A fluorescent probe targeting estrogen receptor and its preparation method and application

Abstract

The present invention relates to the technical field of molecular imaging probes, and more specifically to a fluorescent probe targeting the estrogen receptor, a preparation method, and applications thereof. The fluorescent probe targeting the estrogen receptor is composed of a fluorescein ester, an estrogen receptor recognition group, raloxifene, and an alkane chain. The preparation method comprises the following steps: Step S1, hydrolyzing or hydrogenolyzing the carbamate of raw material I to synthesize an amino intermediate II; Step S2, condensing the amino intermediate II with FITC to obtain a fluorescent probe targeting the estrogen receptor III. The fluorescent probe targeting the estrogen receptor in the present invention can avoid the effects of short emission wavelength and low signal-to-noise ratio during probe application, and can be better used to promote the clinical application of the fluorescent probe targeting the estrogen receptor.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular imaging probes, and in particular to an estrogen receptor-targeted fluorescent probe and a preparation method and application thereof. Background Art

[0002] Cancer has become one of the world's most pressing problems plaguing human life and health. Breast cancer, as a type of cancer, poses a serious threat to women's health. Its high clinical incidence and potential harmfulness make it an important topic in medical research.

[0003] The pathogenesis of breast cancer is complex and usually involves the combined effects of multiple factors, including genetics, environment, hormones, and other factors. Among these factors, hormones, especially estrogen, play an important role in the occurrence and development of breast cancer.

[0004] Estrogen receptor (ER) in breast cancer cells is one of the key markers for breast cancer treatment and prognosis assessment. Estrogen receptor regulates gene transcription by binding to estrogen, affecting cell proliferation and survival, and therefore becomes an important target for targeted therapy.

[0005] Drugs targeting estrogen receptors, such as anti-estrogen therapy, have been widely used clinically, but accurate and rapid detection of estrogen receptor expression levels in breast cancer cells remains a research hotspot. To achieve accurate detection of estrogen receptors, researchers have begun exploring the application of novel molecular probes, particularly fluorescent probes, in this field.

[0006] Fluorescent probes not only provide highly sensitive detection but also enable real-time imaging of biological molecules (such as proteins), and are expected to improve the accuracy of breast cancer diagnosis and the level of personalized treatment in clinical practice. However, existing probes are easily affected by short emission wavelengths and low signal-to-noise ratios when used, making them difficult to promote the clinical translation of estrogen receptor fluorescent probes. Summary of the Invention

[0007] The present invention provides a fluorescent probe targeting estrogen receptors, aiming to solve the problem that fluorescent probes in the prior art are easily affected by short emission wavelengths and low signal-to-noise ratios when used, making them difficult to be used to promote the clinical transformation of estrogen receptor fluorescent probes.

[0008] In a first aspect, the present invention provides an estrogen receptor-targeted fluorescent probe, which is composed of a fluorescein ester, an estrogen receptor recognition group raloxifene, and an alkane chain.

[0009] Preferably, the structural formula of the estrogen receptor-targeting fluorescent probe is as follows:

[0010]

[0011] In a second aspect, the present invention provides a method for preparing a fluorescent probe targeting estrogen receptors, the preparation method is as follows:

[0012]

[0013] Step S1, subjecting the raw material I to hydrolysis or hydrogenolysis of the carbamate to synthesize the amino intermediate II;

[0014] Step S2: performing a condensation reaction on the amino intermediate II and FITC to obtain an estrogen receptor-targeting fluorescent probe III.

[0015] Preferably, the reaction in step S1 is carried out under an argon environment.

[0016] Preferably, the organic solvent used in the reaction in step S1 is dichloromethane.

[0017] Preferably, the reaction in step S1 is carried out at room temperature for 3-5 hours.

[0018] Preferably, in step S2, the molar ratio of the III intermediate II to the FITC is 1:3.

[0019] Preferably, in step S2, the organic solvent used in the condensation reaction is one or more of N,N-dimethylformamide, 1,4-dioxane or tetrahydrofuran.

[0020] Preferably, the base used in the condensation reaction is one or both of N,N-diisopropylethylamine and triethylamine.

[0021] In a third aspect, this embodiment provides the application of the estrogen receptor-targeted fluorescent probe provided in the first aspect, which is applied to protein labeling, protein molecule docking and protein inhibitor molecule screening, cell imaging, and fluorescent dye probes.

