A purine skeleton-based aggregation-induced emission photosensitizer and its preparation method and application

Abstract

The present invention discloses an aggregation-induced luminescence photosensitizer based on a purine skeleton, a preparation method thereof, and an application thereof, and belongs to the field of biochemical technology. The present invention uses purine as the basic skeleton, and by constructing a D-π-A type system, increases the electron push-pull effect and the π conjugated area, effectively improving the active oxygen generation efficiency of the photosensitizer, having a high type I and type II active oxygen generation capacity, and exhibiting an excellent photodynamic ablation effect on cancer cells; in addition, the aggregation-induced luminescence characteristics enable this type of photosensitizer to have cell-free imaging capabilities and more efficient photodynamic therapy capabilities. The present invention solves the problem of unsatisfactory active oxygen generation efficiency of existing photosensitizers. In addition, the present invention also has the characteristics of cheap and readily available raw materials, simple preparation methods, and mild reaction conditions.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and in particular relates to an aggregation-induced luminescence photosensitizer based on a purine skeleton, and a preparation method and application thereof. Background Art

[0002] As an efficient treatment method, photodynamic therapy has been widely used in integrated diagnosis and treatment due to its unique advantages of high temporal and spatial precision, controllability, non-invasiveness, low toxicity and side effects, and its inherent fluorescence can provide real-time monitoring. The principle of photodynamic therapy is that under the irradiation of light of a specific wavelength, the photosensitizer absorbs light energy and undergoes energy level transition. The generated electrons combine with the surrounding oxygen to generate reactive oxygen species ROS (such as OH, 1 O2, superoxide anions, etc.), oxidize biological macromolecules such as lipids, proteins and DNA in cells, and then induce the death of bacteria or tumor cells.

[0003] However, traditional photosensitizers are prone to aggregation under physiological conditions, resulting in a decrease in the efficiency of generating reactive oxygen species and a decrease in fluorescence quantum yield, which is not conducive to practical development and application. However, the introduction of aggregation-induced emission has successfully solved the problem of fluorescence quenching caused by aggregation. Photosensitizers with aggregation-induced emission properties not only have high luminescence efficiency in the aggregated state, but can also efficiently generate reactive oxygen species. They have important application prospects in the field of fluorescence imaging-guided photodynamic therapy. Studies have also shown that aggregation-induced emission photosensitizers can achieve better photodynamic therapy effects by constructing D-π-A structures. The design of this type of photosensitizer has become a research hotspot in the field of photodynamic therapy in recent years.

[0004] However, most current photosensitizers still suffer from suboptimal ROS generation efficiency. Therefore, the development of new and more advantageous photosensitizers is crucial. Purines possess excellent biocompatibility and optical properties in vivo. Furthermore, the large π-conjugated plane and multiple modification sites of the purine ring facilitate the construction of D-π-A type photosensitizers. Furthermore, polynitrogen heterocycles can effectively promote intersystem crossing and enhance ROS generation. These characteristics of the purine ring give it great potential for the construction of highly efficient photosensitizers. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention provides an aggregation-induced emission photosensitizer based on a purine skeleton, and a preparation method and application thereof, to solve the problem of unsatisfactory efficiency of active oxygen generation of photosensitizers in the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is to provide an aggregation-induced emission photosensitizer based on a purine skeleton, characterized in that its structure is shown in formula (I):

[0007]

[0008] Wherein, R1 is C1-C20 alkyl; R2 is Wherein, X is PF6.

[0009] The beneficial effects of the present invention are: the present invention realizes the efficient generation of reactive oxygen species, including type I and type II reactive oxygen species, and at the same time the photosensitizer can quickly kill cancer cells, and has good application prospects in photodynamic anti-tumor.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows.

[0011] Further, R1 is methyl, R2 is

[0012] Furthermore, the structural formula of the aggregation-induced emission photosensitizer based on the purine skeleton is shown in (II):

[0013]

[0014] The beneficial effect of adopting the above further technical solution is: making the compound have a larger conjugated plane, promoting the intersystem crossing process and the generation of active oxygen.

