A high-efficiency production of hydroxyl radical of cyanine photosensitizer dyes, and preparation method and application thereof

Abstract

The application discloses a high-efficiency hydroxyl radical producing merocyanine photosensitizer, a preparation method and application thereof, wherein the structural formula of the merocyanine photosensitizer is shown in the description; the application further specifically discloses a specific preparation method of the merocyanine photosensitizer and application of the merocyanine photosensitizer in preparation of a photodynamic therapy anticancer drug; and the merocyanine photosensitizer prepared by the application can efficiently produce hydroxyl radicals under light irradiation, the merocyanine photosensitizer has good biocompatibility, can enter cells, and can efficiently produce hydroxyl radicals after being activated by light irradiation, and then kill tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of cyanine photosensitive dyes and tumor photodynamic therapy technology, specifically relating to a cyanine photosensitive dye that efficiently generates hydroxyl radicals, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT) is a non-invasive cancer treatment. Its basic principle is to use light of a specific wavelength to excite a photosensitizer enriched in tumor tissue, generating reactive oxygen species (ROS) through photophysical and photochemical processes, thereby inducing tumor cell death. Compared with traditional treatments such as surgery, chemotherapy, and radiotherapy, PDT has significant advantages such as minimal invasiveness, repeatability, and low damage to normal tissues. Cyanide dyes have attracted much attention in the field of PDT due to their near-infrared absorption, good biocompatibility, and ease of modification.

[0003] Based on their mechanism of action, photosensitizers are mainly classified into Type I (which generates free radicals, such as hydroxyl radicals ·OH, through electron transfer) and Type II (which depends on oxygen to generate singlet oxygen). Solid tumors generally present a hypoxic microenvironment, limiting the efficacy of Type II photosensitizers. In contrast, the ·OH generated by Type I photosensitizers has an extremely high oxidation potential, enabling them to efficiently kill tumor cells, and they have lower oxygen dependence, making them an ideal approach to overcome hypoxic drug resistance.

[0004] In the prior art, patent document CN202211599244.7 discloses a coumarin hemicyanine photosensitizer, its preparation method, and its application. To address the problem of low singlet oxygen efficiency in photosensitizers constructed from classic coumarin hemicyanine dyes, a photosensitizer was developed based on structurally rigid coumarin hemicyanine dyes. The developed photosensitizer RCHC-I not only has advantages such as long absorption wavelength and good biocompatibility, but it can also exist in an aggregated state in PBS, greatly improving the singlet oxygen quantum yield. This photosensitizer can generate a large number of reactive oxides under near-infrared light irradiation, thereby killing cancer cells. Patent document CN202410339876.2 discloses an indole salt photosensitive dye, its preparation method, and its application. The photosensitive dye, which has a quinoxaline structure and generates superoxide anion free radicals upon light irradiation, is prepared using hemicyanine as the parent material. The presence of hydrophilic sulfonate groups and indole salts effectively enhances its hydrophilicity. The hemicyanine indole salt structure it contains allows the photosensitive dye to be localized to mitochondria, enabling it to be activated in HepG2 cells to efficiently generate superoxide anion free radicals to kill HepG2 cells. This dye can be used to prepare photodynamic therapy anticancer drugs. Patent document CN202310846068.0 discloses a photosensitizer based on pentamethine cyanine dye, its synthesis method, and its applications. The photosensitizer based on pentamethine cyanine dye is synthesized and its applications in biological staining and photodynamic therapy. The pentamethine cyanine dye in the photosensitizer has excellent intersystem crossing ability and can generate a large number of free radical species under light irradiation, exhibiting near-infrared excitation, mitochondrial localization, and good hypoxia photodynamic effects. Patent document CN202210444767.8 discloses a small molecule of heptamethine indolecyanine-TEMPO chemically coupled with its preparation method and its application in preparing radiation protection agents. The small molecule chemically links TEMPO, which has highly efficient ROS scavenging properties, to a mitochondrial-targeting heptamethine indolecyanine structure, enabling TEMPO to specifically accumulate in mitochondria and efficiently scavenge large amounts of ROS generated in mitochondria due to radiation, thus reducing radiation damage to normal cells. The aforementioned patent documents do not contain any records of cyanine-based photosensitive dyes that can efficiently generate hydroxyl radicals (·OH) after light exposure, nor do they provide any technical inspiration for using cyanine-based photosensitive dyes to activate and efficiently generate ·OH in HepG2 cells to kill HepG2 cells, thereby achieving photodynamic therapy for hypoxic tumors.

