Estrogen receptor positive breast cancer fluorescent probe, preparation method and application

Abstract

The present application provides a kind of estrogen receptor positive breast cancer fluorescent probe by parent nucleus dye nile blue derivative, estrogen receptor (ER) recognition group tamoxifen and alkane chain three parts constitute, the probe has the structure of general formula 1.The probe shows folded conformation in water environment, there is photoinduced electron transfer effect between nile blue derivative and tamoxifen, no obvious fluorescence signal;When the probe recognizes ER protein, the recognition group is combined with the binding site, the conformation of the probe changes into unfolded conformation, and strong fluorescence signal is shown.The excitation wavelength of the probe is about 630 nm, the emission wavelength is about 640-700nm, has stronger tissue penetration ability, lower tissue self-absorption and light scattering, can be targeted to breast cancer cell overexpressed ER protein, and specifically highlights estrogen receptor positive breast cancer.

Description

Technical Field

[0001] This invention relates to the field of fine chemical dyes technology, and in particular to a class of estrogen receptor-positive breast cancer fluorescent probes, their preparation methods, and applications. Background Technology

[0002] In recent years, with rapid social development, people's living standards and quality of life have improved dramatically, but more and more health problems have also emerged. Cancer has become one of the world's most serious health challenges, with breast cancer posing a significant threat to the health of women worldwide. According to the latest global cancer burden data from the International Agency for Research on Cancer (IARC) of the World Health Organization in 2020, China has the highest number of new cancer cases and deaths globally. In 2020, China had 4.57 million new cancer cases, with breast cancer ranking first globally. In 2020, 2.09 million new cancer cases were reported in Chinese women, accounting for 46% of the total, with breast cancer surpassing lung cancer to become the leading cause of cancer incidence. The incidence of breast cancer continues to rise, influenced by multiple factors such as the body's internal environment, living environment, and lifestyle habits. Patients of different races and regions may exhibit different clinical characteristics, and different types of breast cancer patients show differences in treatment plans, prognoses, and survival times. Therefore, scholars are constantly exploring and researching breast cancer. Accurate and rapid classification of breast cancer is key to developing the correct treatment plan.

[0003] Breast cancer classification includes both pathological and molecular classifications. Pathological classification determines the nature of the tumor by observing its cell characteristics under a microscope, typically classifying breast cancer into non-invasive, early-stage invasive, and invasive types. Molecular classification involves analyzing breast cancer cells at the gene and protein levels, grouping them based on gene mutations and protein expression characteristics. One classic method is molecular classification of breast cancer based on the expression levels of three receptor proteins: estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor-2 (HER2). Notably, estrogen receptor (ER)-positive breast cancer, characterized by upregulation and dysfunction of ER, accounts for over 70% of this type of malignancy. Clinically, molecular-level breast cancer classification is crucial for cancer staging and treatment planning; hormone therapy is always recommended for estrogen receptor-positive breast cancer.

[0004] In clinical practice and laboratory research, immunohistochemistry (IHC) is the most commonly used method for determining estrogen receptor (ER) expression levels. It is based on the specific interaction between antibodies and antigens and is performed on frozen tissues or fixed cells. However, immunohistochemistry is limited by its complex procedures, physical invasiveness, and limited sample size, making it unsuitable for evaluation in vivo. In contrast, fluorescence imaging has become an attractive technique for real-time, sensitive, selective, and in vivo visualization of gene expression, protein expression, and molecular interactions in living samples. To date, several fluorescent probes have been reported for detecting estrogen receptor-positive breast cancer, but these probes are still limited by short emission wavelengths and low signal-to-noise ratios. Therefore, overcoming these drawbacks is crucial for the clinical translation of estrogen receptor fluorescent probes. Summary of the Invention

[0005] This invention provides a class of estrogen receptor-positive breast cancer fluorescent probes, their preparation method, and applications, overcoming the problem that existing probes are easily affected by short emission wavelengths and low signal-to-noise ratios, making them difficult to use for promoting the clinical translation of estrogen receptor fluorescent probes.