[0015] The present invention also provides an aggregation-induced emission photosensitizer based on a purine skeleton and a preparation method thereof, comprising the following steps:

[0016] S1: Synthesis of the first intermediate: 2,6-dichloropurine and a base are dissolved in an organic solvent, heated and stirred at 45-55°C for 30 minutes, and then a C1-C20 substituted alkane is added. Heating and stirring are continued for 4-6 hours, and the organic phase obtained by extraction is separated by column chromatography to obtain the first intermediate;

[0017] S2: Synthesis of the second intermediate: dissolving the first intermediate, triphenylamine boronic acid, a base, and a catalyst in a mixed solvent of water and 1,4-dioxane, heating and refluxing at 75-85°C under anaerobic conditions for 6-8 hours, and separating the organic phase obtained by extraction by column chromatography to obtain the second intermediate;

[0018] S3: Synthesis of the third intermediate: dissolving the second intermediate, 4-formylphenylboronic acid, a base, and a catalyst in a mixed solvent of water and 1,4-dioxane, heating and refluxing at 75-85°C under anaerobic conditions for 6-8 hours, and separating the organic phase obtained by extraction by column chromatography to obtain the third intermediate;

[0019] S4: Synthesis of an aggregation-induced emission photosensitizer based on a purine skeleton: The third intermediate and the matrix are dissolved in ethanol, piperidine is added, and the mixture is heated under reflux at 75-80° C. for 10-12 hours. A crude product is obtained by column chromatography, and the crude product is then dissolved in a saturated potassium hexafluorophosphate acetone solution, stirred at room temperature for 1-3 hours, and the acetone is removed. The purine skeleton-based aggregation-induced emission photosensitizer is obtained after extraction; the matrix is ​​one of the compounds represented by formula (III):

[0020]

[0021] Wherein, X is Cl, Br or I.

[0022] The beneficial effects of the present invention are as follows: the present invention introduces triphenylamine as a strong power-supplying group at position 6 of purine, introduces a benzene ring as a bridge ring at position 2 of purine, introduces quinoline salt as an electron-withdrawing group, and changes its electron-withdrawing ability by changing the methyl modification site, constructs a D-π-A type system from a structural perspective, increases the electron push-pull effect and the π conjugated area, and can effectively improve the active oxygen generation efficiency of the photosensitizer.

[0023] Furthermore, in S1, the molar ratio of 2,6-dichloropurine to the C1-C20 substituted alkane is 1:1; in S2, the molar ratio of the first intermediate to triphenylamine boronic acid is 1:1.2; in S3, the molar ratio of the second intermediate to 4-formylphenylboronic acid is 1:1.2; and in S4, the molar ratio of the third intermediate to the substrate is 1:1. Furthermore, in S2, the molar ratio of the first intermediate to triphenylamine boronic acid is 1:1.2.

[0024] The beneficial effect of adopting the above further technical solution is that this ratio can maximize the use of synthetic raw materials and maximize the product yield in each step.

[0025] Furthermore, the reagents used for extraction in S1 are ethyl acetate and water; the reagents used for extraction in S2 are dichloromethane and water; the reagents used for extraction in S3 are dichloromethane and water; and the reagents used for extraction in S4 are dichloromethane and saturated aqueous potassium hexafluorophosphate solution.

[0026] The beneficial effect of adopting the above further technical solution is that the selection of suitable extraction reagents improves the extraction efficiency, selectivity and reagent controllability, which helps to carry out efficient and sustainable chemical processes.

[0027] Furthermore, the S1 column chromatography used silica gel as the stationary phase and ethyl acetate: petroleum ether = 1:1 as the mobile phase; the S2 column chromatography used silica gel as the stationary phase and dichloromethane as the mobile phase; the S3 column chromatography used silica gel as the stationary phase and dichloromethane as the mobile phase; the S4 column chromatography used silica gel as the stationary phase and dichloromethane: methanol = 50:1 as the mobile phase.

