran and stirring at room temperature overnight. After evaporation of the solvent, 0.4 g of sodium acetate anhydrous and 20 ml of acetic anhydride are added and stirred at 75 °C for 6 h. After the mixture is cooled, it is purified to obtain an orange-yellow powder TBZMA.

[0019] Further, in the steps (1) and (2), the conditions for the Suzuki coupling reaction are refluxing for 12 h at 100 °C under nitrogen; in the step (3), the reaction conditions for the reaction of the intermediate 2 with maleic anhydride are 75 °C for 6 h.

[0020] Further, the triphenylamine derivative is 4-boronic acid triphenylamine or a derivative of 4-boronic acid triphenylamine; the catalyst is tetrakis(triphenylphosphine)palladium; the salt is one of K 2 CO 3 、Na 2 CO 3 .

[0021] The present invention also provides an application of the photosensitizer in the preparation of anti-tumor drugs.

[0022] The present invention also provides a preparation method of a nanopolymer containing the photosensitizer according to claim 1, comprising the following steps:

[0023] The photosensitizer and the amphiphilic polymer liposome are dissolved in an organic solvent to obtain a mixed solution; the obtained mixed solution is mixed with water and then ultrasonically treated until uniformly dispersed, and the organic solvent is removed, and the amphiphilic polymer liposome coats the photosensitizer to obtain a nanopolymer containing the photosensitizer.

[0024] Further, the amphiphilic polymer liposome is DSPE-PEG 2000 , and the mass ratio of the photosensitizer to DSPE-PEG 2000 is 1:2.

[0025] The present invention also provides an application of the nanopolymer containing the photosensitizer obtained by the method in the preparation of anti-tumor drugs.

[0026] Compared with the prior art, the present invention has the following advantages and technical effects:

[0027] Maleimide, which has specific recognition for Cys and is a strong electron acceptor itself, is introduced into the molecular skeleton of the photosensitizer. After the photosensitizer reacts with the sulfhydryl group of Cys, the fluorescence signal is enhanced, the photosensitizer is "lit up", the electron donor and the electron acceptor are optimized, the generation of singlet oxygen is inhibited, and thus the generation of radical ROS is promoted, realizing the specific recognition of Cys by the photosensitizer and being "lit up" by fluorescence and improving the generation efficiency of radical ROS, and having a good photodynamic therapy effect on cancer cells.

[0028] By encapsulating the above free radical photosensitizers with amphiphilic polymer liposomes, the prepared AIE photosensitizer nanopolymer (TBZMANPs) has good water dispersibility, higher solubility, faster dissolution rate, longer circulation time in the bloodstream, and enhanced permeability and retention effect in tumor tissues. TBZMANPs can be rapidly phagocytosed by cells and exhibit bright red fluorescence, and can also effectively generate 1 O 2 and O 2 ·- free radical type ROS such as ·OH. The results of cytotoxicity evaluation show that TBZMANPs have good biocompatibility with cells without white light irradiation, but after white light irradiation with appropriate power, most cancer cells are killed. The results of in vivo tumor experiments show that TBZMANPs can effectively inhibit the growth of tumors in mice. Description of the Drawings

[0029] The drawings forming a part of this application are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation of this application. In the drawings:

[0030] Figure 1 is a schematic diagram of the synthesis process of the nanopolymer containing the AIE photosensitizer;

[0031] Figure 2 is a graph showing the ultraviolet absorption spectra and fluorescence spectra of the photosensitizers prepared in Examples 1-4 in a tetrahydrofuran solvent;

[0032] Figure 3 is a graph showing the fluorescence intensity of the photosensitizers prepared in Examples 1-4 in dimethyl sulfoxide / water mixed solvents with different water contents;

[0033] Figure 4 is a graph showing the change amplitude of fluorescence intensity after adding the photosensitizers prepared in Examples 1-4 and irradiating with light in DMSO / aqueous solutions containing H 2 DCF-DA probe, DHR123 probe, HPF probe, ABDA probe;

[0034] Figure 5 is a graph showing the fluorescence change results after adding Cys to the AIE photosensitizers prepared in Examples 1-4 and irradiating with light;

[0035] Figure 6 is a graph showing the change amplitude of fluorescence intensity after adding and irradiating with light with or without Cys to the photosensitizers in DMSO / aqueous solutions containing H 2 DCF-DA probe, DHR123 probe, HPF probe, ABDA probe;

