Targeted estrogen receptor fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of molecular imaging probes, in particular to a targeted estrogen receptor fluorescent probe and a preparation method and application thereof, and the targeted estrogen receptor fluorescent probe is composed of fluorescein ester, an estrogen receptor recognition group raloxifene and an alkane chain; the preparation method comprises the following steps: S1, carrying out carbamate hydrolysis or hydrogenolysis on a raw material I to synthesize an amino intermediate II; step S2, carrying out condensation reaction on the amino intermediate II and FITC (Fluorescein Isothiocyanate), so as to obtain the targeted estrogen receptor fluorescent probe III. The targeting estrogen receptor fluorescent probe provided by the invention can avoid the influence of short emission wavelength and low signal-to-noise ratio when the probe is applied, and can be better used for promoting the clinical transformation of the estrogen receptor fluorescent probe.

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 major problems that plague human life and health. Breast cancer, as a type of cancer, seriously threatens women's life and 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 in clinical practice, but accurate and rapid detection of estrogen receptor expression levels in breast cancer cells remains one of the current research hotspots. In order to achieve accurate detection of estrogen receptors, relevant technicians have begun to explore new molecular probes, especially the application of fluorescent probes in this field.

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

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

[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-targeted fluorescent probe is as follows:

[0010]

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

[0012]

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

[0014] Step S2, subjecting the amino intermediate II to a condensation reaction with FITC to obtain an estrogen receptor-targeting fluorescent probe III.

[0015] Preferably, the reactions in step S1 are all 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, and the reaction time is 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 fluorescent probe targeting estrogen receptor as provided in the first aspect, which is applied to protein labeling, protein molecule docking and screening of protein inhibitor molecules, cell imaging and fluorescent dye probes.

[0022] Compared with the prior art, the targeted estrogen receptor fluorescent probe in the present invention is composed of fluorescein ester, estrogen receptor recognition group raloxifene and alkane chain, so that the probe can avoid being affected by short emission wavelength and low signal-to-noise ratio when used, and can be better used to promote the clinical transformation of estrogen receptor fluorescent probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be described in detail below in conjunction with the accompanying drawings. The above and other aspects of the present invention will become clearer and easier to understand through the detailed description made in conjunction with 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 is the cytotoxicity of the ER recognition probe III in this example Figure 1 ;

[0027] Figure 4 is the cytotoxicity of the ER recognition probe III in this example Figure 2 ;

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

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

[0030] Figure 7 In this embodiment, 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 method for preparing the estrogen receptor targeting fluorescent probe in this embodiment. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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] The 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-targeted 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 carbamate to synthesize the amino intermediate II;

[0039] Step S2, subjecting the amino intermediate II to a condensation reaction with 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 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 3-5h.

[0042] In the 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); the base used in the condensation reaction is one or two of N,N-diisopropylethylamine (DIPEA) and triethylamine.

[0043] The estrogen receptor-targeted fluorescent probe may be referred to as probe or probe III hereinafter. Its excitation wavelength is about 495 nm, its emission wavelength is about 515 nm, it has strong tissue penetration ability, and can specifically illuminate estrogen receptor-positive breast cancer by targeting 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 easily 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 fluorescence 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 probe.

[0046] In order to better understand the estrogen receptor-targeted fluorescent probe in 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 with saturated brine once, and then the organic layers were combined, dried with 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 intermediate II were added to a flask respectively, and then Et3N (5 mL) was added to the mixture and stirred for 12 h in the dark at 25°C or dissolved in 2 mL of anhydrous DMF, and then FITC (1 mmol) and 0.1 mL of DIPEA were added and stirred overnight at 80°C; when the reaction was completed, it was purified by column chromatography (methanol / dichloromethane) to obtain a yellow solid (the estrogen receptor-targeted fluorescent probe III, 0.5 g, 63%).

[0050] It has been verified that the structure of the estrogen receptor-targeted 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-targeted fluorescent probe:

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

[0053] Test method: dilute the probe to a concentration of 2.5 μmol / L, transfer it to a quartz cuvette and mix it evenly 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 of FIG, 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] Cell Counting Kit-8, referred to as CCK-8 kit or CCK8 cell proliferation toxicity detection kit, is a kit based on WST-8 and widely used for rapid and highly sensitive detection of cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT. In the presence of an electron coupling agent, it can be reduced by some dehydrogenases in mitochondria to generate orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells.