[0006] To address the aforementioned issues, this application provides a class of estrogen receptor-targeting fluorescent probes composed of a Nile Blue derivative (a nucleus dye), a tamoxifen estrogen receptor (ER) recognition group, and an alkane chain. In an aqueous environment, the probe exhibits a folded conformation. A photoinduced electron transfer effect exists between the Nile Blue derivative and tamoxifen, resulting in no significant fluorescence signal. When the probe recognizes the ER protein, the recognition group binds to the binding site, causing the probe to transition to an unfolded conformation and exhibit a strong fluorescence signal. The probe has an excitation wavelength of approximately 630 nm and an emission wavelength of approximately 680 nm, exhibiting strong tissue penetration, low tissue self-absorption, and light scattering. It can specifically illuminate estrogen receptor-positive breast cancer cells by targeting the overexpressed ER protein within the breast cancer cells. This allows for rapid and accurate screening of estrogen receptor-positive breast cancer and localization of tumor lesions, providing clear surgical margins for tumor resection. It shows promising clinical translational applications in pan-tumor fluorescence-guided surgery and other biomedical fields.

[0007] Therefore, this application provides a class of estrogen receptor-positive fluorescent probes for breast cancer, having the following general formula 1 structure:

[0008]

[0009] In general formula 1:

[0010] R1 and R2 may be the same or different, and each is independently selected from one of methyl, ethyl, propyl sulfonate, butyl sulfonate, and propionic acid groups; preferably, R1 and R2 are the same and are selected from methyl.

[0011] A method for preparing a class of estrogen receptor-positive fluorescent probes for breast cancer includes the following steps:

[0012] (1) 1-Naphthylamine and ethyl 6-bromohexanoate were refluxed in the first organic solvent for 6-12 h. After the reaction was completed, the mixture was cooled to room temperature and the first organic solvent was removed to obtain the intermediate shown in 2.

[0013] The intermediate shown in 2 was dissolved in 1,4-dioxane, and a strong alkali aqueous solution was added. The mixture was stirred at room temperature for 1-5 hours to obtain the intermediate shown in 3.

[0014] The intermediate shown in 3 was dissolved in a second organic solvent, and then the compound shown in general formula S-1 was added. Concentrated hydrochloric acid was added to the above reaction system under ice bath conditions. After reacting for 10 min, the temperature was raised to 80-90℃ and the reaction was continued for 20-24 h to obtain the compound shown in general formula 4.

[0015] (2) Tamoxifen was dissolved in a third organic solvent, and 1-chloroethyl chloroformate was added under ice bath conditions. The mixture was stirred for 15 min and then heated under reflux for 24-36 h. The reaction was monitored by TLC. The third organic solvent was removed by vacuum distillation, and the oily residue was completely dissolved in a fourth organic solvent and refluxed for 4-8 h. The fourth organic solvent was removed to obtain a white solid compound, TAM.

[0016] (3) Under alkaline conditions, the compound represented by general formula 4 was subjected to an amidation reaction with compound TAM at room temperature to obtain the target compound;

[0017] The reaction process is as follows:

[0018] .

[0019] Furthermore, the first organic solvent is selected from any one or a combination of several of methanol, ethanol, dichloromethane, ethyl acetate, isopropanol and acetone; the strong base is selected from any one of sodium hydroxide, potassium hydroxide, lithium hydroxide and ammonia; and the molar ratio of 1-naphthylamine to ethyl 6-bromohexanoate is 1:(0.8-1.3).

[0020] The second organic solvent is selected from any one or a combination of several of methanol, ethanol, acetonitrile, ethyl acetate, isopropanol and acetone; the molar ratio of the intermediate shown in 3 to the compound shown in general formula S-1 is 1:(0.9-2.0).

[0021] Furthermore, the third organic solvent is selected from any one or a combination of several of 1,2-dichloroethane, dichloromethane, propane, methanol, ethanol, acetonitrile, ethyl acetate, isopropanol, and acetone; the fourth organic solvent is selected from any one or a combination of several of methanol, ethanol, acetonitrile, ethyl acetate, isopropanol, and acetone; the molar ratio of tamoxifen to 1-chloroethyl chloroformate is 1:(0.9-1.5).

