A nucleus-targetable photosensitizer, and a preparation method and application thereof

Abstract

The application discloses a photosensitizer with cell nucleus targeting imaging capability and a preparation method and application thereof. The synthesis method of the photosensitizer comprises the following steps: reacting compound 1 with 1-methylpiperazine under an inert atmosphere at 110-150 DEG C, cooling, extracting, drying, reducing pressure concentration and column chromatography purification after the reaction is completed, and obtaining the photosensitizer. The photosensitizer can effectively generate reactive oxygen under light conditions, and the photodynamic cytotoxicity thereof shows obvious dose dependence, and the killing rate of the photosensitizer to cancer cells is more than 90% when the concentration of the photosensitizer is 10 muM; and the photosensitizer has no obvious toxicity to normal cells under light-avoiding conditions, and exhibits excellent killing selectivity and biological safety. In addition, the photosensitizer has fluorescence imaging capability, can efficiently target the cell nucleus, provides stable fluorescence signals, helps to realize real-time imaging and curative effect evaluation of a photodynamic treatment process, and thus realizes an integrated precise anti-tumor strategy of 'imaging + treatment'.

Description

Technical Field

[0001] This invention belongs to the field of photosensitizer technology, specifically relating to a nuclear-targetable photosensitizer, its preparation method, and its application. Background Technology

[0002] The rapid proliferation and malignant transformation of tumor cells are closely related to biomolecules such as nucleic acids and enzymes within the cell nucleus; therefore, the cell nucleus is considered an ideal target for anti-tumor therapy. Photodynamic therapy (PDT) is a non-invasive treatment that uses photosensitizers (PS) to generate reactive oxygen species (ROS) under specific wavelengths of light to kill cancer cells. However, existing research on photosensitizers targeting the cell nucleus is relatively limited.

[0003] Currently, some imaging agents capable of targeting the cell nucleus have been developed, but they lack killing capabilities. Furthermore, some commercially available materials exhibit dark toxicity at high concentrations during prolonged imaging, hindering long-term tracking. Meanwhile, some photosensitizers can target the cell nucleus for killing, but they lack imaging capabilities, making it difficult to track the imaging process. Additionally, while some photosensitizers possess strong photodynamic therapy effects, they also exhibit extremely high dark toxicity, failing to selectively kill tumor cells. Finally, due to the strict nucleus-entry and nucleus-exit screening mechanisms of the cell nucleus, many photosensitizers struggle to effectively enter the nucleus, thus affecting their therapeutic efficacy.

[0004] Therefore, developing a highly efficient, low-toxicity, and highly targeted nuclear photosensitizer is of great significance for enhancing the anti-tumor effect of PDT. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a compound material (photosensitizer).

[0006] Another object of the present invention is to provide a method for preparing a compound material.

[0007] Another object of the present invention is to provide an application of a compound material as a nuclear staining reagent in fluorescence imaging.

[0008] Another object of the present invention is to provide an application of a compound material in the generation of reactive oxygen species.

[0009] The compound material of the present invention can target the cell nucleus and can be precisely located using fluorescence imaging; and it has photosensitizer properties, that is, it maintains low dark toxicity, has no effect on normal cells, but can generate ROS to kill tumor cells when exposed to light.

[0010] The objective of this invention is achieved through the following technical solutions.

[0011] A compound material with the following structural formula:

[0012] A method for preparing a compound material includes the following steps:

[0013] Compound 1 and 1-methylpiperazine were mixed and stirred at 110–150 °C for 24–36 hours under inert conditions. After cooling to room temperature, the mixture was extracted, dried, purified by vacuum distillation and column chromatography to obtain the compound material.

[0014] The structural formula of compound 1 is as follows:

[0015] The ratio of the molar amount of compound 1 to the volume amount of 1-methylpiperazine is 1:(1-3), where the molar amount is in mmol and the volume amount is in mL.