[0005] Most existing cyanine photosensitizers primarily generate superoxide anions, with relatively few reports on their efficient generation of ·OH. Under normoxic conditions, superoxide anions exhibit photodynamic therapeutic effects; however, in the hypoxic microenvironment of tumors, the generation efficiency of superoxide anions is somewhat limited. ·OH, on the other hand, can be efficiently generated via an oxygen-independent pathway, exhibiting lower oxygen dependence, higher oxidizing power, and a faster reaction rate, resulting in better therapeutic efficacy. Therefore, there is an urgent need to develop a cyanine photosensitizer that can efficiently generate ·OH and possesses good biocompatibility to overcome the limitations of existing photosensitizers in the photodynamic therapy of hypoxic tumors. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a photosensitive dye that generates hydroxyl radicals efficiently and its preparation method. The method uses heptamethrin cyanide as a parent material to prepare a photosensitive dye that can generate ·OH efficiently after light exposure. It can be used to activate and generate ·OH efficiently in HepG2 cells to kill HepG2 cells. This photosensitive dye provides ideas for photodynamic therapy and can be further used to prepare photodynamic therapy anticancer drugs.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A highly efficient cyanine photosensitive dye that generates hydroxyl radicals has the structure shown in Formula I:

[0009]

[0010] Formula I;

[0011] The cyanine photosensitive dye can efficiently generate hydroxyl radicals under light conditions, and at the same time, the cyanine photosensitive dye has good biocompatibility.

[0012] A method for preparing a highly efficient cyanine photosensitive dye that generates hydroxyl radicals, the specific preparation steps of which are as follows:

[0013] Step S1: The compound shown in Formula VII is mixed thoroughly with N,N-dimethylformamide, then 1,3-dibromopropane is added to dissolve it, and the mixture is refluxed at 50-70°C to obtain the compound shown in Formula VI. The structural formulas of the compounds shown in Formula VII and Formula VI are as follows:

[0014]

[0015] Equation VII;

[0016]

[0017] Formula VI;

[0018] Step S2: The compound shown in Formula VI is mixed thoroughly with 1,1,2-trimethyl-1H-benzo[e]indole, then 1,2-dichlorobenzene is added to dissolve it, and the mixture is refluxed at 100-120°C under a protective atmosphere to obtain the compound shown in Formula III, wherein the structural formula of the compound shown in Formula III is:

[0019]

[0020] Formula III;

[0021] Step S3: The compound shown in Formula V is added to the reaction vessel, followed by phosphorus oxychloride containing dichloromethane and N,N-dimethylformamide. The mixture is then refluxed at 70-90°C under a protective atmosphere to obtain the compound shown in Formula IV. This compound can be used directly in the next reaction without purification. The structural formulas of the compounds shown in Formula V and Formula IV are as follows:

[0022]

[0023] Formula V;

[0024]

[0025] Formula IV;

[0026] Step S4: Compounds of Formula III and Formula IV are added to a reaction vessel, followed by the addition of a mixed solvent of toluene and n-butanol, and the mixture is refluxed at 120-140°C to obtain compound II. The structural formula of compound III is:

[0027]

[0028] Formula II;

[0029] Step S5: The compound shown in Formula II and N-methylpiperazine are added to the reaction vessel, acetonitrile is added, and the mixture is refluxed at 50-70°C to obtain the compound shown in Formula I.

[0030] Preferably, in step S1, the molar ratio of the compound shown in formula VII to 1,3-dibromopropane is 10:(1~2).