[0036] Figure 7 It is a test result graph of the size distribution of nano-polymer TBZMANPs;

[0037] Figure 8 It is the fluorescence staining effect diagram of 4T1 cells incubated with TBZMANPs (10 μM);

[0038] Figure 9 It is for 4T1 cells incubated with TBZMA NPs (10 μM) containing 10 μM H 2 The fluorescence staining effect diagram of 4T1 cells with DCF-DA;

[0039] Figure 10 It is the MTT test result graph of the photodynamic therapy of 4T1 cells incubated with TBZMANPs at different culture concentrations;

[0040] Figure 11 It is the calcein-AM / PI live-dead staining effect diagram of 4T1 cells incubated with 10 μM TBZMANPs and subjected to photodynamic therapy;

[0041] Figure 12 It is the fluorescence imaging graph of mice tumors after injecting TBZMANPs, as well as the statistical graphs of tumor growth and body weight changes. Among them, A is the fluorescence imaging graph of different time lengths after injecting TBZMANPs into the tumor, B is the statistical graph of tumor volume changes in different groups, C is the tumor graph of different groups, and D is the statistical graph of body weight changes of mice in different groups. Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0044] In the embodiments of the present invention, room temperature refers to "25 ± 2 °C".

[0045] Unless otherwise specified, the raw materials in the embodiments of the present invention are all obtained through commercial purchase channels.

[0046] Example 1

[0047] This embodiment provides a radical-based photosensitizer with fluorescence "turn-on" properties, which is an aggregation-induced emission photosensitizer (AIE photosensitizer) that can be "turned on" by Cys and improve the generation efficiency of radical reactive oxygen species. Its structural formula is:

[0048]

[0049] The synthesis route is as follows:

[0050]

[0051] The specific preparation process is as follows:

[0052] (1) Synthesis of compound TBZ-Br

[0053] 4,7-Dibromo-2,1,3-benzothiazole (440 mg, 1.5 mmol), 4-(triphenylamino)benzeneboronic acid (520 mg, 1.8 mmol), Pd(PPh 3 ) 4 (68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water is 20 mL:8 mL), and then refluxed at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate. Column chromatography was carried out using DCM and petroleum ether (the volume ratio of DCM to petroleum ether is 1:30) as the eluent to obtain bright yellow powder TBZ-Br (754 mg).

[0054] The characterization data of the obtained TBZ-Br are as follows: 1 H NMR(500MHz,CDCl 3 )δ7.90(d,1H),7.80(d,2H),7.54(d,1H),7.32-7.27(m,4H),7.21-7.14(m,6H),7.10-7.05(m,2H).

[0055] (2) Synthesis of compound TBZ-NH 2

[0056] TBZ-Br (460 mg, 1 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (260 mg, 1.2 mmol), Pd(PPh 3 ) 4 ​(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 20 mL:8 mL), and then refluxed at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate. Column chromatography was carried out using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:30) as the eluent to obtain the yellow powder TBZ-NH 2 (438 mg).

[0057] The obtained TBZ-NH 2 The characterization data were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.08 (d, 2H), 7.92 (d, 4H), 7.49 (d, 2H), 7.32 (t, 4H), 7.20 (s, 2H), 7.08 (d, 8H).

[0058] (3) Synthesis of compound TBZMA

[0059] TBZ-NH 2 (300 mg, 0.64 mmol) and maleic anhydride (125 mg, 1.28 mmol) were added to tetrahydrofuran and stirred at room temperature overnight. After the solvent was evaporated, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate. Column chromatography was carried out using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:20) as the eluent to obtain the orange-yellow powder TBZMA (308 mg).

[0060] The characterization data of the obtained TBZMA were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.08 (d, 2H), 7.92 (d, 4H), 7.49 (d, 2H), 7.32 (t, 4H), 7.20 (s, 2H), 7.08 (d, 8H).

[0061] (4) Synthesis of TBZMA NPs

[0062] As Figure 1 shown, TBZMA (1.0 mg) and DSPE-PEG 2000 (2.0 mg) were fully dissolved in 1 mL of tetrahydrofuran, and then placed in 10 mL of distilled water and sonicated until evenly dispersed. The obtained solution was stirred for 24 h until the tetrahydrofuran was removed to obtain a clear aqueous solution, which was the nano-polymer TBZMA NPs.

