A near-infrared fluorescent diagnostic and therapeutic probe capable of real-time monitoring of ERα protein degradation, and its preparation method and application

Abstract

The present invention relates to the field of medical technology, and specifically discloses a near-infrared fluorescent diagnostic and therapeutic probe that can monitor ERα protein degradation in real time, as well as its preparation method and application. The highly ERα-targeting fluorophore 2-(6-hydroxy-2-(4-hydroxystyryl)-4H-benzopyran-4-subunit) malononitrile is used as a warhead that specifically binds to the target protein, methyl VHL is used as a ligand to recruit E3 ligase, and carbon chains of different lengths are used as linkers to design and synthesize a series of near-infrared fluorescent diagnostic and therapeutic probes that can monitor ERα protein degradation in real time. The diagnostic and therapeutic NIR probe of the present invention can not only effectively degrade ERα protein, but also exhibit excellent anti-proliferative activity. This innovative study successfully integrated PROTAC technology with a fluorescent probe targeting ERα for the first time, realizing real-time visualization of ERα protein degradation in MCF-7 cells. These results provide strong support for the potential application prospects of this type of compound in the treatment of breast cancer and expand the application field of PROTAC technology.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, specifically to a near-infrared fluorescent diagnostic and therapeutic probe for real-time monitoring of ERα protein degradation, as well as its preparation method and application. This invention encompasses cross-disciplinary innovations across multiple fields, including fluorescent molecule design, biomedical imaging, and chemical synthesis. It provides an innovative, highly effective, and broadly applicable method for breast cancer treatment and offers a new strategy for real-time visualization of protein degradation. Background Art

[0002] Breast cancer, a common malignancy in women, poses a serious threat to their health and life. With the continuous advancement of medical technology, treatments for breast cancer are also evolving, but several challenges remain. One of these is the need for precise molecular monitoring and treatment of breast cancer. ERα (estrogen receptor α) plays a crucial role in the development and progression of breast cancer. Therefore, precise monitoring and treatment of ERα has become a key approach in breast cancer treatment. Current monitoring methods primarily include tissue biopsy and serum marker detection. However, these methods have limitations, such as invasiveness, time delay, and limited information acquisition. Fluorescent molecules, particularly near-infrared fluorescent molecules, are an essential component of biomedical imaging. Due to their high sensitivity and real-time monitoring capabilities, they have played a vital role in molecular-level research. However, there are currently few diagnostic and therapeutic probes on the market that can target and monitor ERα degradation in real time.

[0003] Therefore, the present invention aims to provide an innovative fluorescent diagnostic and therapeutic probe, such as Figure 1 As shown, the probe has a high degree of ERα targeting and can monitor the degradation process of ERα protein in real time. The development of this probe aims to provide a more accurate and real-time monitoring fluorescent diagnostic and therapeutic probe to meet the higher requirements of breast cancer treatment and achieve breakthroughs in improving the precision and effectiveness of breast cancer treatment. Summary of the Invention

[0004] The purpose of the present invention is to provide a near-infrared fluorescent diagnostic and therapeutic probe that can monitor the degradation of ERα protein in real time. The probe has a high degree of ERα targeting and can monitor the degradation process of ERα protein in real time.

[0005] Another object of the present invention is to provide a method for preparing a near-infrared fluorescent diagnostic and therapeutic probe that can monitor ERα protein degradation in real time. The highly ERα-targeting fluorophore 2-(6-hydroxy-2-(4-hydroxystyryl)-4H-benzopyran-4-ylidene) malononitrile is used as a warhead that specifically binds to the target protein, methyl VHL is used as a ligand to recruit E3 ligase, and carbon chains of different lengths are used as linkers to design and synthesize a series of near-infrared fluorescent diagnostic and therapeutic probes that can monitor ERα protein degradation in real time.

[0006] Another object of the present invention is to provide an application of a near-infrared fluorescent diagnostic and therapeutic probe prepared by the above-mentioned preparation method in the treatment of breast cancer. When used in vivo, the near-infrared fluorescent diagnostic and therapeutic probe can effectively inhibit the growth of MCF-7 breast cancer cells, achieving a breakthrough in improving the accuracy and effectiveness of breast cancer treatment.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A near-infrared fluorescent diagnostic and therapeutic probe capable of real-time monitoring of ERα protein degradation has a structure shown in the following general formula:

[0009]

[0010] Among them, n=5, 7, 9, 11.