[0016] In the above technical solution, the method for obtaining compound 1 includes: mixing compound 2, compound 3 and an organic solvent to obtain a mixed solution; adding NaH to the mixed solution at -10 to 0°C under an inert environment; stirring at 45 to 85°C for 8 to 24 hours; cooling to room temperature; quenching the reaction with dilute hydrochloric acid; extraction; drying; vacuum distillation; and column chromatography purification to obtain compound 1, wherein the ratio of compound 2, compound 3 and NaH by molar amount is (1 to 2): 2: 2;

[0017] The structural formula of compound 2 is as follows: The structural formula of compound 3 is as follows:

[0018] In the above technical solution, the ratio of the molar amount of compound 2 to the volume fraction of the organic solvent is 1:(5-20), where the molar amount is in mmol and the volume fraction is in mL.

[0019] In the above technical solution, the organic solvent is anhydrous tetrahydrofuran.

[0020] In the above technical solution, the concentration of HCl in the dilute hydrochloric acid is 0.1-2M.

[0021] In the above technical solution, dichloromethane is used for extraction, and the extraction is performed 3 to 4 times with dichloromethane.

[0022] In the above technical solution, anhydrous sodium sulfate is used for drying.

[0023] In the above technical solution, the inert environment is a nitrogen environment or an argon environment.

[0024] The above-mentioned compound materials are used as cell nuclear staining reagents in fluorescence imaging.

[0025] In the above technical solution, the compound material emits light at wavelengths of 420–580 nm under light excitation at 280–460 nm.

[0026] Applications of the aforementioned compound materials in the generation of reactive oxygen species.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The compound material of the present invention capable of nuclear imaging has good reactive oxygen generation ability and significant dose-dependent photodynamic cytotoxicity. Under light conditions, it can generate enough reactive oxygen to effectively induce cell death. When the concentration of the compound material is 10 μM, the cancer cell mortality rate is as high as 80% or more. Under light-protected conditions, it has no obvious toxicity to normal cells and the cell survival rate reaches more than 90%. It has photosensitivity and selectivity.

[0029] (2) The compound material (photosensitizer) of the present invention has imaging function and can generate stable fluorescence signal. Compared with traditional PDT photosensitizer, this nuclear imaging photosensitizer has nuclear targeting ability, can be efficiently enriched in the nuclear region, and can directly act on key biomolecules such as DNA, improving the accuracy and efficiency of photodynamic therapy. This characteristic enables it to perform nuclear fluorescence imaging while performing photodynamic therapy, providing real-time visualization of the treatment process, facilitating the monitoring of the effect of photodynamic therapy, and realizing the integration of "imaging + treatment". Attached Figure Description

[0030] Figure 1 The UV-Vis absorption spectra of the photosensitizers prepared in Example 1 and Comparative Example 1;

[0031] Figure 2 The fluorescence emission spectra of the photosensitizers prepared in Example 1 and Comparative Example 1 in deionized water or acetonitrile are shown, where (A) is Comparative Example 1 and (B) is Example 1.

[0032] Figure 3 The UV-Vis absorbance curves of ABDA solutions with no photosensitizer or with different photosensitizers as a function of light exposure time are shown below. (A) shows the UV-Vis absorbance curve of ABDA solution with photosensitizer prepared in Comparative Example 1 as a function of light exposure time, (B) shows the UV-Vis absorbance curve of ABDA solution with photosensitizer prepared in Example 1 as a function of light exposure time, and (C) shows the UV-Vis absorbance curve of ABDA aqueous solution without photosensitizer as a function of light exposure time.

[0033] Figure 4 Laser confocal microscopy images of HeLa cells co-incubated with the photosensitizers prepared in Example 1 and Comparative Example 1;

[0034] Figure 5 3D fluorescence images of HeLa cells after co-incubation with the photosensitizer prepared in Example 1, wherein (A) shows the localization of Hoechst 33342, (B) shows the image under bright field, (C) shows the localization of the photosensitizer prepared in Example 1, and (D) shows the co-localization of Hoechst 33342 and the photosensitizer prepared in Example 1;

[0035] Figure 6 The cell survival rate of HeLa cells after treatment with (A) COMP light group, (B) COMP light-shielded group, (C) DCOMP light group and (D) DCOMP light-shielded group in Example 4.