[0022] Compared with the existing technology, the estrogen receptor-targeted fluorescent probe in the present invention is composed of fluorescein ester, the estrogen receptor recognition group raloxifene and an alkane chain, which can avoid the influence of short emission wavelength and low signal-to-noise ratio when the probe is used, and is better used to promote the clinical transformation of estrogen receptor fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be described in detail below with reference to the accompanying drawings. The above and other aspects of the present invention will become clearer and easier to understand through the detailed description made with reference to the following drawings. In the accompanying drawings:

[0024] Figure 1is the ultraviolet absorption spectrum of 1ER recognition probe III in this embodiment;

[0025] Figure 2 is the fluorescence emission spectrum of the ER recognition probe III in this embodiment;

[0026] Figure 3 This is one of the cytotoxicity graphs of the ER recognition probe III in this example;

[0027] Figure 4 This is the second cytotoxicity graph of the ER recognition probe III in this example;

[0028] Figure 5 This is a diagram showing that the ER recognition probe III in this embodiment is used to specifically illuminate (ER+) breast cancer cells;

[0029] Figure 6 This is the selective imaging diagram of the ER recognition probe III in this embodiment;

[0030] Figure 7 In this embodiment, the ER recognition probe III is used for breast cancer (ER-) cell sphere imaging:

[0031] Figure 8 4 is a flow chart of the steps of the preparation method of the estrogen receptor targeting fluorescent probe in this embodiment. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] An embodiment of the present invention provides an estrogen receptor-targeted fluorescent probe, which is composed of fluorescein ester (FITC), an estrogen receptor (ER) recognition group raloxifene, and an alkane chain.

[0034] Specifically, the structural formula of the estrogen receptor-targeting fluorescent probe is as follows:

[0035]

[0036] Specifically, the preparation method of the estrogen receptor-targeting fluorescent probe comprises the following steps:

[0037]

[0038] Step S1, subjecting the raw material I to hydrolysis or hydrogenolysis of the carbamate to synthesize the amino intermediate II;

[0039] Step S2: performing a condensation reaction on the amino intermediate II and FITC to obtain an estrogen receptor-targeting fluorescent probe III.

[0040] The amino intermediate II may be referred to as intermediate or intermediate II hereinafter.

[0041] The reaction in step S1 is carried out under an argon environment; the organic solvent used in the cyclization reaction in step S1 is dichloromethane; the reaction in step S1 is carried out at room temperature, and the reaction time is 3-5 hours.

[0042] In step S2, the molar ratio of the amino intermediate II to the FITC is 1:3; the organic solvent used in the condensation reaction is one or more of N,N-dimethylformamide (DMF), 1,4-dioxane or tetrahydrofuran (THF); and the base used in the condensation reaction is one or both of N,N-diisopropylethylamine (DIPEA) and triethylamine.

[0043] The estrogen receptor-targeted fluorescent probe may be referred to as probe or probe III hereinafter. It has an excitation wavelength of approximately 495 nm and an emission wavelength of approximately 515 nm. It has strong tissue penetration ability and can specifically illuminate estrogen receptor-positive breast cancer by targeting the ER protein overexpressed in breast cancer cells. The estrogen receptor-targeted fluorescent probe can selectively image cells with high estrogen receptor expression at the cellular level. At the same time, its synthesis process is simple and easy, the raw materials are cheap and readily available, the preparation cost is low, and it is easy to promote.

[0044] When breast cancer cells are co-incubated with 10 μM of the estrogen receptor-targeted (positive breast cancer) fluorescent probe, a strong fluorescent signal will appear at an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0045] The estrogen receptor-targeted fluorescent probe in the embodiments of the present invention is mainly used for protein labeling, protein molecule docking, protein inhibitor molecule screening, cell imaging, and fluorescent dye probes.

[0046] In order to better understand the estrogen receptor-targeting fluorescent probe of the present invention, a detailed description will be given below.

[0047] Implementation of Probe III Synthesis:

[0048] Under argon (Ar) gas environment, raw material I (1 g, 1.88 mmol) was added to ultra-dry CH2Cl2 (15 mL) solution and cooled to -30 ° C, and then BBr3 (1 Min ether, 15 mL) was added; after being taken out from the ice bath, the mixture was vigorously stirred at room temperature for 3 h, and then quenched with ice water (5 mL), specifically extracted with ethyl acetate 3 times, back-extracted once with saturated brine, and then the organic layers were combined, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure, and then purified by column chromatography to obtain a yellow solid (intermediate II, 0.6 g, 79%).