[0022] Furthermore, the solvent used in the amidation reaction is selected from any one of anhydrous DMF, anhydrous DMSO, anhydrous acetonitrile, and anhydrous dichloromethane; the acid-binding agent used is selected from any one of K2CO3, KOH, Na2CO3, NaOH, DIEA, and triethylamine, and the molar ratio of the compound shown in general formula 4 to compound TAM is 1:(0.9-1.5).

[0023] Applications of a class of estrogen receptor-positive breast cancer fluorescent probes in the fields of biology, medicine, and chemistry.

[0024] Furthermore, it can be used for protein labeling, protein molecular docking and protein inhibitor molecular screening, cell imaging, fluorescent dye probes, tumor phototherapy and tumor fluorescence-guided surgery.

[0025] Furthermore, when breast cancer cells were co-incubated with 250 nM of the estrogen receptor-positive breast cancer fluorescent probe described above, a strong fluorescence signal was observed at an excitation wavelength of 630 nm and an emission wavelength of 640-700 nm.

[0026] The beneficial effects of this invention are:

[0027] This application selects a Nile blue derivative as the fluorescent group, which overcomes the problem of short emission wavelength by leveraging its large absorption coefficient, excellent photostability and near-infrared emission, and achieves a near-infrared window response to the opening of ER protein.

[0028] This application selects tamoxifen as the recognition group because tamoxifen is a selective estrogen receptor modulator (SERM) that can bind to the estrogen receptor (ER) overexpressed in breast cancer cells to specifically target estrogen receptor-positive breast cancer.

[0029] This application connects the fluorophore to the recognition group via an alkane chain, allowing the probe to fold in its free state to quench fluorescence and effectively bind to the active pocket in the presence of the ER protein to release fluorescence, thus achieving a high signal-to-noise ratio. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 ER recognition probe I NMR 1 H and 13 C-spectrum;

[0032] Figure 2 High-resolution mass spectrometry of ER recognition probe I;

[0033] Figure 3 The ultraviolet absorption spectrum of ER recognition probe I;

[0034] Figure 4 The fluorescence emission spectrum of ER recognition probe I;

[0035] Figure 5 ER recognizes the water solubility of probe I;

[0036] Figure 6 ER recognizes the cytotoxicity of probe I;

[0037] Figure 7 ER recognition probe I is used to specifically illuminate (ER+) breast cancer cells;

[0038] Figure 8 ER recognition probe I is used to specifically illuminate (ER+) breast cancer. Detailed Implementation

[0039] The following is in conjunction with the appendix Figure 1-8 The invention will be further described in detail with reference to the embodiments. However, it is not limited to the following embodiments.

[0040] The following describes a type of estrogen receptor-positive fluorescent probe for breast cancer, as shown in Formula 1, with specific examples:

[0041]

[0042] In general formula 1:

[0043] R1 and R2 may be the same or different, and each is independently selected from one of methyl, ethyl, propyl sulfonate, butyl sulfonate, and propionic acid groups.

[0044] The present invention can be synthesized from the compound represented by general formula 1 by the method described below.

[0045] Example

[0046] R1 and R2 can be prepared identically, both selected from a methyl-based estrogen receptor-positive fluorescent probe for breast cancer.

[0047] The reaction process is as follows:

[0048]

[0049] Its preparation method includes the following steps:

[0050] (1) Synthesis of dye NB-COOH

[0051] First, ethyl 6-bromohexanoate (38.41 mmol, 8.57 g) was weighed into a 50 mL three-necked flask equipped with a thermometer, and anhydrous ethanol (17.5 mL) was added. Then, 1-naphthylamine (34.92 mmol, 5 g) was added while stirring at room temperature to obtain a mixture. The mixture was refluxed under N2 protection for 12 h. As the reaction proceeded, the reaction solution turned brown. The reaction was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, the solvent was removed, and a brown oily crude product with the structure shown in Figure 2 was obtained.

[0052] Then, 1,4-dioxane (87.3 mL) was used to completely dissolve the brown oily crude product (11.5 g), followed by the addition of 2M NaOH (0.175 mol, 6.98 g) aqueous solution. The mixture was stirred at room temperature for 3 h to remove 1,4-dioxane. The pH was adjusted to 2-3, and the product was extracted with ethyl acetate, with the product appearing in the ethyl acetate layer. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. Purification by column chromatography (DCM: MeOH = 20:1, v / v) yielded a brown solid compound (7.75 g, 86%) with the structure shown in Figure 3.