[0058] The experimental process is as follows: MCF-7 and MCF 10A cells were cultured in a 96-well plate (0.8×104 cells / well) at 37°C and 5% carbon dioxide for 24 hours until the cells attached; a certain concentration of probe (2.5μM, 5μM, 10μM, 25μM, 50μM, 100μM, 250μM, 500μM, 1000μM) of culture fluid (150μL) was added to different wells, and the cells were incubated for 48h and 72h. For the viability test, 15μL CCK8 was added, and the cells were incubated at 37°C and 5% carbon dioxide for 4h. The absorbance (OD) was measured at a wavelength of 450nm using an enzyme reader, and the 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] Test results such as Figure 3 and Figure 4As shown, analysis shows that no obvious cytotoxic effect was observed in both cell types when cultured with different concentrations of the probe. When the concentration of MCF-7 cells was 0-100 μM, the cell survival rate was above 80%, and when the concentration of MCF-10A cells was 0-100 μM, the survival rate was 70%, indicating that the probe is biosafe.

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

[0062] MCF-7 cells (ER+), MDA-MB-231 cells (ER-), and DU-145 cells (ER-) were inoculated on 35 mm confocal dishes, and the probe was directly dissolved in the culture medium DMEM, and the concentration of the probe was diluted to 10 μmol / L; after the cell abundance reached 90%, the cells were inoculated in a 35 mm glass bottom culture dish, and after 24 hours of attachment, the three cells were incubated with the targeting probe (10 μM) for 30 minutes. In the competition experiment, the probe (10 μM) was incubated at 37 ° C for 10 minutes, and then treated with E2 (100 μM) for 30 minutes; in the cell sphere imaging, MCF-7 cells were formed into cell spheres by adding agarose and matrix gel, and the probe was added and incubated for 30 minutes, 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 (ad), after incubation at 37°C for 30 minutes, the probe was imaged immediately and showed strong fluorescence. In comparison, the competitive binding of ER protein to MCF-7 cells by incubation with the probe and E2 resulted in the disappearance of the fluorescence signal. 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 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 Figure 5 (eh) As shown. The probe (10 μM) was added to MCF-7 cells for 30 minutes, and then DAPI was added to stain the cell nucleus for 10 minutes, and then imaging was performed. The results showed that the probe existed both in the cell membrane and in the cell nucleus. These indicated that the targeting performance of the targeted probe to the estrogen receptor (ER) facilitated the cellular uptake and intracellular retention of the probe, and made the MCF-7 cells overexpressing the ER protein show a significantly stronger fluorescence signal, proving that the targeted probe had a high signal-to-noise ratio.

[0064] ERα targeting probes should have good ERα selectivity. ERα and ERβ are the main isoforms of ER. A favorable ERα targeting probe responds rapidly to ERα-positive cells, but the fluorescence to ERβ-positive cells and other ERα-negative cells is negligible. 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 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 prior art, the targeted estrogen receptor fluorescent probe in this embodiment is composed of fluorescein ester, estrogen receptor recognition group raloxifene and alkane chain, so that the probe can avoid being affected by short emission wavelength and low signal-to-noise ratio when used, and can be better used to promote the clinical transformation of estrogen receptor fluorescent probe.

[0067] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0068] The embodiments of the present invention are described above in conjunction with the accompanying drawings. What is disclosed is only the preferred embodiment of the present invention. However, the present invention is not limited to the above-mentioned specific implementation manner. The above-mentioned specific implementation manner is only illustrative rather than restrictive. Under the enlightenment 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, all of which are within the protection of the present invention.

Claims

1. A fluorescent probe targeting estrogen receptor, characterized in that: The estrogen receptor-targeted fluorescent probe is composed of fluorescein ester, an estrogen receptor recognition group raloxifene and an alkane chain.

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

3. A method for preparing a fluorescent probe targeting estrogen receptor, characterized in that: The preparation method is as follows: Step S1, subjecting the raw material I to hydrolysis or hydrogenolysis of carbamate to synthesize the amino intermediate II; Step S2, subjecting the amino intermediate II to a condensation reaction with FITC to obtain an estrogen receptor-targeting fluorescent probe III.

4. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 3, characterized in that: The reactions in step S1 are all carried out under an argon environment.

5. The method for preparing the estrogen receptor targeting fluorescent probe according to claim 3, characterized in that: The organic solvent used in the reaction in step S1 is dichloromethane.

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

7. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 3, characterized in that: In the step S2, the molar ratio of the amino intermediate II to the FITC is 1:

3.

8. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 3, characterized in that: In the step S2, the organic solvent used in the condensation reaction is one or more of N,N-dimethylformamide, 1,4-dioxane or tetrahydrofuran.

9. The method for preparing a fluorescent probe targeting estrogen receptor according to claim 3, characterized in that: The base used in the condensation reaction is one or both of N,N-diisopropylethylamine and triethylamine.

10. The use of the estrogen receptor targeting fluorescent probe according to claim 1 or 2, characterized in that: The estrogen receptor-targeted fluorescent probe is applied to protein labeling, protein molecule docking, protein inhibitor molecule screening, cell imaging and fluorescent dye probe.

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