[0036] Figure 7 The cell survival rate of 293T cells after treatment in (A) DCOMP group and (B) control group in Example 5. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0038] In the following examples, the structural formula of compound 1 is as follows: The structural formula of compound 2 is The structural formula of compound 3 is

[0039] In the following examples, the structural formula of bis(4-fluorophenyl)methyl ketone is:

[0040] In the following examples, DMEM culture medium, fetal bovine serum, and antibiotics were all purchased from Shenzhen Shangwei Biotechnology Co., Ltd.

[0041] In the following examples, NaH was dispersed in mineral oil and stored at a content of 60 wt%. The mineral oil did not interfere with the preparation process of Example 1.

[0042] In the following examples, the fluorescent dye working solutions used in Examples 2, 3, 4 and 5 are all mixtures of fluorescent dye and DMEM culture medium, and the fluorescent dye is one of the photosensitizers prepared in Example 1 and Comparative Example 1.

[0043] In the following examples, the Hoechst working solution used in Examples 3 and 5 was a mixture of Hoechst 33342 and DMEM medium.

[0044] In the following examples, the cultures were incubated in a carbon dioxide incubator to achieve light protection.

[0045] Example 1

[0046] A method for preparing a photosensitizer (designated: DCOMP, i.e., a compound material) includes the following steps:

[0047] Compound 1 and 1-methylpiperazine were mixed and stirred at 120°C for 24 hours under argon atmosphere. After cooling to room temperature (20-25°C), the mixture was extracted three times with dichloromethane. The organic phases were combined, dried with anhydrous sodium sulfate, and purified by vacuum distillation and column chromatography (the eluent for column chromatography was a mixture of dichloromethane and methanol, with a volume ratio of 20:1). A yellow solid was obtained as the photosensitizer (number: DCOMP). The molar ratio of compound 1 to the volume ratio of 1-methylpiperazine was 1:2. The molar ratio is in mmol, and the volume ratio is in mL.

[0048] The method for obtaining compound 1 includes: mixing compound 2, compound 3, and an organic solvent (anhydrous tetrahydrofuran) to obtain a mixed solution; adding NaH (NaH introduced by mineral oil containing 60 wt% NaH) to the mixed solution at 0°C under argon atmosphere; stirring at 65°C for 12 hours; cooling to room temperature; quenching the reaction with dilute hydrochloric acid (HCl concentration of 1 M) until no gas is produced; extracting three times with dichloromethane; combining the organic phases; drying the organic phase with anhydrous sodium sulfate; purifying by vacuum distillation and column chromatography (the eluent for column chromatography is a mixture of ethyl acetate and hexane, with a volume ratio of ethyl acetate to hexane of 1:5) to obtain compound 1; the molar ratio of compound 2, compound 3, and NaH is 2.17:4.32:4.32, and the molar ratio of compound 2 to the volume ratio of the organic solvent is 2.17:20.

[0049] The structural formula of the photosensitizer prepared in Example 1 is as follows:

[0050]

[0051] The NMR data of the photosensitizer prepared in Example 1 are as follows:

[0052] 1 H NMR (400MHz, CDCl3) δ7.89 (d, J = 9.0 Hz, 4H), 6.91 (d, J = 9.0 Hz, 4H), 6.68 (s, 1H), 3.38–3.36 (m, 8H), 2.60–2.55 (m, 8H), 2.36 (s, 6H).

[0053] 13C NMR (101MHz, CDCl3) δ184.14,153.66,131.22,128.74,125.66,113.99,113.33,90.74,54.73,54.64,47.45,47.05,46.07.