[0063] Example 2

[0064] This example provides a radical photosensitizer with fluorescence "turn-on" properties, which is an aggregation-induced emission photosensitizer (AIE photosensitizer) that is fluorescently turned on by Cys and improves the generation efficiency of radical reactive oxygen species. Its structural formula is:

[0065]

[0066] The synthesis route is as follows:

[0067]

[0068] (1) Synthesis of compound MTBZ-Br

[0069] 4,7-Dibromo-2,1,3-benzothiazole (440 mg, 1.5 mmol), 4-boronic acid-4`,4`-dimethoxytriphenylamine (630 mg, 1.8 mmol), Pd(PPh 3 ) 4 (68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 20 mL:8 mL), and then refluxed at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate. Column chromatography was carried out using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:30) as the eluent to obtain orange-red powder MTBZ-Br (558 mg).

[0070] The characterization data of the obtained MTBZ-Br are as follows: 1 H NMR(400MHz,CD 2 Cl 2 )δ7.88(d,1H),7.75(s,2H),7.52(d,1H),7.31-6.70(m,10H),3.80(s,6H).

[0071] (2) Synthesis of compound MTBZ-NH 2

[0072] MTBZ-Br (500 mg, 0.96 mmol), 4-aminophenylboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh 3 ) 4 ​(68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 20 mL:4 mL), and then refluxed at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate. Column chromatography was carried out using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:25) as the eluent to obtain a red powder MTBZ-NH 2 (362 mg).

[0073] The obtained MTBZ-NH 2 had the following characterization data: 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (d, 2H), 7.74 (dd, 4H), 7.07 (d, 4H), 6.92 (d, 4H), 6.86 (d, 2H), 6.68 (d, 2H), 5.42 (s, 2H), 3.73 (s, 6H).

[0074] (3) Synthesis of compound MTBZMA

[0075] MTBZ-NH 2 (300 mg, 0.56 mmol) and maleic anhydride (120 mg, 1.2 mmol) were added to tetrahydrofuran and stirred at room temperature overnight. After the solvent was evaporated, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate. Column chromatography was carried out using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:20) as the eluent to obtain a red powder MTBZMA (281 mg).

[0076] The obtained MTBZMA had the following characterization data: 1 H NMR (500 MHz, DMSO-d6) δ 8.13 - 8.09 (m, 1H), 7.99 - 7.87 (m, 2H), 7.56 - 7.50 (m, 1H), 7.24 (d, 1H), 7.16 - 7.09 (m, 2H), 7.00 - 6.94 (m, 2H), 6.90 (d, 1H), 3.77 (s, 3H).

[0077] (4) Synthesis of MTBZMANPs

[0078] As Figure 1 shown, MTBZMA (1.0 mg) and DSPE-PEG 2000(2.0 mg) was fully dissolved in 1 mL of tetrahydrofuran, then placed in 10 mL of distilled water, and ultrasonically treated until evenly dispersed. The resulting solution was stirred for 24 h until the tetrahydrofuran was removed, obtaining a clear aqueous solution, which was the nano-polymer MTBZMANPs.

[0079] Example 3

[0080] This example provides a radical-based photosensitizer with fluorescence "turn-on" properties, which is an aggregation-induced emission photosensitizer (AIE photosensitizer) that is fluorescently turned on by Cys and improves the generation efficiency of radical reactive oxygen species. Its structural formula is:

[0081]

[0082] The synthesis route is:

[0083]

[0084] (1) Synthesis of compound TNZ-Br

[0085] 4,9-Dibromonaphtho[2,3-c][1,2,5]thiadiazole (500 mg, 1.45 mmol), 4-triphenylamine boronic acid (520 mg, 1.8 mmol), Pd(PPh 3 ) 4 (68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 20 mL:8 mL), and then refluxed at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried with anhydrous sodium sulfate. Column chromatography was carried out using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:30) as the eluent to obtain red powder TNZ-Br (520 mg).

[0086] The characterization data of the obtained TNZ-Br are as follows: 1 H NMR (500 MHz, CD 2 Cl 2 ) δ 8.33 (d, 1H), 8.07 (d, 1H), 7.64 (t, 1H), 7.55 (d, 2H), 7.48 (t, 1H), 7.41 (t, 4H), 7.19 - 7.11 (m, 8H).