[0053] Compound 3 (3.89 mmol, 1 g) was weighed into a 50 mL flask under ice bath conditions. Acetonitrile (10 mL) was added and stirred until completely dissolved. Then, 2-nitroso-3-(dimethylamino)phenol (6.61 mmol, 1.1 g) was added, and concentrated hydrochloric acid (0.6 mL) was slowly added dropwise to the reaction system. After reacting for 10 min, the reaction temperature was raised to 85 °C and the reaction was continued for 24 h. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 10 / 1, v / v) to obtain a dark blue powder NB-COOH (1.13 g, 72%).

[0054] The structure has been verified to be correct: 1H NMR (400 MHz, MeOD) δ 9.00 (d, J = 7.7 Hz, 1H), 8.45 (bs, 1H), 8.06 – 7.84 (m, 3H), 7.31 (d, J = 6.9 Hz, 1H), 7.19 – 6.75 (m,2H), 3.78 (bs, 2H), 3.39 (s, 6H), 2.44 (bs, 2H), 1.99 (bs, 2H), 1.81 (bs,2H), 1.64 (bs, 2H). MS: m / z calcd for [M]+: 404.1969, found 404.1968.

[0055] (2) Synthesis of the inhibitor TAM

[0056] Tamoxifen (1.16 mmol, 0.43 g) was completely dissolved in 1,2-dichloroethane (8 mL), and 1-chloroethyl chloroformate (1.27 mmol, 0.18 g) was added under ice bath conditions. The mixture was stirred for 15 min and heated under reflux for 24 h. The reaction was monitored by TLC, and the results showed that most of the substance was converted to an intermediate with a higher Rf. The solvent was removed by rotary evaporation under reduced pressure, and the oily residue was completely dissolved in methanol. The mixture was then refluxed for 4 h. After removing methanol, the compound was purified by column chromatography (DCM:MeOH = 20:1, v / v) to give a white solid compound TAM (0.368 g, 89%).

[0057] The structure has been verified to be correct: 1 H NMR (400 MHz, DMSO) δ 9.14 (s, 2H), 7.38 (t, J =7.1 Hz, 2H), 7.29 (d, J = 6.9 Hz, 1H), 7.24 – 7.16 (m, 4H), 7.16 – 7.08 (m,2H), 6.77 (d, J = 8.1 Hz, 2H), 6.66 (d, J = 8.2 Hz, 2H), 4.11 (s, 2H), 3.21(s, 2H), 2.54 (s, 3H), 2.37 (dd, J = 13.9, 6.5 Hz, 3H), 0.84 (t, J = 7.1 Hz,3H). MS: m / z calcd for [M+H] + 358.2166, found 358.2172.

[0058] (3) Synthesis of probe I

[0059] The NB-COOH (0.49 mmol, 0.2 g) synthesized in step (1) and 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.66 mmol, 0.25 g) were completely dissolved in 3 mL of N,N'-dimethylformamide. Then, 0.32 mL of N,N'-diisopropylethylamine was injected under magnetic stirring at 200 rpm. After stirring for 20 min, 2 mL of N,N'-dimethylformamide containing dissolved TAM (0.59 mmol, 0.21 g) was slowly injected into the system. The stirring speed was then increased to 500 rpm and stirred for 3 h to obtain the dark blue powdery fluorescent probe NB-TAM (0.34 g, 92%).