[0054] Comparative Example 1

[0055] A method for preparing a photosensitizer (No.: COMP) includes the following steps:

[0056] Bis(4-fluorophenyl) ketone and 1-methylpiperazine were mixed thoroughly and stirred at 120°C for 24 hours under argon atmosphere. After the reaction was completed, the mixture was cooled to room temperature and extracted three times with dichloromethane. The organic phases were combined, dried with anhydrous sodium sulfate, and purified by vacuum distillation and column chromatography (the eluent for column chromatography was a mixture of chloroform and methanol, with a volume ratio of 20:1). A white solid was obtained as the photosensitizer (number: COMP). The molar ratio of bis(4-fluorophenyl) ketone to the volume ratio of 1-methylpiperazine was 2:2. The molar ratio is in mmol and the volume ratio is in mL.

[0057] The structural formula of the photosensitizer prepared in Comparative Example 1 is as follows:

[0058]

[0059] The NMR data of the photosensitizer prepared in Comparative Example 1 are as follows:

[0060] 1 H NMR (400MHz, CDCl3) δ7.74(d,J=8.9Hz,4H),6.90(d,J=8.9Hz,4H),3.42–3.35(m,8H),2.63–2.56(m,8H),2.37(s,6H).

[0061] 13 C NMR (101MHz, CDCl3) δ193.99,153.46,132.01,128.61,113.56,54.79,47.52,46.07.

[0062] Figure 1 The UV-Vis absorption spectra of the photosensitizers prepared in Example 1 and Comparative Example 1 are shown. The photosensitizer prepared in Comparative Example 1 has an absorption peak at 350 nm, while the photosensitizer prepared in Example 1 has a significant absorption peak at 345 nm, with a significantly higher intensity than that of the photosensitizer prepared in Comparative Example 1. This indicates that the photosensitizer prepared in Example 1 has higher light collection efficiency and a wider spectral coverage, making it suitable for applications such as light-induced ROS generation and fluorescence imaging.

[0063] Based on the excitation wavelengths obtained from the aforementioned UV-Vis absorption spectra, the photosensitizers prepared in Example 1 and Comparative Example 1 were dissolved in solvents and fluorescence emission tests were performed at their respective excitation wavelengths (the excitation wavelength of the photosensitizer in Example 1 was 345 nm, and the excitation wavelength of the photosensitizer in Comparative Example 1 was 350 nm). The solvents were deionized water or acetonitrile, and the concentration of the photosensitizer prepared in Example 1 or Comparative Example 1 in the solvent was 2 mM. The fluorescence emission spectra are as follows: Figure 2 As shown, by Figure 2 It was found that when the solvent was acetonitrile (ACN), the photosensitizer prepared in Comparative Example 1 emitted fluorescence at 515 nm, and the photosensitizer prepared in Example 1 showed a slight blue shift compared to Comparative Example 1; however, when the solvent was water, the fluorescence of both was almost completely quenched. The photosensitizer prepared in Example 1 exhibits fluorescence quenching in an aqueous phase, which can help reduce background fluorescence and improve targeting, while fluorescence may be restored under specific conditions in the cell nucleus, enabling efficient imaging and precise treatment.

[0064] 9,10-Anthracenediyl-bis(methylene)dicarboxylic acid (ABDA), as an indicator, can react with singlet oxygen, leading to its structural degradation and a corresponding decrease in absorbance. Therefore, this invention utilizes ABDA to evaluate the reactive oxygen species (ROS) induced by photosensitizers under light irradiation. By monitoring the decrease in absorbance over time, the ROS generation capacity and reaction kinetics can be quantitatively assessed, directly reflecting the photocatalytic activity of the photosensitizer in photodynamic therapy.

