Nile blue-PARP inhibitor dye and its synthesis method and application

Abstract

The present invention discloses a Nile blue-PARP inhibitor dye, a synthesis method, and an application thereof. A photosensitizer dye that can be used for both cell imaging and photodynamic therapy is designed and synthesized using Nile blue dye as a matrix. This type of dye retains the high molar extinction coefficient, fluorescence imaging, and reactive oxygen species generation efficiency of Nile blue dye, can effectively improve the killing ability against breast cancer cells, especially BRCA-mutated breast cancer cells, and has a higher lethality. At the same time, it reduces the dark toxicity of the photosensitizer in normal cells and induces ferroptosis in breast cancer cells, thereby achieving extensive killing of breast cancer. The dye has broad application prospects in the treatment of breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of organic dyes, in particular to a Nile blue-PARP inhibitor dye and a synthesis method and application thereof. Background Art

[0002] At present, cancer is one of the diseases that pose the greatest threat to human life and health. According to data from the American Cancer Society: In 2023, the number of new cases of breast cancer in the United States is expected to be approximately 290,000, ranking first in the incidence of cancer among women and is the most common malignant tumor. Studies have shown that women with BRCA (Breast Cancer Susceptibility Gene) mutations have a much higher risk of cancer than normal women and face serious health risks. Chemotherapeutic drugs such as cisplatin and doxorubicin are typical DNA-damaging drugs that kill cancer cells by breaking the double-stranded DNA in tumor cells and are widely used in the treatment of breast cancer. However, the DNA damage repair pathways present in cells will reduce the effectiveness of drugs in killing cancer cells, leading to failure of cancer treatment. At the same time, due to the poor targeting of such drugs, they have large toxic side effects and are prone to cause immune deficiency and other problems. Therefore, there is an urgent need to develop more accurate, efficient and safe treatment models for breast cancer.

[0003] Normal cells contain poly ADP-ribose polymerase (PARP) and BRCA, which can repair DNA damage through base excision or homologous recombination. For cancer cells carrying BRCA mutations, when the PARP repair pathway is blocked, cancer cells lacking the BRCA repair pathway die from DNA damage. This method is called "synthetic lethality", which only kills cancer cells with BRCA mutations without damaging normal cells. Although PARP inhibitors combined with DNA-damaging drugs have achieved certain therapeutic effects, they have limitations such as the tendency of cells to develop drug resistance, high drug dosages, and large drug toxicity and side effects during treatment. In addition, the treatment effect on breast cancer patients who do not carry BRCA mutations is poor, which limits their large-scale clinical application. Summary of the Invention

[0004] In view of the limitations of the existing technology of PARP inhibitors combined with DNA-damaging drugs, such as the easy development of drug resistance in cells during treatment, high dosage, and large toxic and side effects of drugs, as well as poor therapeutic effects on breast cancer patients without BRCA mutations, which further limits their large-scale clinical application, the present invention provides a Nile blue-PARP inhibitor dye that retains the excellent fluorescence properties and efficient photodynamic effect of Nile blue dye. When used in combination with PARP inhibitors, it can reduce toxicity in normal cells while improving the ability to damage breast cancer cells, especially having a higher lethality to BRCA mutation breast cancer cells. It also rationally constructs a combined treatment system based on PARP inhibitor therapy, thereby achieving the optimal therapeutic effect in breast cancer cells, which has important research significance.

[0005] In order to achieve the above-mentioned object, the technical solution of the present invention is: a Nile blue-PARP inhibitor dye, the structure of which is shown in the general formula I:

[0006]

[0007] In the general formula I,

[0008] R1 is selected from one of the PARP inhibitors: olaparib, niraparib, rucaparib, veliparib and their derivatives;

[0009] X is selected from one of oxygen, sulfur, selenium and tellurium, preferably oxygen or sulfur;

[0010] Y - Selected from halogen ions, ClO4 - 、BF4 - One of the following, preferably a halogen ion or ClO4 - .

[0011] n is selected from any positive integer, and is selected from 1, 2 or 3. More preferably, n is 2, in which case the alkyl chain is extended, which has little effect on the performance of the final product.