[0028] The beneficial effect of adopting the above further technical solution is that the selection of appropriate column chromatography stationary phase and mobile phase can improve the separation effect, speed and stability of column chromatography and minimize the damage to the column packing.

[0029] Application of purine skeleton-based aggregation-induced emission photosensitizers in the preparation of fluorescent imaging agents or cancer therapeutic drugs.

[0030] The beneficial effects of the present invention are as follows: by constructing a D-π-A type system, the present invention effectively improves the reactive oxygen species generation efficiency of the photosensitizer, has a high type I and type II reactive oxygen species generation capacity, and exhibits excellent photodynamic ablation effect on cancer cells. In addition, the aggregation-induced luminescence characteristics enable this type of probe to have cell-free imaging capabilities and more efficient photodynamic therapy capabilities; compared with traditional photosensitizers, the aggregation-induced luminescence photosensitizer based on the purine skeleton provided by the present invention has a stronger photodynamic effect and improves the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The synthetic route diagram of the preparation method of the present invention;

[0032] Figure 2 is the hydrogen spectrum of the photosensitizer of Example 1;

[0033] Figure 3 is the carbon spectrum of the photosensitizer of Example 1;

[0034] Figure 4 This is a high-resolution mass spectrum of the photosensitizer of Example 1;

[0035] Figure 5 is the hydrogen spectrum of the photosensitizer of Example 2;

[0036] Figure 6 The carbon spectrum of the photosensitizer of Example 2;

[0037] Figure 7 This is a high-resolution mass spectrum of the photosensitizer of Example 2;

[0038] Figure 8 is the ultraviolet absorption spectrum of the photosensitizer of Example 1 in PBS solution;

[0039] Figure 9 1 is a linear relationship diagram between the concentration of the photosensitizer in PBS solution and the absorbance value of Example 1;

[0040] Figure 10 is the UV absorption spectrum of the photosensitizer of Example 2 in PBS solution;

[0041] Figure 11 is a linear relationship diagram between the concentration and absorbance of the photosensitizer in PBS solution of Example 2;

[0042] Figure 12 is the normalized fluorescence emission spectrum of the photosensitizer of Example 1 in different solvents;

[0043] Figure 13 is the normalized fluorescence emission spectrum of the photosensitizer of Example 1 in toluene / dimethyl sulfoxide solutions with different ratios;

[0044] Figure 14 is the normalized fluorescence emission spectrum of the photosensitizer of Example 2 in different solvents;

[0045] Figure 15 is the normalized fluorescence emission spectrum of the photosensitizer of Example 2 in toluene / dimethyl sulfoxide solutions at different ratios;

[0046] Figure 16 This is the singlet oxygen detection experiment of the photosensitizer in ABDA solution of Example 1;

[0047] Figure 17 This is the singlet oxygen detection experiment of the photosensitizer in ABDA solution of Example 2;

[0048] Figure 18 This is the hydroxyl radical detection experiment of the photosensitizer in HPF solution of Example 1;

[0049] Figure 19 This is the hydroxyl radical detection experiment of the photosensitizer in HPF solution of Example 2;

[0050] Figure 20 This is a CCK-8 cytotoxicity experiment of the photosensitizers of Examples 1 and 2 under dark and light conditions;

[0051] Figure 21 This is the photodynamically induced cell ablation imaging under the action of the photosensitizer of Examples 1 and 2. DETAILED DESCRIPTION

[0052] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0053] Example 1

[0054] A method for preparing an aggregation-induced emission photosensitizer based on a purine skeleton, the steps are as follows Figure 1 As shown, the specific steps include:

[0055] S1: Synthesis of the first intermediate: 2,6-dichloro-9-methylpurine

[0056] The synthetic route is as follows:

[0057]

[0058] 2,6-Dichloropurine (4.75 g, 25 mmol) and potassium carbonate (1.5 eq) were stirred in dimethyl sulfoxide (15 mL) at 50°C for 30 minutes. When bubbling almost ceased, iodomethane (1 eq) was added and stirring continued at 50°C for 5 hours. After the reaction was complete, the organic phase was extracted with ethyl acetate and water, and the extraction was repeated three times. The organic phase was dried over anhydrous sodium sulfate to remove all water, and then evaporated and concentrated under vacuum to obtain the crude product. The crude product was separated by column chromatography using silica gel as the stationary phase and ethyl acetate:petroleum ether = 1:1 as the mobile phase to obtain the first intermediate (Compound 1) as a white solid with a yield of 60%.