[0087] (2) Synthesis of compound TNZ-NH 2

[0088] ​TNZ-Br (500 mg, 1 mmol), 4-aminophenylboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh 3 ) 4 (68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 1.8 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 20 mL:8 mL). Then, the mixture was refluxed at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and the organic layer was dried over anhydrous sodium sulfate. Column chromatography was performed using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:25) as the eluent to obtain a purple-red powder TNZ-NH 2 (186 mg).

[0089] The characterization data of the obtained TNZ-NH 2 were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.01 (d, 1H), 7.94 (d, 1H), 7.47 (d, 2H), 7.40 - 7.23 (m, 8H), 7.18 - 7.01 (m, 8H), 6.75 (d, 2H), 5.38 (s, 2H).

[0090] (3) Synthesis of compound TNZMA

[0091] TNZ-NH 2 (300 mg, 0.58 mmol) and maleic anhydride (120 mg, 1.2 mmol) were added to tetrahydrofuran and stirred at room temperature overnight. After the solvent was evaporated, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and the organic layer was dried over anhydrous sodium sulfate. Column chromatography was performed using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:20) as the eluent to obtain a red-black powder TNZMA (176 mg).

[0092] The characterization data of the obtained TNZMA were as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.09 - 8.05 (m, 1H), 7.94 - 7.90 (m, 1H), 7.79 - 7.74 (m, 2H), 7.62 - 7.56 (m, 4H), 7.53 - 7.49 (m, 2H), 7.40 (dd, 4H), 7.28 (s, 2H), 7.23 - 7.17 (m, 6H), 7.14 (t, 2H).

[0093] (4) Synthesis of TNZMANPs

[0094] As Figure 1 shown, TNZMA (1.0 mg) and DSPE-PEG 2000 (2.0 mg) were fully dissolved in 1 mL of tetrahydrofuran, and then placed in 10 mL of distilled water. After ultrasonic treatment until evenly dispersed, the resulting solution was stirred for 24 h until the tetrahydrofuran was removed, obtaining a clear aqueous solution, which was the nano-polymer TNZMANPs.

[0095] Example 4

[0096] This example provides a radical-based photosensitizer with fluorescence "turn-on" properties, which is an aggregation-induced emission photosensitizer (AIE photosensitizer) that is fluorescently turned on by Cys and improves the generation efficiency of radical reactive oxygen species. Its structural formula is:

[0097]

[0098] The synthesis route is:

[0099]

[0100] (1) Synthesis of compound MTNZ-Br

[0101] 4,9-Dibromonaphtho[2,3-c][1,2,5]thiadiazole (500 mg, 1.45 mmol), 4-boronic acid-4`,4`-dimethoxytriphenylamine (630 mg, 1.8 mmol), Pd(PPh 3 ) 4 (68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 20 mL:8 mL), and then refluxed at 100 °C under nitrogen for 12 h. After the mixture was cooled, it was extracted three times with dichloromethane (DCM), then washed with brine, and then the organic layer was dried over anhydrous sodium sulfate. Then, DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:30) were used as eluents for column chromatography to obtain red powder MTNZ-Br (518 mg).

[0102] The characterization data of the obtained MTNZ-Br are as follows: 1 H NMR (500 MHz, CD 2 Cl 2 ) δ 8.47 - 8.24 (m, 1H), 8.14 - 8.00 (m, 1H), 7.61 - 7.49 (m, 1H), 7.43 - 7.35 (m, 3H), 7.21 (t, 4H), 7.04 (dd, 2H), 6.90 (d, 4H), 3.81 (d, 6H).

[0103] (2) Compound MTNZ-NH 2 Synthesis

[0104] MTNZ-Br (500 mg, 0.88 mmol), 4-aminophenylboronic acid pinacol ester (260 mg, 1.2 mmol), Pd(PPh 3 3)2 4 (68 mg, 0.06 mmol) and anhydrous potassium carbonate (2.4 g, 18 mmol) were added to a mixed solvent of tetrahydrofuran and water (the volume ratio of tetrahydrofuran to water was 20 mL:8 mL). Then, the mixture was refluxed at 100 °C under nitrogen for 12 h. After cooling, the mixture was extracted three times with dichloromethane (DCM), washed with brine, dried over anhydrous sodium sulfate, and then column chromatography was performed using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:25) as the eluent to obtain a purple-red powder MTNZ-NH 2 (168 mg).