[0012] The synthesis method of Nile Blue-PARP inhibitor dye, the process of which is as follows:

[0013]

[0014] Its synthesis method comprises the following steps:

[0015] (1) In an aqueous solution, N,N-diethyl-p-phenylenediamine is used as a raw material and added to an aqueous aluminum sulfate solution. NaS2O3 and ZnCl2 are added sequentially under stirring in an ice bath, and KMnO4 is added dropwise. After reacting for 2-4 hours, the mixture undergoes nucleophilic substitution, nitro reduction, oxidation reaction, etc., and then is filtered to obtain a precipitate. The compound represented by formula Z-1 is obtained by extraction, concentration, and purification.

[0016] Alternatively, 3-hydroxy-N,N-diethylaniline is added to a mixed solution of concentrated hydrochloric acid and ultrapure water, the reaction temperature is -5-0°C, and a sodium nitrite aqueous solution is slowly added. The mixture is stirred for 2-4 hours, allowed to stand, and a light yellow solid is obtained by filtration. The solid is washed with sodium acetate and concentrated to obtain the compound represented by formula Z-2.

[0017] (2) Add 1-naphthylamine and ethyl bromate to an organic solvent at 80-100°C and react for 15-24 hours. After the reaction is completed, reduce pressure, concentrate, and purify to obtain a compound represented by formula Z-3. Then, add a base to the above product and stir at room temperature for 4-8 hours. After the reaction is completed, acidify the reaction mixture with acid, extract, concentrate, and purify to obtain an intermediate product Z-4 containing different alkyl chain substitutions;

[0018] (3) In an organic solvent, at 50-100°C, the intermediate product Z-1 or Z-2 is subjected to a condensation reaction with Z-4 under the catalysis of an inorganic base. The reaction is filtered, an acid is added to adjust the pH to 3-4, the reaction is extracted, concentrated, and purified to obtain Nile blue dye NBS. At room temperature, Nile blue dye NBS and an organic base are added to the organic solution, stirred at room temperature for 0.5-1 h, and then R1 inhibitor is added to react for 1-2 h. The reaction is then extracted, dried, concentrated, and purified to obtain a blue solid product I.

[0019] In the above step (1), the extraction solvent is selected from any one of methanol, ethanol, acetonitrile, ethyl acetate, ether, acetone, and propanol, or a mixed solvent of several combinations thereof;

[0020] In the above step (2), the polar solvent is selected from one or a mixed solvent of ethanol, petroleum ether, dichloromethane, and ethyl acetate; the inorganic base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, and sodium ethoxide;

[0021] In the above step (3), the organic solvent is selected from any one of ethanol, methanol, dichloromethane, and DMF, or a mixed solvent of several combinations thereof; the inorganic base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, and silver carbonate; the polar solvent is selected from any one of methanol, dichloromethane, acetonitrile, ether, acetone, and isopropanol, or a mixed solvent of several combinations thereof; and the organic base is selected from any one of triethylamine, pyridine, and HATU.

[0022] Furthermore, in step (2), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the compound represented by formula Z-4 is 1:1.5;

[0023] In the step (3), the molar ratio of the reaction of Nile blue dye NBS and R1 inhibitor is 1:1.5.

[0024] The application of the Nile Blue-PARP inhibitor dye in the treatment of breast cancer, specifically, includes application in bioimaging, combined therapy or photodynamic therapy.

[0025] In summary, the present invention has the following beneficial effects:

[0026] 1. Due to the introduction of PARP inhibitors, the Nile blue-PARP inhibitor dye described in this application can be rapidly taken up by breast cancer cells and has the excellent fluorescence properties, high molar extinction coefficient and efficient reactive oxygen species generation capacity of traditional Nile blue dye.

[0027] 2. Experimental tests have shown that the Nile Blue-PARP inhibitor dye described in this application has low dark toxicity in normal cells. At a dose of 2.0 μM, the survival rate remains above 80%, while the survival rate in breast cancer cells is only about 50%. It has a high lethality and its killing ability is further enhanced under low-dose light irradiation (5 mW, 5 min), especially for BRCA mutation breast cancer cells. IC 50 0.35μM.

[0028] 3. The Nile blue-PARP inhibitor dye described in this application retains the excellent fluorescence properties and efficient photodynamic effect of Nile blue dye. When used in combination with a PARP inhibitor, it can reduce toxicity in normal cells while increasing its ability to damage breast cancer cells. In particular, it has a higher lethality to BRCA mutant breast cancer cells and has good application prospects in the treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0030] Figure 1 Absorption and fluorescence spectra of the examples NBSO and NBSV of the present invention and the comparative example NBS-C in different reagents.