[0049] Under an argon atmosphere, FITC (1 mmol) and the above-mentioned intermediate II were added to a flask respectively, and then Et3N (5 mL) was added to the mixture and stirred in the dark at 25°C for 12 h or dissolved in 2 mL of anhydrous DMF, and then FITC (1 mmol) and 0.1 mL of DIPEA were added and stirred at 80°C overnight; when the reaction was completed, it was purified by column chromatography (methanol / dichloromethane) to obtain a yellow solid (the estrogen receptor-targeting fluorescent probe III, 0.5 g, 63%).

[0050] It has been verified that the structure of the estrogen receptor-targeting fluorescent probe is correct: 1 H NMR (400MHz, Methanol-d4) δ7.87 (d, J=2.2Hz, 1H), 7.69-7.65 (m, 2H), 7.55 (dd, J= 8.2,2.3Hz,1H),7.37(d,J=8.8Hz,1H),7.22(d,J=2.2Hz,1H),7.16-7.11(m,4H),7 .08(d,J=8.2Hz,1H),6.90-6.86(m,2H),6.82(dd,J=8.8,2.3Hz,1H),6.62-6.55(m ,6H),4.25(t,J=5.4Hz,2H),4.01(d,J=5.4Hz,2H),3.30(p,J=1.6Hz,3H).MS:calcd for C 44 H 30 N2O9S2[M+H]:795.14, found 795.14.

[0051] Testing the physicochemical properties of the estrogen receptor-targeting fluorescent probe:

[0052] The estrogen receptor-targeting fluorescent probe is hereinafter referred to as probe or targeting probe; the synthesized probe is tested by ultraviolet absorption spectroscopy and fluorescence spectroscopy respectively.

[0053] Test method: The probe was diluted to a concentration of 2.5 μmol / L and transferred to a quartz cuvette for uniform mixing for absorption and fluorescence emission spectrum testing. All tests were completed at 25°C.

[0054] like Figure 1 As shown in the UV absorption spectrum in , the absorption peak of the probe in 10 mM PBS (pH = 7.4) appears at around 495 nm, and the peak of the fluorescence emission appears at around 515 nm.

[0055] Test cytotoxicity assessment:

[0056] The CCK8 assay was used to evaluate the cytotoxicity of the probe.

[0057] The Cell Counting Kit-8, also known as the CCK-8 kit or CCK8 Cell Proliferation and Toxicity Assay Kit, is a rapid, highly sensitive assay based on WST-8. WST-8 is a compound similar to MTT. In the presence of an electron coupling reagent, it is reduced by mitochondrial dehydrogenases to produce an orange-yellow formazan. The color darkens with increasing cell proliferation and cytotoxicity, while the color becomes lighter with increasing cytotoxicity. For the same cell population, the color intensity is linearly correlated with cell number.

[0058] The experimental process is as follows: MCF-7 and MCF 10A cells were cultured in 96-well plates (0.8×104 cells / well) at 37°C and 5% carbon dioxide for 24 hours until the cells attached; culture medium (150 μL) containing a certain concentration of probe (2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 250 μM, 500 μM, 1000 μM) was added to different wells, and the cells were incubated for 48 hours and 72 hours. For viability testing, 15 μL of CCK8 was added and the cells were incubated at 37°C and 5% carbon dioxide for 4 hours. The absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader, and cell viability (100%) was calculated using the following formula:

[0059] Cell viability = (OD 加药 -OD 空白 ) / (OD 0加药 -OD 空白 )×100%; the drug-added group and blank group were the drug-free group and blank culture medium group, respectively.

[0060] The test results are as follows Figure 3 and Figure 4As shown, analysis shows that no obvious cytotoxic effect was observed in both cell lines when cultured with different concentrations of the probe. For MCF-7 cells, when the concentration was 0-100 μM, the cell survival rate was above 80%, and when the concentration was 0-100 μM, the survival rate was 70% for MCF-10A cells, indicating that the probe is biosafe.

[0061] The test probe is used to specifically illuminate (ER+) breast cancer cells:

[0062] MCF-7 cells (ER+), MDA-MB-231 cells (ER-), and DU-145 cells (ER-) were seeded onto 35 mm confocal dishes. The probe was dissolved directly in DMEM culture medium to a concentration of 10 μmol / L. After reaching 90% cell confluence, the cells were seeded onto 35 mm glass-bottomed culture dishes. After 24 hours of adherence, the three cell types were incubated with the targeted probe (10 μM) for 30 minutes. In the competition experiment, the cells were incubated with the probe (10 μM) at 37°C for 10 minutes, followed by treatment with E2 (100 μM) for 30 minutes. For spheroid imaging, MCF-7 cells were formed into spheroids by adding agarose and Matrigel, then incubated with the probe for 30 minutes. The cells were then transferred to a 35 mm confocal dish and observed under a confocal scanning microscope.