[0060] The structure has been verified to be correct: 1 H NMR (400 MHz, DMSO) δ 10.53 (s, 1H), 8.79 – 8.67(m, 2H), 7.95 (dd, J = 13.9, 7.0 Hz, 1H), 7.87 – 7.78 (m, 2H), 7.40 – 7.31(m, 2H), 7.27 (t, J = 7.2 Hz, 1H), 7.22 – 6.98 (m, 9H), 6.80 (s, 1H), 6.70 (d, J = 8.6 Hz, 1H), 6.65 (d, J = 8.6 Hz, 1H), 6.57 (t, J = 7.7 Hz, 2H), 3.96(t, J = 5.0 Hz, 1H), 3.90 (t, J = 5.6 Hz, 1H), 3.72 (s, 2H), 3.61 (t, J = 4.8Hz, 1H), 3.54 (t, J = 5.6 Hz, 1H), 3.25 (s, 6H), 2.98 (s, 1.5H), 2.81 (s,1.5H), 2.40 – 2.26 (m, 4H), 1.81 – 1.72 (m, 2H), 1.62 – 1.52 (m, 2H), 1.48 –1.37 (m, 2H), 0.83 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 173.28,157.86, 156.61, 156.34, 155.55, 151.77, 147.71, 143.51, 143.42, 142.11,142.01, 141.31, 141.26, 138.18, 138.11, 135.76, 135.55, 132.52, 131.67,130.94, 130.22, 129.70, 129.66, 129.22, 129.18, 128.71, 128.68, 128.31,128.25, 127.13, 126.61, 124.42, 123.60, 113.77, 113.64, 96.21, 93.98, 65.54, 48.64, 44.70, 40.95, 36.83, 33.43, 32.11, 28.33, 26.38, 24.78, 13.71. See also Figure 1 .

[0061] MS: m / z calcd for [M] + : 743.3956, found 743.4314. See also Figure 2 .

[0062] Test case

[0063] Accurately weigh the vacuum-dried dye using a 0.01 g / L balance, prepare a 1 mmol / L DMSO probe stock solution in a brown sample vial, and store it in a 4 °C refrigerator for later use.

[0064] Test Example 1: Study on the physicochemical properties of probe I

[0065] The probe I synthesized in Example 1 was tested by ultraviolet absorption spectroscopy and fluorescence spectroscopy, respectively.

[0066] Test method: 7.5 μL of the probe stock solution was measured using a micropipette and dissolved in a quartz cuvette containing 3 mL of the test solvent. The mixture was thoroughly mixed to obtain a probe concentration of 2.5 μmol / L, which was used for absorption and fluorescence emission spectroscopy. All tests were performed at 25 °C.

[0067] See test results Figure 3-4 The results showed that probe I exhibited a significant solvent effect.

[0068] like Figure 3As shown in the ultraviolet absorption spectrum, the absorption peak position of probe I is different in different solvents; in particular, it exhibits a broad absorption peak in PBS buffer, with the peak value appearing at around 620 nm.

[0069] like Figure 4 The fluorescence spectra shown indicate that the emission peak of probe I in different solvents is around 670 nm, thus proving that probe I achieved the goal of increasing the emission wavelength. Meanwhile, the fluorescence intensity of probe I varies significantly in different solvents; in particular, it exhibits extremely low fluorescence intensity in both PBS buffer and cell culture medium. The test results indicate that the probe has a weak background signal, which is beneficial for achieving a high signal-to-noise ratio, consistent with expectations. The optical properties in the aqueous phase are mainly due to the folded conformation of probe I in this state, and the photoinduced electron transfer effect between the parent dye Nile Blue and tamoxifen ligands.

[0070] Test Example 2: Investigation of Water Solubility

[0071] The probe I synthesized in Example 1 was tested using ultraviolet absorption spectroscopy.

[0072] Micropipette was used to measure 3, 6, 9, 12, 15, 18, 24, 30, 45, 60, and 90 μL of probe stock solution, respectively, and dissolved in quartz cuvettes containing 3 mL of PBS buffer solution. The mixtures were thoroughly mixed, and the absorbance at different concentrations of probe I (1, 2, 3, 4, 5, 6, 8, 10, 15, 20, and 30 μM) was measured. All tests were performed at 25 °C. The results are shown below. Figure 5 As shown.

[0073] from Figure 5 It can be seen that the absorbance increases with the increase of the concentration of probe I in the solution. In the range of 0-20 μM, the absorbance at 600 nm has a good linear relationship with the probe concentration, which indicates that probe I has good water solubility.

[0074] Test Example 3: Cytotoxicity Assessment

[0075] The cytotoxicity of probe I was assessed using the MTT assay.

[0076] The principle is as follows: Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. Dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its absorbance value at a wavelength of 570 nm using an ELISA reader can indirectly reflect the number of living cells.