[0031] Preferably, in step S2, the molar ratio of the compound shown in formula VI to 1,1,2-trimethyl-1H-benzo[e]indole is 1:(1~2).

[0032] Preferably, in step S3, the molar ratio of the compound shown in formula V to phosphorus oxychloride and N,N-dimethylformamide is 1:1~3:4~6.

[0033] Preferably, in step S4, the molar ratio of the compound shown in Formula III to the compound shown in Formula IV is 2~3:1, and the volume ratio of toluene to n-butanol in the mixed solvent is 7:3.

[0034] Preferably, in step S5, the molar ratio of the compound shown in Formula II to N-methylpiperazine is 1:(1~3).

[0035] This invention provides the application of the cyanine photosensitive dye of Formula I, which efficiently generates hydroxyl radicals, in the preparation of photodynamic therapy anticancer drugs. The cyanine photosensitive dye can efficiently generate ·OH under light conditions, thereby killing tumor cells.

[0036] Preferably, the cyanine photosensitive dye has good amphiphilicity, and can efficiently generate ·OH in both solution and HepG2 cells to achieve tumor treatment. Specifically, the cyanine photosensitive dye does not generate ·OH in solution under non-light conditions, but generates a large amount of ·OH under light conditions. In HepG2 cells, the cyanine photosensitive dye is non-toxic to cells under non-light conditions, but the cell survival rate decreases under light conditions.

[0037] The cyanine photosensitive dye of Formula I provided by this invention has a long emission wavelength, a large Stokes shift, and good fluorescence properties.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects: The cyanine photosensitive dye of Formula I prepared by the present invention has good water-oil amphiphilicity, i.e., biocompatibility, can enter cells, and efficiently generate ·OH after photoactivation, thereby killing tumor cells, and can be used to further prepare photodynamic therapy anticancer drugs. Attached Figure Description

[0039] Figure 1 The 1H NMR spectrum of the compound shown in Formula VI in Example 1 is shown.

[0040] Figure 2 The 1H NMR spectrum of the compound shown in Formula III in Example 1.

[0041] Figure 3 The 1H NMR spectrum of the compound shown in Formula II in Example 1.

[0042] Figure 4 The 1H NMR spectrum of the compound (Cy-NPZ) shown in Formula I in Example 1.

[0043] Figure 5 The solvent effect of the compound shown in Formula I includes absorption and emission spectra.

[0044] Figure 6 The water solubility of the compound shown in Formula I.

[0045] Figure 7 The pH stability of the compound represented by Formula I.

[0046] Figure 8 This refers to the anti-interference properties of the compound shown in Formula I.

[0047] Figure 9 The graph shows the total reactive oxygen species (ROS) generated in the solution of the compound represented by Formula I.

[0048] Figure 10 Superoxide radicals (O2) are generated in the solution of the compound shown in Formula I. .- Test chart.

[0049] Figure 11 The graph shows the test results for the generation of hydroxyl radicals (·OH) in the solution of the compound shown in Formula I.

[0050] Figure 12 The results are electron paramagnetic spectra of the compound shown in Formula I in solution, obtained by detecting the generation of hydroxyl radicals.

[0051] Figure 13 The cytotoxicity of the compound shown in Formula I. Detailed Implementation

[0052] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0053] Example 1

[0054] The synthesis steps of the cyanine photosensitive dye Cy-NPZ are as follows:

[0055] (1) Synthesis of the compound shown in Formula VI

[0056] The compound of formula VII (20 mmol) and K₂CO₃ (20 mmol) were dissolved in 15 mL of N,N-dimethylformamide, and then 1,3-dibromopropane (2 mmol) was added. The mixture was stirred at 60 °C until TCL spotting was complete. The organic layer was dried over anhydrous Na₂SO₄ and filtered. The filtrate was concentrated and purified by PE and DCM column chromatography to obtain a pale yellow oil, the compound of formula VI, in 73% yield. Characterization by 1H NMR: 1¹H NMR (600 MHz, CDCl₃) δ 7.75 (s, 1H), 4.13 (t, J = 6.9 Hz, 2H), 3.37 (t, J = 6.0 Hz, 2H), 2.42 (s, 3H), 2.31 (dt, J = 12.8, 6.6 Hz, 2H). The ¹H NMR spectrum of the obtained product is shown below. Figure 1 As shown. The structural formulas of the compounds shown in Formula VII and Formula VI are as follows:

[0057]

[0058] Equation VII;

[0059]

[0060] Formula VI.