[0105] The characterization data of the obtained MTNZ-NH 2 were as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 8.00 (dd, 2H), 7.61 (dd, 2H), 7.45 - 7.36 (m, 4H), 7.30 (dd, 3H), 7.17 (d, 3H), 7.03 - 6.92 (m, 5H), 6.76 (d, 2H), 6.54 (d, 1H), 3.74 (s, 6H).

[0106] (3) Synthesis of compound MTNZMA

[0107] MTNZ-NH 2 (300 mg, 0.52 mmol) and maleic anhydride (120 mg, 1.2 mmol) were added to tetrahydrofuran and stirred at room temperature overnight. After evaporation of the solvent, anhydrous sodium acetate (0.4 g) and acetic anhydride (20 ml) were added and stirred at 75 °C for 6 h. After cooling, the mixture was extracted three times with dichloromethane (DCM), washed with brine, dried over anhydrous sodium sulfate, and then column chromatography was performed using DCM and petroleum ether (the volume ratio of DCM to petroleum ether was 1:20) as the eluent to obtain a red-black powder MTNZMA (172 mg).

[0108] The characterization data of the obtained MTNZMA were as follows: 11H NMR (500 MHz, DMSO-d6) δ 8.11 - 8.04 (m, 1H), 7.94 - 7.87 (m, 1H), 7.84 - 7.72 (m, 2H), 7.69 - 7.36 (m, 7H), 7.29 - 7.16 (m, 5H), 7.05 - 6.90 (m, 6H), 3.84 - 3.72 (m, 6H).

[0109] (4) Synthesis of MTNZMANPs

[0110] As Figure 1 shown, MTNZMA (1.0 mg) and DSPE-PEG 2000 (2.0 mg) were fully dissolved in 1 mL of tetrahydrofuran, then placed in 10 mL of distilled water, and ultrasonicated until evenly dispersed. The resulting solution was stirred for 24 h until the tetrahydrofuran was removed, obtaining a clear aqueous solution, which was the nanopolymer MTNZMANPs.

[0111] Experiment 1

[0112] Test the ultraviolet absorption spectra and fluorescence spectra of the photosensitizers prepared in Test Examples 1 - 4 in a tetrahydrofuran solvent. As can be seen from Figure 2 it, the maximum absorption peaks of TBZMA, MTBZMA, TNZMA, and MTNZMA are at 438 nm, 458 nm, 500 nm, and 520 nm respectively, while the maximum fluorescence peaks of TBZMA, MTBZMA, TNZMA, and MTNZMA are at 605 nm, 663 nm, 658 nm, and 578 nm respectively. The Stokes shifts of TBZMA, MTBZMA, TNZMA, and MTNZMA are 167 nm, 205 nm, 158 nm, and 58 nm respectively, preliminarily indicating that the molecules of TBZMA, MTBZMA, and TNZMA all have large Stokes shifts.

[0113] Experiment 2

[0114] Test the fluorescence intensities of the photosensitizers prepared in Test Examples 1 - 4 in dimethyl sulfoxide / water mixed solvents with water contents (v / v) of 0, 20%, 40%, 60%, and 80% respectively. The fluorescence intensity increases and fluorescence effects are as Figure 3 shown.

[0115] Experiment 3

[0116] Test the change amplitudes of the fluorescence intensities after irradiating the DMSO / aqueous solutions containing H 2 DCF-DA ( Figure 4 abbreviated as DCFH in it), DHR123 probe, HPF probe, and ABDA probe and adding the photosensitizers prepared in Examples 1 - 4 respectively. The results are as Figure 4 shown, among which,Figure 4 A shows the fluorescence intensity change amplitude diagrams after adding TBZMA, MTBZMA, TNZMA, and MTNZMA to the DMSO / aqueous solution containing the H 2 DCF-DA probe and irradiating with light, λ ex = 488 nm; Figure 4 B shows the fluorescence intensity change amplitude diagrams after adding TBZMA, MTBZMA, TNZMA, and MTNZMA to the DMSO / aqueous solution containing the DHR123 probe and irradiating with light, λ ex = 490 nm; Figure 4 C shows the fluorescence intensity change amplitude diagrams after adding TBZMA, MTBZMA, TNZMA, and MTNZMA to the DMSO / aqueous solution containing the HPF probe and irradiating with light, λ ex = 490 nm; Figure 4 D shows the fluorescence intensity change amplitude diagrams after adding TBZMA, MTBZMA, TNZMA, and MTNZMA to the DMSO / aqueous solution containing the ABDA probe and irradiating with light, λ abs = 400 nm. It can be seen from Figure 4 that among the four AIE photosensitizers of TBZMA, MTBZMA, TNZMA, and MTNZMA, all generate reactive oxygen species, and all generate radical-type reactive oxygen species. Among them, the three AIE photosensitizers of MTBZMA, TNZMA, and MTNZMA only generate radical-type reactive oxygen species, while TBZMA generates both radical-type reactive oxygen species and singlet oxygen, and TBZMA has the highest efficiency of generating reactive oxygen species.