[0077] The experimental procedure is as follows: the inoculation density in the 96-well plate is 1×10⁻⁶. 5Three cell lines (MCF-7, MCF 10A, and MDA-MB-231) were cultured for 24 h until cell attachment. 2.5, 5, 10, 15, and 20 μL of probe I stock solution were pipetted into 1.5 mL centrifuge tubes containing 1 mL of the corresponding culture medium and mixed thoroughly. Probe I concentrations (2.5, 5, 10, 15, and 20 μM) were added to different wells, and cells were incubated for another 24 h. For viability testing, 10 μL of MTT (5 mg / mL) was added to each well, and cells were incubated for 4 h. The culture medium was then carefully removed, and the blue-violet crystals were dissolved in 150 μL of DMSO. The optical density (OD) was measured at 570 nm using a microplate reader (Tristar 5 LB942, Berthold), and the absorbance was subtracted from the absorbance of the cell-free blank volume at 630 nm. Relative cell viability (100%) was calculated using the following formula:

[0078] Cell viability = (OD) 实验组 OD 空白对照 ) / (OD 阴性对照 OD 空白对照 () × 100%, where the negative control and blank control are the no-drug group and the blank culture medium group, respectively.

[0079] Test results are as follows Figure 6 As shown, when cultured with different concentrations of probe I, the three cell types still exhibited good survival rates. Even when the concentration was increased to 20 μM, the cell survival rate remained high, indicating that this type of estrogen receptor-targeting fluorescent probe has excellent biocompatibility and does not produce toxic side effects on cells within the working concentration range. Therefore, it can be applied in the fields of biology and medicine.

[0080] Test Example 4: Probe I was used to specifically illuminate (ER+) breast cancer cells.

[0081] MCF-7 cells (ER+), MCF 10A cells (ER-), and MDA-MB-231 cells (ER-) were seeded onto 35 mm confocal dishes. First, 100 μL of probe I stock solution was pipetted into a 1.5 mL centrifuge tube containing 0.9 mL DMSO and mixed thoroughly. Then, 2.5 μL of the diluted probe I stock solution was pipetted into a 1.5 mL centrifuge tube containing 1 mL of the corresponding culture medium and mixed thoroughly, yielding a probe concentration of 0.25 μmol / L. Once the cell abundance reached 60%, the three cell types were incubated with target probe I (250 nM) for 30 min. As a control, MCF-7 cells were pretreated with the ER inhibitor tamoxifen (5 μM) and then co-incubated with the 250 nM probe for 30 min. Cell fluorescence signals were acquired using an Olympus FV3000 laser scanning confocal fluorescence microscope (excitation wavelength 630 nm, emission wavelength reception range 640-700 nm).

[0082] As shown in Figure 7, the fluorescence intensity of MCF-7 cells stained with probe I was significantly higher than that of MDA-MB-231 (8.26-fold) and MCF 10A (18.15-fold) cells, while the fluorescence of the control group was suppressed due to tamoxifen pretreatment. These data indicate that the targeting performance of probe I on the estrogen receptor (ER) facilitates cellular uptake and intracellular retention of the probe, resulting in significantly stronger fluorescence signals in ER-overexpressing MCF-7 cells. This demonstrates that probe I has a high signal-to-noise ratio and can be used to effectively distinguish between (ER+) breast cancer cells, (ER-) normal breast cells, and breast cancer cells.

[0083] Test Example 5: Probe I was used to specifically illuminate (ER+) breast cancer.

[0084] MCF-7 cells were subcutaneously injected into the axilla of nude mice to construct xenograft breast tumors. Vacuum-dried dye was precisely weighed using a 0.01% balance to prepare a 5 mmol / L DMSO probe stock solution in a brown sample vial. 2 μL of the probe I stock solution was pipetted into a 1 mL centrifuge tube containing 98 μL of PBS buffer solution and mixed thoroughly to obtain a probe concentration of 100 μmol / L. The tumors were allowed to grow to 200 mm. 3 In the experimental group, 100 μL of probe I was injected subcutaneously near the tumor. In the control group, tamoxifen (100 nmol / 100 μL PBS) was first injected subcutaneously near the tumor. After 6 hours of pretreatment, 100 μL of probe I was injected subcutaneously near the tumor. Fluorescence imaging was performed on both groups of mice using a NightOWL II LB983 small animal imaging system. The results are as follows: Figure 8 As shown.