[0061] (2) Synthesis of the compound shown in Formula III

[0062] 1,1,2-Trimethyl-1H-benzo[e]indole (1.27 g, 6.05 mmol) and the compound shown in Formula VI (1 g, 4.03 mmol) were mixed in 1,2-dichlorobenzene and stirred at 110 °C for 12 h under argon protection. The reaction mixture was cooled to room temperature and added to acetone. The precipitate was collected and washed with acetone to give the compound shown in Formula III, which could be used directly in the next reaction without purification, with a yield of 65.72%. Characterization by 1H NMR: 1 ¹H NMR (600 MHz, MeOD) δ 8.33 (d, J = 8.5 Hz, 1H), 8.26 (t, J = 4.4 Hz, 2H), 8.17 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.81 (t, J = 7.1 Hz, 1H), 7.72 (t, J = 7.2 Hz, 1H), 4.78–4.72 (m, 2H), 4.42–4.37 (m, 2H), 2.55 (t, J = 8.0 Hz, 2H), 2.50 (s, 3H), 1.86 (s, 6H). The ¹H NMR spectrum of the obtained product is shown below. Figure 2 As shown. The structural formula of the compound shown in Formula III is:

[0063]

[0064] Formula III.

[0065] (3) Synthesis of the compound shown in Formula IV

[0066] POCl3 (17.5 mL, 115 mmol, 2.3 eq) was added to 20 mL of DCM, mixed, and then added to a constant pressure dropping funnel. Under a N2 atmosphere, the mixture was added dropwise to 20 mL of DCM containing DMF (20 mL, 273 mmol, 5.5 eq), and the mixture was stirred in an ice bath for 30 min. After the reaction, cyclohexanone (5.3 mL, 50 mmol, 1 eq), the compound shown in Formula V, was added to the mixture, and the mixture was refluxed in an oil bath at 80 °C for 3 h. The mixture was then poured into ice water and allowed to stand overnight. After filtration, washing, and drying, a yellow product, the compound shown in Formula IV, was obtained. No purification was required for the next reaction, with a yield of 60%. The structural formula of the compound shown in Formula IV is:

[0067]

[0068] Formula IV.

[0069] (4) Synthesis of the compound shown in Formula II

[0070] Compound III (1.02 g, 2.7 mmol) and compound IV (200 mg, 1.16 mmol) were dissolved in a mixed solvent of methanol and n-butanol (toluene:n-butanol = 7 mL:3 mL). The reactants were heated to 130 °C and refluxed for 6 h. After the reaction was complete, the solvent was removed by vacuum evaporation, and the crude product was purified by silica gel column chromatography to give the green solid, compound II, denoted as Cy-Cl, in 60% yield. Characterization by 1H NMR: 1 H NMR (600 MHz, CDCl3) δ 8.42(d, J = 13.9 Hz, 2H), 8.36 (s, 2H), 8.08 (d, J = 8.5 Hz, 2H), 7.94 (d, J =8.8 Hz, 2H), 7.89 (d, J = 8.3 Hz, 2H), 7.65 (d, J = 8.9 Hz, 2H), 7.58 (t, J =7.6 Hz, 2H), 7.44 (t, J = 7.5 Hz, 2H), 6.30 (d, J = 14.0 Hz, 2H), 4.72–4.59(m, 8H), 2.70 (s, 4H), 2.48 (s, 6H), 1.99 (d, J = 9.5 Hz, 12H), 1.86 (dd, J = 21.5, 15.5 Hz, 6H). The proton NMR spectrum of the obtained product is shown below. Figure 3 As shown. The structural formula of the compound shown in Formula II is:

[0071]

[0072] Formula II.