[0059] 1 H NMR (400MHz, Chloroform-d) δ8.07 (s, 1H), 3.90 (s, 3H).

[0060] S2: Synthesis of the second intermediate: 2-chloro-6-triphenylamine-9-methylpurine

[0061] The synthetic route is as follows:

[0062]

[0063] Compound 1 (1.01 g, 5 mmol), triphenylamine boronic acid (1.2 eq), tetrakistriphenylphosphine palladium (0.05 eq), and 2 mL of 2 M potassium carbonate solution were dissolved in 10 mL of 1,4-dioxane and refluxed under a nitrogen atmosphere for 8 hours. TLC spot plate monitoring was used. Once the reaction was nearly complete, the reaction mixture was extracted with dichloromethane and water. The organic phase was separated and extracted three times. The organic phase was dried over anhydrous sodium sulfate to remove all water, and then evaporated and concentrated under vacuum to obtain the crude product. The crude product was separated by column chromatography using silica gel as the stationary phase and dichloromethane as the mobile phase to obtain the second intermediate (compound 2) as a yellow solid in a 70% yield.

[0064] 1 H NMR (400MHz, Chloroform-d) δ 8.62 (d, J = 8.8 Hz, 2H), 7.99 (s, 1H), 7.28 (t, J = 7.8 Hz, 4H), 7.18-7.12 (m, 5H), 7.12-7.06 (m, 3H), 3.87 (s, 3H).

[0065] S3: Synthesis of the third intermediate: 4-(6-triphenylamine-9-methylpurine)-2-benzaldehyde

[0066] The synthetic route is as follows:

[0067]

[0068] Compound 2 (411 mg, 1 mmol), 4-formylphenylboronic acid (1.2 eq), tetrakistriphenylphosphine palladium (0.05 eq), and 2 mL of potassium carbonate solution (2 M) were dissolved in 10 mL of 1,4-dioxane and refluxed under a nitrogen atmosphere for 8 hours. TLC spot plate monitoring was performed. Once the reaction was almost complete, the reaction mixture was extracted with dichloromethane and water, and the organic phase was separated. This extraction was repeated three times; the organic phase was dried over anhydrous sodium sulfate to remove all water, and then evaporated and concentrated under vacuum to obtain a crude product. The crude product was separated by column chromatography using silica gel as the stationary phase and dichloromethane as the mobile phase to obtain the third intermediate (compound 3) as a yellow solid with a yield of 80%.

[0069] 1 H NMR(400MHz,Chloroform-d)δ10.10(s,1H),8.80(dd,J=11.0,8.5Hz,4H),8.07(s,1H),8.00(d, J=8.2Hz,2H),7.29(t,J=7.8Hz,4H),7.19(t,J=7.9Hz,6H),7.09(t,J=7.3Hz,2H),3.98(s,3H).

[0070] S4: Synthesis of a purine-based aggregation-induced emission photosensitizer: (E)-4-(4-(6-triphenylamine-9-methylpurine)-2-phenylvinyl)-1-methylquinoline-1-hexafluorophosphate

[0071] The synthetic route is as follows:

[0072]

[0073] Compound 3 (241 mg, 0.5 mmol) and 1,4-dimethylquinoline-1-iodide (143 mg, 0.5 mmol) were added to anhydrous ethanol (10 mL), and piperidine (0.05 mL) was added dropwise to the stirred solution. The mixture was then heated under reflux at 78°C for approximately 12 hours. After the reaction was complete based on TLC, the solvent was removed and the mixture was separated by column chromatography using a mobile phase of dichloromethane:methanol = 50:1 to afford a dark brown solid. The solid was then dissolved in 10 mL of acetone saturated with potassium hexafluorophosphate. After stirring at room temperature for 2 hours, the acetone was removed under reduced pressure to afford the crude product. The crude product was extracted with dichloromethane and a saturated potassium hexafluorophosphate aqueous solution, and the organic solvent was evaporated to dryness, yielding the purine-based aggregation-induced emission photosensitizer 1 (compound 4) as a dark brown solid in a 35% yield.