[0085] from Figure 8 As can be seen, significant fluorescence was observed in the tumor lesions of the experimental group mice 30 minutes after injection of probe I, and the fluorescence signal could be maintained for about 1 hour; weaker fluorescence signals were observed in the tumor lesions of the control group mice 2 hours after administration. These results indicate that probe I has a high signal-to-noise ratio and can illuminate breast cancer lesions by specifically binding to ER protein.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 class of estrogen receptor-positive fluorescent probes for breast cancer, characterized in that, It has the following general formula 1 structure: In general formula 1: R1 and R2 are the same, and both are methyl groups.

2. The method for preparing a class of estrogen receptor-positive breast cancer fluorescent probes according to claim 1, characterized in that, Includes the following steps: (1) React 1-naphthylamine with ethyl 6-bromohexanoate in a first organic solvent for 6-12 h. After the reaction is complete, add 1,4-dioxane and a strong base aqueous solution and continue the reaction for 1-5 h to obtain the intermediate shown in general formula 3. The intermediate shown in general formula 3 was dissolved in a second organic solvent, and then the compound shown in general formula S-1 was added. Concentrated hydrochloric acid was added to the above reaction system under ice bath conditions. After the reaction time was set, the temperature was raised to 80-90℃ and the reaction was continued for 20-24 h to obtain the compound shown in general formula 4. (2) Under alkaline conditions, the compound represented by general formula 4 was subjected to an amidation reaction with compound TAM at room temperature to obtain the target compound; The reaction process is as follows: 。 3. The preparation method according to claim 2, characterized in that, The compound TAM was prepared by the following method: tamoxifen was dissolved in a third organic solvent, 1-chloroethyl chloroformate was added under ice bath, and the mixture was heated under reflux for 24-36 h. The third organic solvent was then removed to obtain a residue. The residue was dissolved in a fourth organic solvent and refluxed for 4-8 h to remove the fourth organic solvent, thus obtaining the compound TAM.

4. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of 1-naphthylamine to ethyl 6-bromohexanoate is 1:(0.8-1.3). The molar ratio of the intermediate shown in Formula 3 to the compound shown in Formula S-1 is 1:(0.9-2.0).

5. The preparation method according to claim 2, characterized in that, In step (1), the first organic solvent is selected from any one or a combination of several of methanol, ethanol, dichloromethane, ethyl acetate, isopropanol and acetone; the strong base is selected from any one of sodium hydroxide, potassium hydroxide, lithium hydroxide and ammonia. The second organic solvent is selected from any one or a combination of several of methanol, ethanol, acetonitrile, ethyl acetate, isopropanol, and acetone.

6. The preparation method according to claim 3, characterized in that, The third organic solvent is selected from any one or a combination of several of 1,2-dichloroethane, dichloromethane, propane, methanol, ethanol, acetonitrile, ethyl acetate, isopropanol, and acetone; the fourth organic solvent is selected from any one or a combination of several of methanol, ethanol, acetonitrile, ethyl acetate, isopropanol, and acetone; the molar ratio of tamoxifen to 1-chloroethyl chloroformate is 1:(0.9-1.5).

7. The preparation method according to claim 2, characterized in that, The solvent used in the amidation reaction is selected from any one of anhydrous DMF, anhydrous DMSO, anhydrous acetonitrile, and anhydrous dichloromethane; the base used is selected from any one of K2CO3, KOH, Na2CO3, NaOH, DIEA, and triethylamine; and the molar ratio of the compound shown in general formula 4 to compound TAM is 1:(0.9-1.5).

8. The application of the estrogen receptor-positive breast cancer fluorescent probe of claim 1 in non-disease diagnosis and treatment in the fields of biology, medicine, and chemistry.

9. The application according to claim 8, characterized in that, Used for protein labeling, protein docking and screening of protein inhibitor molecules, cell imaging, and fluorescent dye probes.

10. The application according to claim 8, characterized in that, When breast cancer cells cultured in vitro were co-incubated with a 250 nM estrogen receptor-positive breast cancer fluorescent probe, a strong fluorescence signal was observed at an excitation wavelength of 630 nm and an emission wavelength of 640-700 nm.

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