[0117] Experiment 4

[0118] Test the fluorescence change after adding Cys and irradiating with light for the AIE photosensitizers prepared in Test Examples 1-4. The results are as Figure 5 shown. It is found that only when the photosensitizer TBZMA is added with Cys and irradiated with light, there is an obvious fluorescence enhancement. The subsequent experiment uses the photosensitizer TBZMA.

[0119] Experiment 5

[0120] Test the fluorescence intensity change after adding the photosensitizer TBZMA with or without Cys and irradiating with light in the DMSO / aqueous solution containing the H 2 DCF-DA probe, DHR123 probe, HPF probe, or ABDA probe. The results are as Figure 6 shown. It can be seen from the figure that after adding Cys to the photosensitizer TBZAM, ·OH and 1 O 2 are quenched, and only O 2 ·- is generated, realizing the conversion of ROS.

[0121] Experiment 6

[0122] The size distribution of nano-polymer TBZMANPs was measured using a laser particle size analyzer. The results are as Figure 7 shown. The size of nano-polymer TBZMANPs mainly concentrates around 118 nm, indicating that it has a high surface area to volume ratio, thus ensuring higher solubility and faster dissolution rate. Such small particles also have a longer circulation time in the bloodstream and enhanced permeability and retention effects in tumor tissues.

[0123] Experiment 7

[0124] 4T1 cells were incubated with 10 μM TBZMANPs, and the fluorescence staining effects were observed at incubation times of 0.5 h, 3 h, 4 h, and 6 h respectively. The results are Figure 8 shown. It can be seen that the photosensitizer TBZMANPs can quickly enter 4T1 cells.

[0125] Experiment 8

[0126] 4T1 cells containing 10 μM H 2 DCF-DA were incubated with 10 μM TBZMANPs, and the fluorescence staining effects were observed at incubation times of 0 s, 10 s, 20 s, and 40 s respectively. The results are as Figure 9 shown. TBZMANPs can generate effective reactive oxygen species in 4T1 cells.

[0127] Experiment 9

[0128] The photodynamic therapy effects of 4T1 cells incubated with TBZMANPs at culture concentrations of 0 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, and 32 μM were tested. The results are as Figure 10 shown. When the culture concentration of TBZMANPs was 1 μM, after white light irradiation, the cell viability was only 20%, indicating that TBZMANPs have good photodynamic therapy effects on cancer cells.

[0129] Experiment 10

[0130] 4T1 cells were incubated with 10 μM TBZMANPs and subjected to photodynamic therapy, and then calcein-AM / PI live-dead staining was performed. The results are as Figure 11 shown. Incubation of 4T1 cells with TBZMANPs has no biological dark toxicity to cells. After white light irradiation, only PI staining is observed, indicating that the photosensitizer has good cellular phototoxicity. The photosensitizer TBZMANPs has good photodynamic therapy effects on cancer cells.

[0131] Experiment 11

[0132] TBZMANPs were injected into the tumors of tumor-bearing mice, and in vivo fluorescence imaging was used to observe the tumor growth. The results are as Figure 12 shown. As can be seen from Figure 12 A, after injecting TBZMANPs into the tumor for 2 h, obvious fluorescence imaging was observed in the tumor, and the signal-to-noise ratio was low, indicating that TBZMANPs had good in vivo fluorescence imaging effects. As can be seen from Figure 12 B and Figure 12 C, it can be seen that the tumors in the non-photodynamic treatment group of TBZMANPs without light illumination proliferated rapidly, while after photodynamic treatment, the growth of tumors could be effectively inhibited, indicating that TBZMANPs still had good effects in the treatment of in vivo tumors. In addition, Figure 12 D shows that the body weight growth trends of the mice in all experimental groups were the same, indicating that TBZMANPs did not cause adverse effects on the mice and had good biosafety.