[0074] The hydrogen spectrum, carbon spectrum and high resolution mass spectrum of the prepared photosensitizer are as follows Figure 2 、 Figure 3 and Figure 4 shown.

[0075] 1 H NMR (400MHz, DMSO-d6) δ9.40(d,J=6.8Hz,1H),9.10(d,J=8.2Hz,1H),8.90(d,J =8.9Hz,2H),8.73(d,J=8.4Hz,2H),8.62(s,1H),8.57(d,J=6.5Hz,1H),8.49-8. 43(m,2H),8.32-8.25(m,2H),8.17(d,J=8.4Hz,2H),8.12-8.08(m,1H),7.44-7 .38(m,4H),7.21-7.16(m,6H),7.13(d,J=8.9Hz,2H),4.57(s,3H),3.96(s,3H).

[0076] 13 C NMR (101MHz, DMSO-d6) δ156.6,153.8,152.8,152.4,150.4,148.6,147.6,146.8,142.7,140.2,139.2,137.4,135. 5,131.2,130.3,129.8,129.5,129.2,128.8,128.7,126.9,126.8,125.7,124.8,121.1,119.8,117.0,45.2,30.1.

[0077] HRMS(ESI):m / z:Calcd for C 42 H 33 N6 + :621.2761; [M-PF6] + Found:621.2759.

[0078] Example 2

[0079] This embodiment is substantially the same as embodiment 1, except that 1,4-dimethylquinoline-1-iodide in S4 is replaced with 1,2-dimethylquinoline-1-iodide. The synthetic route is as follows:

[0080]

[0081] The purine skeleton-based aggregation-induced emission photosensitizer 2 (compound 5) was obtained as a dark red solid with a yield of 35%.

[0082] The hydrogen spectrum, carbon spectrum and high resolution mass spectrum of the prepared photosensitizer are as follows Figure 5 、 Figure 6 and Figure 7 shown.

[0083] 1 H NMR (400MHz, DMSO-d6) δ9.04(d,J=9.0Hz,1H),8.84(d,J=8.9Hz,2H),8.67(d,J=8.5Hz,2H), 8.57(t,J=4.6Hz,2H),8.52(d,J=9.1Hz,1H),8.29(d,J=8.1Hz,1H),8.23(d,J=15.8Hz,1H),8 .15(d,J=8.6Hz,1H),8.10(d,J=8.5Hz,2H),7.96(d,J=16.0Hz,1H),7.90(t,J=7.5Hz,1H),7 .37(t,J=7.9Hz,4H),7.14(d,J=8.4Hz,6H),7.08(d,J=8.9Hz,2H),4.56(s,3H),3.90(s,3H).

[0084] 13 C NMR (101MHz, DMSO-d6) δ156.4,153.8,152.4,150.4,147.7,146.8,146.6,144.7,140.8,139.6,136.7,135.4,131. 2,130.5,130.3,129.9,129.5,129.3,128.7,128.7,128.3,125.7,124.8,121.7,121.0,120.5,119.7,40.5,30.1.

[0085] HRMS(ESI):m / z:Calcd for C 42 H 33 N6 + :621.2761; [M-PF6] + Found:621.2721.

[0086] Experimental Example 1

[0087] UV Absorption Spectra and Molar Extinction Coefficient Experiments: The purine-based aggregation-induced emission photosensitizers prepared in Examples 1 and 2 were each prepared as a 5 mM DMSO stock solution. PBS solutions were then prepared at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM concentrations. After thorough shaking, the working solutions were added to clean UV cuvettes and absorbance was measured within the 250-700 nm range to obtain the corresponding UV-visible absorption spectra. According to the Lambert-Beer law (A = εbc), ΔA and Δc at the wavelength of maximum absorption were substituted into the formula to calculate the corresponding molar absorptivity ε.