[0031] Figure 2 Ultraviolet absorption attenuation curves of the active oxygen scavenger DPBF in dichloromethane for examples NBSO and NBSV of the present invention and comparative example NBS-C.

[0032] Figure 3 The present invention also shows an experiment on the uptake of NBSO in HCC1937 cells.

[0033] Figure 4 Lysosomal localization of NBSO in HCC1937 cells according to the present invention.

[0034] Figure 5 The toxicity of NBSO and NBSV to different cells was tested in the examples of the present invention.

[0035] Figure 6 The damage experiment of NBSO on MCF-7 cells in the embodiment of the present invention.

[0036] Figure 7 The present invention shows an apoptosis experiment of HCC1937 cells with NBSO. DETAILED DESCRIPTION

[0037] Hereinafter, the present invention will be described in further detail.

[0038] Unless otherwise specified, the terms used herein have the following meanings.

[0039] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0040] The term "alkyl group" used in the present invention includes straight-chain alkyl groups and branched-chain alkyl groups.

[0041] The term "MTT" as used in the present invention refers to a method for detecting cell survival and growth.

[0042] The structure of the Nile Blue-PARP inhibitor dye described in the present application is shown in Formula I:

[0043]

[0044] In the general formula I,

[0045] R1 is selected from one of the PARP inhibitors: olaparib, niraparib, rucaparib, veliparib and their derivatives;

[0046] X is selected from one of oxygen, sulfur, selenium and tellurium, preferably oxygen or sulfur;

[0047] Y - Selected from halogen ions, ClO4 - 、BF4 - One of the following, preferably a halogen ion or ClO4 - .

[0048] n is selected from any positive integer, preferably 1, 2 or 3, more preferably, n is 2.

[0049] The Nile Blue-PARP inhibitor dye represented by the general formula I is described in detail below with reference to the examples.

[0050] Example

[0051] The general synthesis process of the compounds synthesized in Examples 1-5 of the present invention is as follows:

[0052]

[0053] Example 1

[0054] One class of Nile Blue-PARP inhibitor dyes has the following structural formula:

[0055]

[0056] Its synthesis method comprises the following steps:

[0057] (1) Synthesis of the compound represented by formula Z-1

[0058] N,N-diethyl-p-phenylenediamine (2.00 g, 12.18 mmol) was used as the raw material and added to an aqueous solution of aluminum sulfate (8.22 g, 13.04 mmol). NaS2O3 (4.42 g, 28 mmol) and ZnCl2 (1.74 g, 12.78 mmol) were added in sequence under stirring in an ice bath, and KMnO4 (0.53 g, 3.36 mmol) was added dropwise within 20 min. After reacting for 2 h, the nucleophilic substitution, nitro reduction, and oxidation reactions were completed. A large amount of gray-black precipitate was obtained by filtration and washed with a small amount of methanol. It was placed in 10 mL of methanol and refluxed for 10 min. The gray-green solid product was filtered out, 1.50 g, and the yield was 44%. It can be directly used in the next reaction without further purification.

[0059] (2) Synthesis of the compound represented by formula Z-4

[0060] 1-Naphthylamine (2.00 g, 13.90 mmol) and ethyl 5-bromopentanoate (3.00 g, 14.35 mmol) were added to ethanol (15 mL) and refluxed for nearly 18 hours, and continuously monitored by TLC. After the reaction was completed, the solvent was evaporated under reduced pressure to obtain a crude intermediate. 1,4-dioxane (4 mL) was then added to the crude intermediate to dissolve the crude product, and sodium hydroxide (1 M, 1.5 mL) was added to the solution, and the resulting mixture was stirred at room temperature for 5 hours. After the reaction was completed, the reaction mixture was acidified to a pH of 3 with dilute hydrochloric acid, extracted with dichloromethane 3×15 mL, and the organic phase was washed with water and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was separated by silica gel column chromatography (V 石油醚 :V 乙酸乙酯 =2:1) ​​to obtain a solid powder, 2.04 g, with a yield of 54%.