[0133] The above is only a preferred specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A free radical photosensitizer with fluorescent "lighting" characteristics, characterized in that: The structural formula is:

2. A method for preparing a photosensitizer as claimed in claim 1, characterized in that: The following steps are involved: (1) Using 4,7-dibromo-2,1,3-benzothiazole and a triphenylamine derivative as raw materials, the intermediate 1 was obtained by Suzuki coupling reaction; (2) subjecting the obtained intermediate product 1 and 4-aminophenylboronic acid pinacol ester to a Suzuki coupling reaction to obtain an intermediate product 2; (3) The intermediate product 2 is reacted with maleic anhydride to obtain the final product TBZMA.

3. The preparation method according to claim 2, characterized in that: In the step (1), the reaction of 4,7-dibromo-2,1,3-benzothiazole with the triphenylamine derivative requires the addition of a catalyst and a salt, and the molar ratio of 4,7-dibromo-2,1,3-benzothiazole, the triphenylamine derivative, the catalyst and the salt is 1:(1-1.2):(0.05-0.1):12; In the step (2), the reaction between the intermediate product 1 and 4-aminophenylboronic acid pinacol ester requires the addition of a catalyst and a salt, and the molar ratio of the intermediate product 1, 4-aminophenylboronic acid pinacol ester, the catalyst and the salt is 1:(1-1.2):(0.05-0.1):18; In the step (3), the reaction of the intermediate product 2 with maleic anhydride requires the addition of anhydrous sodium acetate and acetic anhydride, and the molar ratio of the intermediate product 2, maleic anhydride, anhydrous sodium acetate and acetic anhydride is 1:(1.8-2.2):(7.6-7.8):(330-335).

4. The preparation method according to claim 3, characterized in that: The detailed process of the step (1) includes: adding 1.5 mmol of 4,7-dibromo-2,1,3-benzothiazole, 1.8 mmol of 4-boric acid triphenylamine, 40.06 mmol of Pd(PPh3) and 18 mmol of anhydrous potassium carbonate to a mixed solvent of tetrahydrofuran and water, wherein the volume ratio of tetrahydrofuran to water is 20 mL:8 mL, and reacting to obtain TBZ-Br; The detailed process of the step (2) includes: adding 1 mmol of TBZ-Br, 1.2 mmol of 4-aminophenylboronic acid pinacol ester, 40.06 mmol of Pd(PPh3) and 18 mmol of anhydrous potassium carbonate into a mixed solvent of tetrahydrofuran and water, wherein the volume ratio of tetrahydrofuran to water is 20 mL:8 mL, and reacting to obtain TBZ-NH2; The detailed process of step (3) includes: adding 0.64 mmol of TBZ-NH2 and 1.28 mmol of maleic anhydride to tetrahydrofuran and stirring at room temperature overnight; after the solvent evaporates, adding 0.4 g of anhydrous sodium acetate and 20 ml of acetic anhydride and stirring at 75° C. for 6 h; cooling the mixture and purifying it to obtain orange-yellow powder TBZMA.

5. The preparation method according to claim 2, characterized in that: In the steps (1) and (2), the Suzuki coupling reaction conditions are 100° C. and reflux under nitrogen for 12 h; in the step (3), the reaction conditions of the intermediate 2 and maleic anhydride are 75° C. and the reaction is for 6 h.

6. The preparation method according to claim 2, characterized in that: The triphenylamine derivative is 4-boric acid triphenylamine or a derivative of 4-boric acid triphenylamine; the catalyst is tetrakis triphenylphosphine palladium; and the salt is one of K2CO3 and Na2CO3.

7. Use of the photosensitizer according to claim 1 in preparing drugs for treating tumors.

8. A method for preparing a nanopolymer containing the photosensitizer according to claim 1, characterized in that: The following steps are involved: The photosensitizer and amphiphilic polymer liposome are dissolved in an organic solvent to obtain a mixed solution; the mixed solution is mixed with water and then ultrasonically treated until uniformly dispersed, the organic solvent is removed, and the amphiphilic polymer liposome encapsulates the photosensitizer to obtain a nano polymer containing the photosensitizer.

9. The method for preparing a nano polymer containing a photosensitizer according to claim 8, characterized in that: The amphiphilic polymer liposome is DSPE-PEG 2000 , the photosensitizer and DSPE-PEG 2000 The mass ratio is 1:

2.

10. Use of the nanopolymer containing photosensitizer obtained by the method according to claims 8-9 in preparing drugs for treating tumors.

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