[0073] (5) Synthesis of the cyanine photosensitive dye (Cy-NPZ) shown in Formula I

[0074] The compound shown in Formula II (100 mg, 0.11 mmol) was added to a 50 mL round-bottom flask and dissolved in 10 mL of acetonitrile. After partial dissolution, N-methylpiperazine (22.50 mg, 0.22 mmol) was added, and the mixture was then reacted at 60 °C for 6 h. Evaporation of the solvent yielded a deep blue solid, namely the cyanine photosensitive dye shown in Formula I (Cy-NPZ), in 10% yield. Characterization by 1H NMR spectroscopy: 1 HNMR (600 MHz, DMSO) δ 8.45 (s, 2H), 8.18 (d, J = 8.6 Hz, 2H), 7.96 (t,J = 8.8 Hz, 4H), 7.69 (d, J = 13.4 Hz, 2H), 7.65 (d, J = 9.0 Hz, 2H), 7.57-7.52 (m, 2H), 7.40-7.36 (m, 2H), 5.77 (d, J = 13.5 Hz, 2H), 4.23-4.20 (m,4H), 4.20-4.16 (m, 4H), 3.68 (s, 4H), 2.62 (s, 4H), 2.37 (d, J = 6.2 Hz, 4H), 2.35 (s, 9H), 2.21–2.13 (m, 4H), 1.87 (s, 12H), 1.66 (s, 2H). The proton NMR spectrum of the obtained product is shown below. Figure 4 As shown. The structural formula of the anthocyanin photosensitive dye shown in Formula I is:

[0075]

[0076] Formula I.

[0077] Solvent effect of the compound shown in Formula I:

[0078] The cyanine photosensitive dye Cy-NPZ synthesized in Example 1 was prepared into a 6 mM DMSO solution. Test solutions with a final concentration of 10 μM were prepared in different solvents (PBS, dimethyl sulfoxide, acetonitrile, methanol, acetone, ethanol, and dichloromethane). The absorption and emission spectra of the molecule in different solvents were measured using a UV-Vis and fluorescence spectrophotometer. The results are as follows. Figure 5 As shown. Experimental results show that the cyanine photosensitive dye represented by Formula I has a relatively long emission wavelength, which is beneficial for phototherapy of cancer.

[0079] Water solubility of the compound shown in Formula I:

[0080] Use a pipette to transfer 1 μL of the above stock solution and dilute it into 3 mL of water, adding it in gradient concentrations each time, stirring and ensuring thorough dissolution, while ensuring that the final volume of DMSO added is less than one-thousandth of the total volume. Measure its absorbance value; the test results are as follows. Figure 6 As shown. The results indicate that the cyanine photosensitive dye represented by Formula I has good solubility, which is a prerequisite for its use in imaging and treatment in organisms.

[0081] pH stability of the compound shown in Formula I:

[0082] High-purity water was prepared into aqueous solutions with different pH values ​​using concentrated hydrochloric acid and sodium hydroxide. The cyanine photosensitive dye shown in Formula I was added to four-way cuvettes containing 3 mL of aqueous solutions with different pH values ​​to prepare a 10 μM test solution. Its fluorescence emission spectrum was measured using a fluorescence spectrophotometer. A scatter plot of Cy-NPZ fluorescence intensity versus pH was obtained by plotting pH value on the x-axis and the maximum fluorescence intensity corresponding to the emission wavelength on the y-axis. The results are shown below. Figure 7 As shown. Experimental results show that within the physiological range (pH=4~10), the cyanine photosensitive dye represented by Formula I exhibits stable fluorescence intensity under pH=4~10 conditions, unaffected by pH.