[0061] (3) Synthesis of target product

[0062] Compound Z-1 (0.50 g, 1.65 mmol) and compound Z-4 (0.61 g, 2.51 mmol) were dissolved in 20 mL of methanol, and the reaction was refluxed for 40 min. Silver carbonate (1 g, 3.60 mmol) was slowly added to the reflux reaction mixture. A strong color change was observed after complete addition, and the color changed from gray to a dark blue solution within 0-5 minutes. After heating for 30 min, the reaction flask was cooled to room temperature. The reaction mixture was filtered and evaporated to obtain a dark blue crude product. The crude product was redissolved with 25 mL of dichloromethane, washed with a saturated sodium carbonate solution, dried over sodium sulfate, and the organic phase was filtered and acidified with 0.4 mL of concentrated hydrochloric acid. The solvent was removed by rotary evaporation under reduced pressure, and the crude product obtained was separated by silica gel column chromatography (V 二氯甲烷 :V 甲醇 =10:1) to obtain a blue solid powder, named NBS-C, 0.26 g, with a yield of 36%;

[0063] The prepared NBS-C compound (0.10 g, 0.23 mmol) and HATU (0.09 g, 0.25 mmol) were dissolved in 10 mL of DMF and stirred at room temperature for half an hour. Then, Olaparib (0.13 g, 0.34 mmol) was added and reacted for 1 hour. TLC continuously monitored the generation of new blue spots. After the reaction was completed, 20 mL of water was added and the mixture was extracted with dichloromethane (15 mL×3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. The product was separated and purified by silica gel column (V 二氯甲烷 :V 甲醇 =20:1) to obtain a blue solid product, named NBSO, 0.12 g, with a yield of 67%, and its structure was identified by nuclear magnetic resonance spectroscopy:

[0064] The NMR hydrogen spectrum data of NBSO is: 1 H NMR (400MHz, DMSO-d6) δ12.60(s,1H),9.81(s,1H),9.01(d,J=7.8Hz,1H),8.52(d,J =6.7Hz,1H),8.25(d,J=7.6Hz,1H),8.04-7.78(m,6H),7.58(d,J=4.7Hz,1H),7.42( d,J=8.7Hz,3H),7.33(s,1H),7.23(s,1H),4.32(s,2H),3.76-3.50(m,10H),3.37(s ,2H),3.15(d,J=15.9Hz,2H),2.40(s,2H),1.85–1.60(m,4H),1.23(t,J=6.9Hz,6H).

[0065] The NMR carbon spectrum data of NBSO is: 13C NMR (101MHz, DMSO-d6) δ171.12,164.46,159.85,158.02,155.59,153.50,151.34,145.30,140. 23,137.38,135.32,134.06,133.95,133.71,132.64,132.33,132.25,132.04,131.85,131.74, 130.06,129.53,129.40,128.35,126.54,125.91,125.26,124.61,124.07,123.88,123.60,117.93,116.51,116.30,105.88,103.55,45.61,44.34,41.64,36.89,32.29,28.35,22.56,13.13.

[0066] Example 2

[0067] One class of Nile Blue-PARP inhibitor dyes has the following structural formula:

[0068]

[0069] The only difference from Example 1 is step (3), specifically: Veliparib (0.67 g, 0.28 mmol) was used to replace Olaparib (0.13 g, 0.34 mmol), and the product was separated and purified on a silica gel column (V dichloromethane: V methanol = 20:1) to obtain a blue solid product, named NBSV, 0.80 g, with a yield of 53%, and the structure was identified by nuclear magnetic resonance spectroscopy:

[0070] The H NMR spectrum data of the product are: 1H NMR (400MHz, DMSO-d6) δ12.41(s,1H),9.73(s,1H),9.27(d,J=3.4Hz,1H),8.94(d,J=7.9Hz,1H),8.40(d,J=8.2H z,1H),7.96(d,J=9.4Hz,1H),7.89(t,J=7.6Hz,1H),7.78(t,J=7.2Hz,1H),7.71(d,J=6.8Hz,1H),7.63(d,J=3.4H z,1H),7.51-7.44(m,2H),7.41-7.31(m,2H),7.15(t,J=7.8Hz,1H),3.83(d,J=9.5Hz,2H),3.70-3.60(m,6H),2. 44(d,J=2.9Hz,2H),2.14-1.95(m,4H),1.86(s,3H),1.81-1.73(m,2H),1.67-1.60(m,2H),1.23(t,J=6.9Hz,6H).

[0071] The NMR carbon spectrum data of the product are: 13 C NMR(101MHz,DMSO-d6)δ170.39,166.26,160.22,152.95,150.86,140.65, 136.86,134.64,133.52,133.16,132.12,131.34,129.50,124.72,124.06, 123.03,121.88,121.26,117.45,114.50,105.36,62.28,48.01,45.12,43. 93,41.51,38.24,34.08,32.46,27.91,23.08,22.52,21.40,20.92,12.65.