[0088] like Figures 8-11 As shown, both Example 1 and Example 2 have a wide absorption range of 350-500nm, and have good absorption at 405nm, which is a good match for the laser confocal imaging system excited at 405nm. As the concentration of the photosensitizer increases, its absorption spectrum also changes accordingly and increases proportionally. By taking the values ​​at the respective maximum absorption wavelengths to draw the concentration-absorbance curve, it can be seen that its absorbance increases linearly with increasing concentration, and the linear correlation coefficients are all over 0.99, indicating that the photosensitizer can still be completely dissolved in PBS at high concentrations and has good solubility under physiological conditions. In addition, according to the Lambert-Beer law, it can be calculated that the molar extinction coefficients of Example 1 and Example 2 at their maximum ultraviolet absorption wavelengths are 3.33×10 4 and 5.96×10 4 M -1 cm -1 , indicating that the photosensitizers all have strong light absorption ability.

[0089] Experimental Example 2

[0090] Fluorescence Spectra and Aggregation-Induced Emission Properties: The purine-based aggregation-induced emission photosensitizers prepared in Examples 1 and 2 were prepared into 5 mM DMSO stock solutions. 2 μL of each DMSO stock solution was added to 2 mL of PBS, dimethyl sulfoxide, acetonitrile, ethanol, acetone, chloroform, tetrahydrofuran, and toluene. The solutions were transferred to clean fluorescence cuvettes, and the fluorescence emission spectra of the probes in different solvents were measured using a fluorescence spectrophotometer.

[0091] 2 μL of the DMSO stock solution of Example 1 and Example 2 was respectively added to 2 mL of a toluene / dimethyl sulfoxide mixed solution containing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% toluene content. The solutions were respectively transferred to clean fluorescence cuvettes, and the fluorescence spectra of the solution state and the aggregated state were measured using a fluorescence spectrophotometer.

[0092] like Figure 12 and Figure 14As shown, both Example 1 and Example 2 have a strongest emission peak at around 400-500nm, and when the probe is in a toluene solution, an obvious emission peak appears at 690nm and 640nm respectively, which may be caused by the aggregation-induced luminescence characteristics of the molecule. We speculate that the emission peak at short wavelengths is emitted by the purine core group, and the entire molecular group emits very weak or even no light when in solution. However, when they are in the poor solvent toluene, molecular aggregation occurs and thus the process of restricted intramolecular motion is activated, resulting in a weaker emission peak at long wavelengths, even in the near-infrared region. After determining that toluene is a poor solvent, we selected dimethyl sulfoxide as a good solvent, prepared toluene / dimethyl sulfoxide mixed solutions with toluene content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, and tested their spectra. As shown Figure 13 and Figure 15 As shown, the long-wavelength fluorescence of Examples 1 and 2 in dimethyl sulfoxide solutions is weak or even absent. However, as the toluene content increases, the fluorescence intensity increases dramatically. When the toluene content is increased to 100%, the fluorescence intensity of the aggregated states of Examples 1 and 2 is 11 times that of their respective solution states, demonstrating the significant aggregation-induced emission properties of the photosensitizers.

[0093] Experimental Example 3

[0094] Singlet oxygen detection experiment: The singlet oxygen detection reagent 9,10-anthryl-bis(methylene)dimalonic acid (ABDA) was prepared into a 10 mM DMSO stock solution. 2 μM of the purine skeleton-based aggregation-induced emission photosensitizer prepared in Example 1 and Example 2 were added to the PBS solution containing 100 μM ABDA, respectively. The photosensitizer was illuminated using a white light source (20 mW / cm 2 ) The mixed solution was irradiated and the absorption spectra of the solution were recorded after irradiation for 0, 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, and 60 seconds respectively to obtain the absorption curve of the ABDA solution. The results are as follows Figures 16-17 The results show that the photosensitizer can quickly generate singlet oxygen in a short illumination time, consume ABDA and reduce its absorption, indicating that both Example 1 and Example 2 have high efficiency in generating type II reactive oxygen species.