[0083] Anti-interference properties of the compound shown in Formula I:

[0084] Add 3.0 mL of PBS (pH=7.40) solution and the stock solution of the photosensitizer to a four-way cuvette to make the final concentration of the photosensitizer 10 μM. Then add 20 equivalents of the interfering agent. Finally, plot the ratio of the fluorescence intensity (F) of the photosensitizer at the maximum wavelength after adding the interfering agent to the fluorescence intensity (F0) of the photosensitizer at the maximum wavelength without the interfering agent to obtain the anti-interference data. The interfering amino acids, ions, and biomolecules involved in this experiment included: glutamic acid, lysine, DL-threonine, glutamine, glycine-DL-phenylalanine, glycyl-L-tyrosine, D-methionine, L-cysteine, glycine, DL-leucine, arginine, hypoxanthine, L-aspartic acid, valine, SnCl2, CdCl2, FeCl2•4H2O, NaHSO4, MgSO4, Zn(NO3)2, CaCl2·6H2O, K2HPO4•3H2O, CuSO4, Hg(NO3)2, AgNO3, KCl, ZnNO3, NaCl, β-amylase, lipase, proteinase K, trypsin, bovine serum albumin, pepsin, hemoglobin, and RNA. The results are as follows: Figure 8As shown in the figure. Experimental results show that the fluorescence intensity of the cyanine photosensitive dye Cy-NPZ, represented by Formula I, is not significantly affected in the presence of ions, amino acids, and biomacromolecules, demonstrating that this photosensitive dye has excellent anti-interference properties.

[0085] The total reactive oxygen species (ROS) generated by the compound shown in Formula I in solution was tested.

[0086] In the experimental group, the cyanine photosensitive dye Cy-NPZ (10 μM) shown in Formula I was added to a cuvette containing 3 mL of PBS (pH=7.40) buffer solution, followed by the addition of a certain volume of DCFH-DA (50 μM) and thorough mixing. The cuvette was then placed under a 690 nm laser (20 mW / cm²). 2 Irradiation was performed under the following conditions (1 min each time, for a total of 7 min), and the fluorescence intensity (e) of DCFH-DA was scanned using a fluorescence spectrophotometer. x =488nm,e m =525nm) changes with illumination time. The control group was identical to the experimental group except that it did not contain the cyanine photosensitizing dye Cy-NPZ. The results were plotted with illumination duration on the x-axis and the F-F0 ratio (F is the fluorescence intensity after illumination, and F0 is the fluorescence intensity without illumination) on the y-axis to obtain the total reactive oxygen species production capacity of the cyanine photosensitizing dye Cy-NPZ. The results are as follows. Figure 9 As shown. The results indicate that the cyanine photosensitive dye Cy-NPZ, represented by Formula I, can generate total reactive oxygen species in the aquatic environment of organisms.

[0087] The compound shown in Formula I generates superoxide radicals (O2) in solution. .- )test:

[0088] In the experimental group, the cyanine photosensitive dye Cy-NPZ (10 μM) shown in Formula I was added to a cuvette containing 3 mL of PBS (pH=7.40) buffer solution, followed by the addition of a certain volume of DHR123 (50 μM) and thorough mixing. The cuvette was then placed under a 690 nm laser (20 mW / cm²). 2 Irradiation was performed under the following conditions (1 min each time, for a total of 7 min), and the fluorescence intensity (e) of DHR123 was scanned using a fluorescence spectrophotometer. x =488nm, e m =525nm) changes with illumination time. The control group was identical to the experimental group except that it did not contain the cyanine photosensitive dye Cy-NPZ. The results were plotted with illumination duration on the x-axis and the F-F0 ratio (F is the fluorescence intensity after illumination, and F0 is the fluorescence intensity without illumination) on the y-axis to obtain the superoxide radical generation capacity of the cyanine photosensitive dye Cy-NPZ. The results are as follows. Figure 10As shown. The results indicate that the cyanine photosensitive dye Cy-NPZ, represented by Formula I, can generate superoxide radicals in the aquatic environment of organisms.