[0065] The photosensitizers prepared in Example 1 or Comparative Example 1 were dispersed in PBS buffer (pH = 8.4) containing 50.0 μM ABDA (the PBS buffer containing 50.0 μM ABDA was used as the ABDA solution), and then dispersed in PBS buffer (pH = 8.4) containing 50.0 μM ABDA as the ABDA solution. The photosensitizers were then dispersed in PBS buffer (pH = 8.4) containing 2 Irradiate with white light for 10 minutes, and measure the UV-Vis absorbance curve every 1 minute during the irradiation period. ABDA aqueous solution (ABDA concentration of 50.0 μM) is then used with 60 mW / cm² light. 2 White light irradiation for 10 minutes served as a control. During irradiation, the UV-Vis absorbance curve was measured every 1 minute. The test results are as follows: Figure 3 As shown, the absorbance of the photosensitizer prepared in Comparative Example 1 decreased slowly over time, indicating that the reactive oxygen species generation of the photosensitizer prepared in Comparative Example 1 was limited; while the absorbance of the photosensitizer prepared in Example 1 decreased significantly, indicating that its reactive oxygen species generation efficiency was higher, suggesting that the photosensitizer prepared in Example 1 has potential in photodynamic therapy.

[0066] Example 2

[0067] Cell imaging experiments:

[0068] S1, Cell Culture: HeLa cells (derived from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) were seeded at a density of 20,000 cells / well into 8-well plates. 400 μL of complete culture medium was added to each well, consisting of DMEM, 10% (v / v) fetal bovine serum, and 1% (v / v) antibiotics. The cells were cultured in the dark at 37°C in an incubator containing 5% (v / v) carbon dioxide for 16 hours, until the cells covered 70-80% of the bottom area of ​​each well.

[0069] S2, Fluorescent Staining: Discard the complete culture medium and add 400 μL of fluorescent dye working solution to each well of cells in S1. The fluorescent dye working solution is a mixture of fluorescent dye and DMEM culture medium. The concentration of fluorescent dye in the fluorescent dye working solution is 2.5 μM. The fluorescent dye is one of the photosensitizers prepared in Example 1 and Comparative Example 1. Incubate for 30 minutes in a dark incubator at 37°C with 5% (volume fraction) carbon dioxide. After incubation, wash the cells once with PBS buffer.

[0070] S3, CLSM Imaging: Place the 8-well plate on the platform of a confocal microscope, select a 63× objective lens, and use a confocal laser scanning microscope (CLSM) to observe its intracellular localization. A schematic diagram of the confocal microscopy imaging is shown below. Figure 4 As shown, the first column is the image under 405nm wavelength laser illumination, the second column is the image under bright field illumination, and the third column is the channel overlay image of the first and second columns.

[0071] Depend on Figure 4 As can be seen, the photosensitizers prepared in Example 1 and Comparative Example 1 were effectively imaged within HeLa cells, with fluorescence signals mainly concentrated in the nucleus or perinuclear region, indicating that the photosensitizers prepared in Example 1 and Comparative Example 1 have nuclear targeting properties. Bright-field imaging showed intact cell morphology, and no significant cytotoxicity was observed. Channel overlay diagrams further verified the intracellular distribution of the photosensitizers prepared in Example 1 and Comparative Example 1.

[0072] Example 3

[0073] HeLa cells were replaced with MCF-7 cells (derived from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). The cell imaging experiment was performed according to steps S1 and S2 of Example 2 (the fluorescent dye used in step S2 was the photosensitizer prepared in Example 1). The cells obtained in S2 were incubated with 400 μL of Hoechst working solution in a dark, 37°C incubator containing 5% (v / v) carbon dioxide for 30 minutes to stain the cells. The Hoechst working solution was a mixture of Hoechst 33342 (a commercial nuclear dye, purchased from Beyotime) and DMEM medium, with a Hoechst 33342 concentration of 2.5 μM. After staining, the cells were washed once with PBS buffer. Fluorescence images were acquired using a confocal laser scanning microscope, such as... Figure 5 As shown, (A) shows the localization of Hoechst 33342, (B) shows the image under bright field, (C) shows the localization of the photosensitizer prepared in Example 1, and (D) shows the co-localization of Hoechst 33342 and the photosensitizer prepared in Example 1.

[0074] Depend on Figure 5 It is evident that the photosensitizer prepared in Example 1 possesses nuclear-targeted imaging capabilities, laying the foundation for its application in nuclear-targeted biomedical research.