[0072] Test Case

[0073] In order to demonstrate that the Nile blue-PARP inhibitor dye prepared in Examples 1-2 is a constantly-on photosensitizer that can be rapidly taken up by breast cancer cells, inherits the excellent fluorescence properties, high molar extinction coefficient, and efficient reactive oxygen species generation capacity of Nile blue dye, and causes almost no damage to normal cells and other cancer cells in the absence of light, and can be used to treat breast cancer, especially BRCA-mutated breast cancer cells, the following tests were performed on the Nile blue-PARP inhibitor dye synthesized in the above examples:

[0074] Test Example 1 UV-visible absorption spectrum and fluorescence spectrum measurement

[0075] The compounds NBSO, NBSV and NBS-C were accurately weighed with a 1 / 10,000 balance after vacuum drying, and a 5 mmol / L DMSO dye stock solution was prepared in a brown sample bottle and stored in a refrigerator at 4°C for later use.

[0076] The above compounds were prepared into 10 μM concentration test solutions. The absorption and fluorescence spectra of NBS-C, NBSO and NBSV (10 μM) in different solvents were measured using a fluorescence-absorption spectrometer. The maximum absorption and emission wavelengths were recorded. The test results are shown in Figure 1 .

[0077] Figure 1 (a) is the absorption curve of NBS-C and NBSO; Figure 1 (b) Fluorescence curves of NBS-C and NBSO; Figure 1 (c) is the NBSV absorption curve; Figure 1 (d) is the NBSV fluorescence curve.

[0078] from Figure 1 It can be seen that in the methanol and PBS systems, NBSO, NBSV and NBS-C all exhibit excellent optical absorption and fluorescence emission properties in the NIR range, and the absorption and emission peak spectra of the two are basically consistent, indicating that the spectral performance of the dye molecules is not affected after inhibitor modification.

[0079] Test Example 2 Active Oxygen Generation Capacity Test

[0080] Compounds NBSO, NBSV and NBS-C were added to quartz dishes containing 3 mL of aqueous solution, and a certain amount of DPBF was added to make the absorbance at 405 nm approximately equal to 1. Using near-infrared light (660 nm, 0.5 mW / cm 2 ) Irradiate the test quartz dish and test the UV absorption every 1 minute. The test results are shown in Figure 2 .

[0081] Figure 2 (a) is the absorption curve of NBS-C; Figure 2 (b) is the absorption curve of NBSO; Figure 2 (c) is the absorption curve of NBSV; Figure 2 (d) is the linear relationship between the absorbance value of DPBF at 415 nm and the illumination time.

[0082] Depend on Figure 2It can be seen that under 660nm light irradiation, the absorbance of the DPBF solution containing NBS-C at 415nm decreased rapidly. Under the same conditions, the absorbance of the solutions containing NBSO and NBSV at 415nm also decreased significantly, and the rates of the two were almost the same, indicating that the introduction of Olaparib and Veliparib did not affect the ability of the photosensitizer to produce ROS.

[0083] Test Example 3 Cellular Uptake Test

[0084] HCC1937 cells were seeded onto confocal cell culture dishes and incubated in a cell culture incubator for 24 hours. Prior to imaging, NBS-C and the compound NBSO were added to the cell culture dishes and incubated. Single-photon laser scanning confocal microscopy (Leica) was used to image the cells at different times to determine the cellular uptake of the compound NBSO and the comparison molecule NBS-C. The test results are shown in the table. Figure 3 .

[0085] from Figure 3 It can be seen that the fluorescence signal intensity of NBSO in the cell gradually increased from 0 to 120 minutes, reached the maximum value at 30 minutes, and remained basically unchanged within 120 minutes. The fluorescence signal of the comparison molecule NBS-C was weaker, indicating that NBSO was more easily absorbed by cancer cells.

[0086] Test Example 4: Subcellular Organelle Localization Experiment

[0087] When the density of MCF-7 cells in the confocal culture dish reached 50%, 1 μM of the compound NBSO was added and incubated for 30 minutes. Then, the commercial lysosomal dye Lyso Tracker™ Green DND was added and incubated for 20 minutes. After washing twice with PBS, new culture medium was added and the cells were observed under a laser confocal scanning microscope. The test results are shown in Figure 4 .