[0089] Test on the generation of hydroxyl radicals (·OH) in solution by the compound shown in Formula I:

[0090] In the experimental group, the cyanine photosensitive dye Cy-NPZ (10 μM) shown in Formula I was added to a cuvette containing 3 mL of PBS (pH=7.40) buffer solution, followed by the addition of a certain volume of HPF (50 μM) and thorough mixing. The cuvette was then placed under a 690 nm laser (20 mW / cm²). 2 Irradiation was performed under the following conditions (1 min each time, for a total of 7 min), and the fluorescence intensity of the HPF was scanned using a fluorescence spectrophotometer (ep). x =490nm, e m =515nm) changes with illumination time. The control group was identical to the experimental group except that it did not contain the cyanine photosensitive dye Cy-NPZ. The results were plotted with illumination duration on the x-axis and the F-F0 ratio (F is the fluorescence intensity after illumination, and F0 is the fluorescence intensity without illumination) on the y-axis to obtain the hydroxyl radical generation capacity of the cyanine photosensitive dye Cy-NPZ. The results are shown below. Figure 11 As shown. The results indicate that the cyanine photosensitive dye Cy-NPZ, represented by Formula I, can generate hydroxyl radicals in the aquatic environment of organisms.

[0091] Electron paramagnetic spectroscopy detection of hydroxyl radicals generated by the compound shown in Formula I:

[0092] Electron paramagnetic resonance (EPR) spin trapping experiments were performed using 5,5-dimethyl-1-pyrrololine N-oxide (DMPO). 2.5 μL of cyanine photosensitive dye Cy-NPZ stock solution and 1.13 μL of DMPO trapping agent were added to 100 µL of high-purity water. The EPR spectra of the cyanine photosensitive dye Cy-NPZ were obtained under both dark and light (white light, 210 s) conditions. The results are shown below. Figure 12 As shown. The results indicate that the cyanine photosensitive dye Cy-NPZ, represented by Formula I, can generate a large number of hydroxyl radicals upon photoexcitation.

[0093] Cytotoxicity of the compound shown in Formula I:

[0094] HepG2 cells were used as the research subject. The nutrient medium used for cell culture in this experiment was prepared by adding 10 wt% fetal bovine serum and 1 wt% trypsin to DMEM basic nutrient medium, and cultured at 37°C in an incubator containing 5.0 wt% / vol CO2. 100 μL of HepG2 cells were cultured at 5 × 10⁻⁶ cells / mL. 4Inoculate the cells at a density of 100 cells / mL into 96-well plates and incubate for 24 hours. Construct a photosensitizer / dark toxicity test system by preparing photosensitizer culture media solutions according to a pre-defined concentration gradient. Remove the existing culture media from the 96-well plate, add 100 μL of the photosensitizer culture media solution to the corresponding well, and add 100 μL of photosensitizer-free culture media to the remaining wells as a blank control. Phototoxicity is assessed by incubating the 96-well plate in the incubator for another 3 hours after adding the photosensitizer, and then placing the 96-well plate under a laser lamp (690 nm laser, light power density 100 mW / cm²). 2 Irradiate the plates for 10 min, then incubate the 96-well plates in the same way as the dark toxicity plates for 24 h. After incubation, add 20 μL (5 mg / mL) of diphenyltetrabromoazole bromide (MTT) prepared with PBS buffer (pH=7.20) to each well and incubate for another 4 h to allow succinate dehydrogenase in the mitochondria of surviving cells to convert MTT to formazan. After incubation, aspirate all the solution from the 96-well plates, add 150 μL LDMSO to each well to dissolve the formazan, and measure the absorbance of each well at 490 nm using a microplate reader. Finally, calculate the data. The results are as follows: Figure 13 As shown in the figure. Experimental results showed that without light irradiation, the survival rate of HepG2 cells treated with Cy-NPZ was around 90%; at a concentration of 12 μM, after 10 min of light irradiation, the cell survival rate of Cy-NPZ molecules decreased to 30%. Therefore, the cyanine photosensitive dye Cy-NPZ shown in Formula I exhibits good killing ability against HepG2 cells under 690 nm light irradiation after entering the cells, and is expected to be used for the elimination of cancer cells in organisms.