[0075] Example 4

[0076] A549 cells (derived from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) were used at a rate of 5 × 10⁻⁶. 3 Cells were seeded at a density of 10 cells / well in 96-well plates, with 200 μL of complete culture medium added to each well. The plates were incubated for 16 hours in a dark, 37°C incubator containing 5% (v / v) carbon dioxide, until the cells covered 70-80% of the bottom area of ​​each well. The complete culture medium was then removed using a pipette. The following experimental groups were set up (each group had 3 replicates to reduce data error):

[0077] DCOMP Illumination Group: Add 200 μL of DMEM medium (corresponding to the following) to the cells in the 96-well plates. Figure 6 Medium concentration of "0" or fluorescent dye working solution, at 70mW / cm 2Irradiate under white light at a certain intensity for 10 minutes, then continue incubation for 24 hours in a dark, 37°C incubator containing 5% (volume fraction) carbon dioxide. Pipette out all liquid from the wells, add 200 μL of MTT reagent (MTT reagent is obtained by mixing thiazolyl blue and sterile PBS solution (pH=7.4), filtered through a 0.22 μm filter for sterilization; the concentration of thiazolyl blue in the MTT reagent is 0.5 mg / mL; thiazolyl blue (MTT) was purchased from Bid Pharmaceuticals; MTT reagent is stored at 4°C in the dark) and continue incubation for 4 hours. Pipette out all liquid from the wells, then add 100 μL of DMSO to dissolve. Finally, measure the absorbance at 492 nm using a microplate reader. Cell viability was calculated based on absorbance values. The fluorescent dye in the working solution was the photosensitizer prepared in Example 1, and the concentrations of the fluorescent dye in the working solution were 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively. Cell viability in the DCOMP illumination group was as follows: Figure 6 As shown in (C).

[0078] COMP illumination group: Essentially the same as the DCOMP illumination group, the only difference being the fluorescent dye. The COMP illumination group used the photosensitizer prepared in Comparative Example 1. The cell viability of the COMP illumination group was as follows: Figure 6 As shown in (A).

[0079] DCOMP light-protected group: Add 200 μL of LMEM medium or fluorescent dye working solution to the cells in the above 96-well plates. Incubate for 24 hours in a light-protected, 37°C incubator containing 5% (v / v) carbon dioxide. Pipette the liquid from the wells, add 200 μL of MTT reagent, and continue incubation for 4 hours. Pipette the liquid from the wells again, then add 100 μL of LDMSO to dissolve the cells. Finally, measure the absorbance at 492 nm using a microplate reader. Calculate cell viability based on the absorbance value. The fluorescent dye working solution used in the DCOMP light-protected group is the same as that used in the DCOMP light-illuminated group. The cell viability of the DCOMP light-protected group is as follows: Figure 6 As shown in (D).

[0080] COMP light-protected group: Essentially the same as the DCOMP light-protected group, the only difference being the fluorescent dye working solution. The fluorescent dye working solution used in the COMP light-protected group is the same as that used in the COMP light-illuminated group. The cell viability of the COMP light-protected group is as follows: Figure 6 As shown in (B).

[0081] Depend on Figure 6It can be seen that the photosensitizer prepared in Example 1 continuously generates reactive oxygen species under light conditions. These reactive oxygen species destroy biomolecules such as nucleic acids and enzymes in cancer cells, thereby exhibiting a significant killing effect on cancer cells. Furthermore, as the concentration of the photosensitizer increases, the cell survival rate decreases significantly, demonstrating significant dose-dependent photodynamic cytotoxicity. At 10 μM, it already showed a good killing effect, with a cancer cell mortality rate exceeding 80%. At 20 μM, the cell survival rate of cancer cells dropped to below 10%, proving its effective photosensitizing killing effect. Under light-protected conditions (DCOMP light-protected group), the photosensitizer prepared in Example 1 exhibited lower cytotoxicity, proving that it possesses photosensitizer properties. In contrast, the photosensitizer prepared in Comparative Example 1 showed a weaker photodynamic effect under light conditions. Even at the highest concentration of 20 μM, the cell survival rate of cancer cells remained above 80%, indicating that it still could not effectively induce cancer cell death under light.