[0088] like Figure 4 As shown, the red fluorescence of NBSO overlaps well with the green fluorescence of Lyso Tracker™ Green DND, with a Pearson correlation coefficient of 0.912, indicating that NBSO can be well localized in lysosomes after entering the cell. Mitochondria are beneficial for subsequent photodynamic therapy.

[0089] Test Example 5: Phototoxicity and Dark Toxicity

[0090] The toxicity of the dye was evaluated by MTT assay. The culture medium containing 0, 0.1, 0.3, 0.5, 1.0, 1.5, and 2.0 μM NBS-C, Olaparib, compound NBSO, and NBSV was added, and the dye was incubated for 30 min. The dye was then detected by irradiation at 660 nm (5 mW cm -2 ) After 5 minutes of light exposure (light group) or 5 minutes of dark protection (dark group), the cells were placed in a cell culture incubator and incubated for 24 hours. The DMEM medium was then removed with a pipette, and the cells were washed once with 100 μL of PBS. 100 μL of 5 mg / mL MTT solution was added to each well and incubated for 4 hours. The medium was then removed with a pipette, and 100 μL of dimethyl sulfoxide was added to each well. The 96-well plate was gently shaken to dissolve the formazan crystals. The absorbance of each well solution at 490 nm was measured using a multifunctional microplate reader (Bio-Tek Synergy H1) to calculate the cell survival rate. Each experimental group was repeated 6 times. The test results are shown in [ 15 ]. Figure 5 .

[0091] The cell viability was calculated using the following formula:

[0092] Cell viability = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 ) (1)

[0093] OD 实验组 Refers to the absorbance value of the microwell containing the drug at 490nm, OD 对照组 Refers to the absorbance value of the microwell containing only culture medium at 490nm, OD 空白组 Refers to the average absorbance value of microwells containing DMSO at 490nm.

[0094] Figure 5 (a) Cell survival rate after incubation with different concentrations of NBSO; Figure 5 (b) Survival rate of MCF-7 cells after incubation with different concentrations of drugs; Figure 5 (c) Survival rate of HCC1937 cells after incubation with different concentrations of drugs; Figure 5 (d) Survival rate of MCF-7 cells after incubation with different concentrations of NBSV.

[0095] like Figure 5As shown in the data, under light-proof conditions, no decrease in cell survival rate was observed in 4T1 and normal MCF-10A cells after treatment with NBSO, and low-dose Olaparib had almost no killing ability against them. However, the cell survival rates of MCF-7 and HCC1937 cells decreased after the same treatment, especially the HCC1937 cell survival rate was less than 50%, and after illumination at 0.3 μM it was less than 20%, indicating that Nile blue-PARP inhibitor is beneficial for reducing the drug dose and has a high killing ability against breast cancer cells, especially for cells with BRCA mutations, which can kill to the greatest extent.

[0096] Test Example 6 Cell Damage Experiment

[0097] MCF-7 cells were inoculated on a cell culture dish and after the cells were fully grown, they were treated with different methods: blank group, NBSO, NBSO+light (5mW cm -2 , 5 min). Prepare the working solution according to the Calcein AM / PI double staining kit instructions to stain the cells, and collect fluorescence signals under confocal microscope. The test results are shown in Figure 6 .

[0098] like Figure 6 As shown, only green (AM) fluorescence signal was detected in the blank group, indicating that the cell viability was good, while in the illumination group, obvious red (PI) fluorescence signal could be observed in the cells, indicating that the cell apoptosis entered the late stage and the chromosomes were degraded.

[0099] Test Example 7 Cell apoptosis experiment

[0100] HCC1937 cells were pre-cultured and incubated with NBSO for 30 minutes, then protected from light and exposed to light. The cells were then washed twice with PBS and incubated again to induce apoptosis. The RPMI medium was then removed and 500 μL of binding buffer containing 5 μL Annexin V and 10 μL PI was added. The cells were incubated at 37°C for 30 minutes and then imaged under a laser confocal microscope. The test results are shown in the table. Figure 7 .