[0063] The results are as follows Figure 5 As shown in Figures ad, after incubation at 37°C for 30 minutes, the probe showed strong fluorescence immediately after imaging. In comparison, by incubating MCF-7 cells with the probe and E2 for competitive binding to ER protein, the fluorescence signal disappeared. These results indicate that the probe can penetrate the cell membrane, specifically label the ER protein in the cell, and make it possible to image the localization of the ER protein. However, a good ERα targeting probe should have good ERα labeling ability and stain both the cell membrane and the nucleus. Therefore, the distribution of the probe in MCF-7 cells was explored, and the results are shown in Figures ad. Figure 5 As shown in Figure eh. The probe (10 μM) was added to MCF-7 cells for 30 minutes, and then DAPI was added to stain the cell nuclei for 10 minutes before imaging. The results showed that the probe was present both on the cell membrane and in the cell nucleus. These results indicate that the targeting performance of the targeted probe to the estrogen receptor (ER) facilitates the cellular uptake and intracellular retention of the probe, resulting in MCF-7 cells overexpressing ER protein showing a significantly stronger fluorescence signal, demonstrating that the targeted probe has a high signal-to-noise ratio.

[0064] ERα-targeted probes should have good ERα selectivity. ERα and ERβ are the main isoforms of ER. A favorable ERα-targeted probe responds rapidly to ERα-positive cells, but has negligible fluorescence in ERβ-positive cells and other ERα-negative cells. To test the ERα selectivity of the probe, we performed live cell imaging on DU-145 ERβ-positive breast cancer cells and MDA-MB-231 triple-negative breast cancer cells. Figure 6 No signal was observed in DU-145 cells, and only weak fluorescence was observed in MDAMB-2 31 cells, indicating that these probes have good ERα selectivity and can be used to effectively distinguish (ER+) breast cancer cells, (ER-) normal breast cells, and breast cancer cells.

[0065] MCF-7 cell spheres were incubated with the targeting probe (10 μM) for 30 min, and then DAPI was added and incubated for 10 min. Figure 7 As shown, the estrogen receptor fluorescent probe can effectively label both planar breast cancer cells and spherical breast cancer cells. The above results indicate that the probe has a high signal-to-noise ratio and can illuminate breast cancer lesions by specifically binding to ER proteins.

[0066] Compared with the existing technology, the estrogen receptor-targeted fluorescent probe in this embodiment is composed of fluorescein ester, the estrogen receptor recognition group raloxifene and an alkane chain, which can avoid the influence of short emission wavelength and low signal-to-noise ratio when the probe is used, and is better used to promote the clinical transformation of the estrogen receptor fluorescent probe.

[0067] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0068] The embodiments of the present invention are described above in conjunction with the accompanying drawings. What is disclosed is only a preferred embodiment of the present invention. However, the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms and equivalent changes without departing from the scope of protection of the purpose of the present invention and the claims, which are all within the protection of the present invention.

Claims

1. A fluorescent probe targeting estrogen receptor, characterized in that: The structural formula of the estrogen receptor-targeted fluorescent probe is as follows:

2. A method for preparing a fluorescent probe targeting estrogen receptor, characterized in that: The preparation method is as follows: Step S1, reacting raw material I with BBr3 to synthesize amino intermediate II; Step S2: performing a condensation reaction on the amino intermediate II and FITC to obtain an estrogen receptor-targeting fluorescent probe III.

3. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 2, wherein: The reaction in step S1 is carried out under an argon environment.

4. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 2, wherein: The organic solvent used in the reaction in step S1 is dichloromethane.

5. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 2, wherein: The reaction in step S1 is carried out at room temperature for 3-5 hours.

6. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 2, wherein: In the step S2, the molar ratio of the amino intermediate II to the FITC is 1:

3.

7. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 2, wherein: In step S2, the organic solvent used in the condensation reaction is one or more of N,N-dimethylformamide, 1,4-dioxane or tetrahydrofuran.

8. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 2, wherein: The base used in the condensation reaction is one or both of N,N-diisopropylethylamine and triethylamine.

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