[0095] Experimental Example 4

[0096] Hydroxyl radical detection experiment: Hydroxylphenyl fluorescein (HPF), a hydroxyl radical detection reagent, was prepared into a 10 mM DMSO stock solution. 2 μM of the purine skeleton-based aggregation-induced emission photosensitizers prepared in Example 1 and Example 2 were added to a PBS solution containing 5 μM HPF, respectively. The 2 μM purine skeleton-based aggregation-induced emission photosensitizers were illuminated using a white light source (20 mW / cm 2) The mixed solution was irradiated, and the fluorescence spectra of the solution were recorded after irradiation for 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, and 180 seconds, respectively, to obtain the fluorescence spectrum of the HPF solution. The results are as follows: Figures 18-19 The results show that the photosensitizer can quickly generate hydroxyl radicals within a short illumination time, consuming HPF to enhance its fluorescence, indicating that both Example 1 and Example 2 have high efficiency in generating type I reactive oxygen species.

[0097] Experimental Example 5

[0098] CCK-8 cytotoxicity test: Human liver cancer cells (HepG2) in the logarithmic growth phase were seeded in 96-well plates, with about 10,000 cells seeded in each well. The cells were cultured at 37°C and 5% CO2 for 24 hours using DMEM medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin-streptomycin, 1000KU / L). After the cells were completely attached, the purine skeleton-based aggregation-induced luminescence photosensitizer prepared in Example 1 and Example 2 with different concentration gradients of 0μM, 1.25μM, 2.5μM, 5μM, 10μM, and 20μM were added, and 3 replicates were set for each concentration. The light group and the dark group were set separately. One hour after the light group cells were added with the photosensitizer, the cells were illuminated with a white light source (20mW / cm 2 ) cells were irradiated for 10 minutes, while the dark group was protected from light. Then the cells were cultured for 24 hours and the cell survival rate was detected using CCK-8 cytotoxicity kit. Figure 20 As shown in the figure, under dark conditions, the photosensitizer has a higher cell survival rate at a concentration of <10μM, while the cells after adding the photosensitizer and irradiating with light have a lower cell survival rate, indicating that the photosensitizer produces reactive oxygen species under light and plays a role in killing cancer cells.

[0099] Experimental Example 6

[0100] Photodynamically induced cancer cell ablation imaging experiment: HepG2 cells were transferred to a glass-bottomed dish with a diameter of about 3.5 cm and incubated for 24 hours until the appropriate density was reached before the imaging experiment. After removing the old culture medium, serum-free DMEM culture medium containing 5 μM of the purine skeleton-based aggregation-induced emission photosensitizer prepared in Example 1 and Example 2 was added and incubated for 15 minutes. Then, confocal imaging was performed. The cells were continuously scanned for 20 minutes using a confocal laser with a power of 50% and a wavelength of 405 nm, and images of cell morphological changes at different times were recorded. Figure 21 As shown, the cells treated with the photosensitizer showed changes such as cell expansion and deformation, cell membrane rupture, and cell nucleus shrinkage under the action of 405nm laser, indicating that the photosensitizers prepared in Example 1 and Example 2 can effectively induce the ablation of cancer cells through photodynamic therapy.

[0101] In summary, the present invention uses purine as the basic skeleton, introduces triphenylamine as a strong power-supplying group, and introduces quinolyl salt as an electron-withdrawing group, and changes its electron-withdrawing ability by changing the methyl modification site, thereby constructing a D-π-A type system from a structural perspective, increasing the electron push-pull effect and the π conjugated area, effectively improving the active oxygen generation efficiency of the photosensitizer, and having a high type I and type II active oxygen generation capacity. In addition, the aggregation-induced emission property enables this type of probe to have cell-free imaging capabilities and more efficient photodynamic therapy capabilities, making it an excellent photosensitizer with broad application prospects.