[0101] like Figure 7As shown, when cells were treated with NBSO, a small amount of fluorescence signal was captured in the FITC and PI channels in the dark. Compared with the control group, some cells also showed significant morphological changes, indicating that NBSO can induce cell death in the dark, possibly due to the PARP inhibitor. After irradiation with 660nm light, cell morphology changed significantly, and the fluorescence signal in the FITC channel increased significantly. Annexin V specifically binds to phosphatidylserine and is anchored to the cell membrane, leading to cell apoptosis. A significant PI signal was also observed in the nucleus, indicating that the apoptotic process has entered a late stage.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Nile blue-PARP inhibitor dye, characterized in that Its structure is shown in general formula I: In the general formula I, R1 is selected from a PARP inhibitor, wherein the PARP inhibitor is selected from one of Olaparib and Veliparib; X is selected from one of oxygen, sulfur, selenium and tellurium; Y - Selected from halogen ions, ClO4 - 、BF4 - One of the following; n is selected from any positive integer.

2. The Nile Blue-PARP inhibitor dye according to claim 1, wherein X is selected from oxygen or sulfur; Y - Selected from halide ions or ClO4 - ; n is selected from 1, 2 or 3.

3. The method for synthesizing the Nile Blue-PARP inhibitor dye according to any one of claims 1 to 2, characterized in that: The process is as follows: Its synthesis method comprises the following steps: (1) reacting 1-naphthylamine and ethyl bromate in a polar solvent at 80-100° C. for 15-24 hours, then adding a base and continuing the reaction for 4-8 hours. After the reaction is complete, the reaction mixture is acidified with an acid, extracted, concentrated, and purified to obtain an intermediate product represented by formula Z-4 containing different alkyl chain substitutions; (2) In an organic solution at 50-100°C, the compound represented by formula Z-1 or formula Z-2 and the compound represented by formula Z-4 are subjected to condensation reaction under the catalysis of an inorganic base, and the mixture is filtered, - Nile blue dye NBS was obtained by acid treatment, extraction, concentration and purification; (3) Add the Nile blue dye NBS synthesized in step (2) and an organic base to an organic solvent, stir at room temperature for 0.5-1 h, then add the R1 inhibitor and react for 1-2 h. After extraction, drying, concentration, and purification, the compound of formula I is obtained.

4. The method for synthesizing Nile Blue-PARP inhibitor dye according to claim 3, characterized in that: The compound represented by formula Z-1 is synthesized by the following method: In an aqueous solution, N,N-diethyl-p-phenylenediamine is used as a raw material and added to a 41wt% aqueous solution of aluminum sulfate. NaS2O3 and ZnCl2 are added sequentially while stirring in an ice bath, and 13wt% KMnO4 is added dropwise. After reacting for 2-4h, the precipitate is obtained by filtration, and the compound represented by formula Z-1 is obtained by extraction, concentration, and purification.

5. The method for synthesizing Nile Blue-PARP inhibitor dye according to claim 3, characterized in that: The compound represented by formula Z-2 is synthesized by the following method: Add 3-hydroxy-N,N-diethylaniline to a mixed solution of concentrated hydrochloric acid and ultrapure water, the reaction temperature is -5-0°C, and 18wt% sodium nitrite aqueous solution is slowly added. Stir and react for 2-4h, let stand, filter, wash with sodium acetate, and concentrate to obtain the compound represented by formula Z-2.

6. The method for synthesizing Nile Blue-PARP inhibitor dye according to claim 3, characterized in that: In step (2), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the compound represented by formula Z-4 is 1:1.5; In the step (3), the molar ratio of the reaction of Nile blue dye NBS and R1 inhibitor is 1:1.

5.

7. The method for synthesizing Nile Blue-PARP inhibitor dye according to claim 3, characterized in that: In step (2), a Y - Adjust the pH to 3-4 with acid.

8. The method for synthesizing Nile Blue-PARP inhibitor dye according to claim 3, characterized in that: In step (1), the polar solvent is selected from one or a combination of ethanol, petroleum ether, dichloromethane, and ethyl acetate; the base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, and sodium ethoxide; In step (2), the organic solution is selected from any one of ethanol, methanol, dichloromethane, and DMF, or a mixed solvent of several combinations thereof; the inorganic base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, and silver carbonate; In step (3), the organic solvent is selected from any one of methanol, dichloromethane, acetonitrile, ether, acetone, isopropanol or a mixed solvent of several combinations thereof; the organic base is selected from any one of triethylamine, pyridine and HATU.

9. The method for synthesizing Nile Blue-PARP inhibitor dye according to claim 4, characterized in that: In the synthesis step of the compound represented by formula Z-1, the extraction solvent is selected from any one of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, and propanol, or a mixed solvent of several combinations thereof.

10. Use of the Nile blue-PARP inhibitor dye according to any one of claims 1-2 in the treatment of breast cancer.

Images