[0082] Therefore, the photosensitizer prepared in Example 1 exhibits strong photodynamic cytotoxicity and good biocompatibility under light irradiation, and is suitable for wide application to a variety of tumor cell types to a certain extent.

[0083] Example 5

[0084] DCOMP group: Referring to the "DCOMP light-protected group" in Example 4, A549 cells were replaced with human embryonic kidney cells (293T cells) for the experiment. The cell viability of the DCOMP group was as follows: Figure 7 As shown in (a).

[0085] Control group: The control group was basically the same as the "DCOMP light-protected group" in Example 4, except for the cells used and the added fluorescent dye working solution. In the control group, 200 μL of DMEM culture medium (corresponding to 293T cells) was added to the cells (cells were 293T cells) in the 96-well plate. Figure 7 (b) The concentration of Hoechst 33342 in the Hoechst working solution was 0, and the concentrations of Hoechst 33342 in the Hoechst working solution were 1.25 μM, 2.5 μM, 5 μM, 10 μM and 20 μM, respectively. The cell viability of the control group was as follows: Figure 7 As shown in (b).

[0086] Depend on Figure 7It was found that Hoechst 33342 in the control group was cytotoxic to normal cells. Compared with the control group, the photosensitizer prepared in Example 1 had no significant toxicity to normal cells in the range of 0-10 μM under no-light conditions, and the cell survival rate was over 95%. This indicates that the photosensitizer prepared in Example 1 is safer than traditional photosensitizers when co-incubated for a long time (24 h). Combined with Examples 4-5, while imaging tumor cells, 10 μM of the photosensitizer prepared in Example 1 can be selected to kill tumor cells under light conditions, while having no toxicity to normal cells under no-light conditions. This can avoid non-specific toxic damage in vivo and improve the potential for clinical application.

[0087] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A compound material, characterized in that, Its structural formula is 2. A method for preparing a compound material, characterized in that, Includes the following steps: Compound 1 and 1-methylpiperazine were mixed and stirred at 110–150 °C for 24–36 hours under inert conditions. After cooling to room temperature, the mixture was extracted, dried, purified by vacuum distillation and column chromatography to obtain the compound material. The structural formula of compound 1 is as follows: The ratio of the molar amount of compound 1 to the volume amount of 1-methylpiperazine is 1:(1-3), where the molar amount is in mmol and the volume amount is in mL.

3. The preparation method according to claim 2, characterized in that, The method for obtaining compound 1 includes: mixing compound 2, compound 3 and an organic solvent to obtain a mixed solution; adding NaH to the mixed solution at -10 to 0°C under an inert environment; stirring at 45 to 85°C for 8 to 24 hours; cooling to room temperature; quenching the reaction with dilute hydrochloric acid; extraction; drying; vacuum distillation; and column chromatography purification to obtain compound 1. The ratio of compound 2, compound 3 and NaH by molar amount is (1 to 2): 2:

2. The structural formula of compound 2 is as follows: The structural formula of compound 3 is as follows:

4. The preparation method according to claim 3, characterized in that, The ratio of the molar amount of compound 2 to the volume fraction of the organic solvent is 1:(5-20), where the molar amount is in mmol and the volume fraction is in mL.

5. The preparation method according to claim 3, characterized in that, The organic solvent is anhydrous tetrahydrofuran.

6. The preparation method according to claim 3, characterized in that, The concentration of HCl in the dilute hydrochloric acid is 0.1–2 M.

7. The preparation method according to claim 2, characterized in that, The inert environment is a nitrogen environment or an argon environment.

8. The application of the compound material as described in claim 1 in nuclear staining.

9. The application according to claim 8, characterized in that, The compound material emits light at wavelengths of 420–580 nm when excited by light in the range of 280–460 nm.

10. The use of the compound material as described in claim 1 in the generation of reactive oxygen species.

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