[0102] Although the specific embodiments of the present invention are described in detail in conjunction with the accompanying drawings, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. An aggregation-induced emission photosensitizer based on a purine skeleton, characterized in that: Its structure is shown in formula (I): （Ⅰ） Wherein, R1 is C1-C20 alkyl; R2 is or , where X is PF6.

2. The purine skeleton-based aggregation-induced emission photosensitizer according to claim 1, characterized in that: R1 is methyl.

3. The purine skeleton-based aggregation-induced emission photosensitizer according to claim 1, characterized in that: Its structural formula is shown in (II): or （Ⅱ）。 4. The method for preparing the purine skeleton-based aggregation-induced emission photosensitizer according to any one of claims 1 to 3, wherein: The following steps are involved: S1: Synthesis of the first intermediate: 2,6-dichloropurine and a base are dissolved in an organic solvent, heated and stirred at 45-55°C for 30 minutes, and then a C1-C20 substituted alkane is added. Heating and stirring are continued for 4-6 hours, and the organic phase obtained by extraction is separated by column chromatography to obtain the first intermediate; S2: Synthesis of the second intermediate: dissolving the first intermediate, triphenylamine boronic acid, a base, and a catalyst in a mixed solvent of water and 1,4-dioxane, heating and refluxing at 75-85°C under anaerobic conditions for 6-8 hours, and separating the organic phase obtained by extraction by column chromatography to obtain the second intermediate; S3: Synthesis of the third intermediate: dissolving the second intermediate, 4-formylphenylboronic acid, a base, and a catalyst in a mixed solvent of water and 1,4-dioxane, heating and refluxing at 75-85°C under anaerobic conditions for 6-8 hours, and separating the organic phase obtained by extraction by column chromatography to obtain the third intermediate; S4: Synthesis of an aggregation-induced emission photosensitizer based on a purine skeleton: The third intermediate and the matrix are dissolved in ethanol, piperidine is added, and the mixture is heated under reflux at 75-80°C for 10-12 hours. The crude product is separated by column chromatography, and then the crude product is dissolved in a saturated potassium hexafluorophosphate acetone solution, stirred at room temperature for 1-3 hours, and then the acetone is removed. The purine skeleton-based aggregation-induced emission photosensitizer is obtained after extraction; the matrix is ​​one of the compounds represented by formula (III): or （Ⅲ）； wherein Y is Cl, Br or I.

5. The method for preparing the purine skeleton-based aggregation-induced emission photosensitizer according to claim 4, characterized in that: The molar ratio of 2,6-dichloropurine to C1-C20 substituted alkane in S1 is 1:1; the molar ratio of the first intermediate to triphenylamine boronic acid in S2 is 1:1.2; the molar ratio of the second intermediate to 4-formylphenylboronic acid in S3 is 1:1.2; and the molar ratio of the third intermediate to the substrate in S4 is 1:

1.

6. The method for preparing the purine skeleton-based aggregation-induced emission photosensitizer according to claim 4, characterized in that: The reagents used for extraction in S1 are ethyl acetate and water; the reagents used for extraction in S2 are dichloromethane and water; the reagents used for extraction in S3 are dichloromethane and water; the reagents used for extraction in S4 are dichloromethane and saturated potassium hexafluorophosphate aqueous solution.

7. The method for preparing the purine skeleton-based aggregation-induced emission photosensitizer according to claim 4, characterized in that: The S1 column chromatography used silica gel as the stationary phase and ethyl acetate: petroleum ether = 1:1 as the mobile phase; the S2 column chromatography used silica gel as the stationary phase and dichloromethane as the mobile phase; the S3 column chromatography used silica gel as the stationary phase and dichloromethane as the mobile phase; the S4 column chromatography used silica gel as the stationary phase and dichloromethane: methanol = 50:1 as the mobile phase.

8. Use of the purine skeleton-based aggregation-induced emission photosensitizer according to any one of claims 1 to 3 in the preparation of fluorescent imaging agents or cancer therapeutic drugs.

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