[0095] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A highly efficient cyanine photosensitive dye that generates hydroxyl radicals, characterized in that... The structure is shown in Equation I: Equation I; The cyanine photosensitive dye can efficiently generate hydroxyl radicals under light conditions, and at the same time, the cyanine photosensitive dye has good biocompatibility.

2. A method for preparing a cyanine photosensitive dye that efficiently generates hydroxyl radicals as described in claim 1, characterized in that... The specific preparation steps are as follows: Step S1: The compound shown in Formula VII is mixed thoroughly with N,N-dimethylformamide, then 1,3-dibromopropane is added to dissolve it, and the mixture is refluxed at 50-70°C to obtain the compound shown in Formula VI. The structural formulas of the compounds shown in Formula VII and Formula VI are as follows: Equation VII; Formula VI; Step S2: The compound shown in Formula VI is mixed thoroughly with 1,1,2-trimethyl-1H-benzo[e]indole, then 1,2-dichlorobenzene is added to dissolve it, and the mixture is refluxed at 100-120°C under a protective atmosphere to obtain the compound shown in Formula III, wherein the structural formula of the compound shown in Formula III is: Formula III; Step S3: The compound shown in Formula V is added to the reaction vessel, followed by phosphorus oxychloride containing dichloromethane and N,N-dimethylformamide. The mixture is then refluxed at 70-90°C under a protective atmosphere to obtain the compound shown in Formula IV. This compound can be used directly in the next reaction without purification. The structural formulas of the compounds shown in Formula V and Formula IV are as follows: Formula V; Formula IV; Step S4: Compounds of Formula III and Formula IV are added to a reaction vessel, followed by the addition of a mixed solvent of toluene and n-butanol, and the mixture is refluxed at 120-140°C to obtain compound II. The structural formula of compound III is: Formula II; Step S5: The compound shown in Formula II and N-methylpiperazine are added to the reaction vessel, acetonitrile is added, and the mixture is refluxed at 50-70°C to obtain the compound shown in Formula I.

3. The method for preparing the cyanine photosensitive dye that efficiently generates hydroxyl radicals according to claim 1, characterized in that: In step S1, the molar ratio of the compound shown in formula VII to 1,3-dibromopropane is 10:(1~2).

4. The method for preparing the cyanine photosensitive dye that efficiently generates hydroxyl radicals according to claim 1, characterized in that: In step S2, the molar ratio of the compound shown in formula VI to 1,1,2-trimethyl-1H-benzo[e]indole is 1:(1~2).

5. The method for preparing a cyanine photosensitive dye that efficiently generates hydroxyl radicals according to claim 1, characterized in that: In step S3, the molar ratio of the compound shown in formula V to phosphorus oxychloride and N,N-dimethylformamide is 1:1~3:4~6.

6. The method for preparing the cyanine photosensitive dye that efficiently generates hydroxyl radicals according to claim 1, characterized in that: In step S4, the molar ratio of the compound shown in Formula III to the compound shown in Formula IV is 2~3:1, and the volume ratio of toluene to n-butanol in the mixed solvent is 7:

3.

7. The method for preparing a cyanine photosensitive dye that efficiently generates hydroxyl radicals according to claim 1, characterized in that: In step S5, the molar ratio of the compound shown in Formula II to N-methylpiperazine is 1:(1~3).

8. The application of the cyanine photosensitive dye that efficiently generates hydroxyl radicals as described in claim 1 in the preparation of photodynamic therapy anticancer drugs, wherein the cyanine photosensitive dye can efficiently generate hydroxyl radicals under light conditions, thereby killing tumor cells.

9. The application according to claim 8, characterized in that: The cyanine photosensitive dye exhibits good amphiphilicity, capable of efficiently generating hydroxyl radicals in both solution and HepG2 cells, thus achieving tumor treatment. Specifically, the cyanine photosensitive dye does not generate hydroxyl radicals in solution under non-light conditions, but generates a large number of hydroxyl radicals under light conditions. In HepG2 cells, the cyanine photosensitive dye is non-toxic under non-light conditions, but cell survival rate decreases under light conditions.

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