[0011] Accordingly, the present invention also provides a method for preparing the aforementioned near-infrared fluorescent diagnostic and therapeutic probe capable of real-time monitoring of ERα protein degradation, comprising:

[0012] First, compound 1 was coupled with compound 2 under piperidine catalysis to obtain intermediate 3;

[0013] Subsequently, intermediate 3 was deprotected under strongly acidic conditions to form carboxylic acid 4;

[0014] Finally, carboxylic acid 4 was condensed with VHL ligand 5 in the presence of EDCI and HOBt to obtain the target compounds 6a-6d;

[0015] The synthetic routes of compounds 6a-6d are as follows:

[0016]

[0017] (a) 2a-2d, toluene, piperidine, 120℃, 12h; (b) TFA, DCM, 0℃ to rt, 2h; (c) 5(VHL Ligand), EDCI, HOBt, DIPEA, DMF, 0℃ to rt, 12h.

[0018] Optionally, the compound 2 includes but is not limited to: tert-butyl 6-(2-formyl-5-(methoxymethoxy)phenoxy)hexanoate (2a), tert-butyl 8-(2-formyl-5-(methoxymethoxy)phenoxy)octanoate (2b), tert-butyl 10-(2-formyl-5-(methoxymethoxy)phenoxy)decanoate (2c), tert-butyl 12-(2-formyl-5-(methoxymethoxy)phenoxy)dodecanoate (2d), etc.

[0019] Furthermore, the preparation method comprises the following specific steps:

[0020] Under N₂ protection, 2-(6-hydroxy-2-(4-hydroxyphenylvinyl)-4H-chromen-4-ylidene)malononitrile (1.0 equiv.) and compound 2 (1.05-1.2 equiv.) were added to 15 ml of toluene solution, followed by the dropwise addition of equal molar amounts of piperidine and acetic acid (0.02 equiv.). The reaction mixture was heated to 120°C and allowed to react for 12 hours. The mixture was then concentrated to an appropriate volume and extracted with dichloromethane. The combined organic layers were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to yield the crude product. Compound 3 was then purified by column chromatography (dichloromethane / methanol = 100:1) to yield compound 3.

[0021] Compound 3 (1.0 equiv.) was added to an appropriate amount of anhydrous dichloromethane at 0-5°C, followed by dropwise addition of trifluoroacetic acid and stirring. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was then filtered and purified by column chromatography (dichloromethane / methanol = 50:1) to afford compound 4.

[0022] Under nitrogen protection, compound 4 (1.0 equiv.) was dissolved in DMF at 0-5°C, and EDCI (1.2-1.5 equiv.) and HOBt (1.2-1.5 equiv.) were added. VHL ligand 5 (1.0 equiv.) and DIPEA (2.0-4.0 equiv.) were then added sequentially. The reaction was stirred for 10 minutes, then warmed to room temperature and stirred for 12 hours. After completion of the reaction, the mixture was poured into water and extracted with dichloromethane. The organic layers were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Compound 6 was obtained by purification via column chromatography (dichloromethane / methanol = 150:1).

[0023] Furthermore, the compound 6 is:

[0024] (2S,4R)-1-((S)-2-(6-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6a),

[0025] (2S,4R)-1-((S)-2-(8-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6b),

[0026] (2S,4R)-1-((S)-2-(10-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)decylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6c),

[0027] (2S,4R)-1-((S)-2-(12-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)dodecanoyl)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6d).

[0028] Preferably, the compound 6 is: (2S,4R)-1-((S)-2-(10-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)decylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6c).

[0029] A near-infrared fluorescence diagnostic and therapeutic probe for treating breast cancer, comprising the following components:

[0030] A highly ERα-targeting fluorophore, 2-(6-hydroxy-2-(4-hydroxyphenylvinyl)-4H-benzopyran-4-ylidene)malononitrile, is used to specifically bind to the ERα protein;

[0031] (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, used as a ligand to recruit E3 ligases;

[0032] A series of linkers with different carbon chain lengths to adjust the molecular structure;

[0033] A structure that can emit fluorescence in the near-infrared band can be used to monitor the degradation of ERα protein in real time.

[0034] Furthermore, the length of the linker ranges from C6 to C 12 In order to meet the regulation of the overall performance of the molecule,

[0035] Correspondingly, the present invention also provides a use of the aforementioned near-infrared fluorescent diagnostic and therapeutic probe in the treatment of breast cancer, wherein the near-infrared fluorescent diagnostic and therapeutic probe can effectively inhibit the growth of MCF-7 breast cancer cells when used in vivo.

[0036] When used in vivo, the near-infrared fluorescent diagnostic and therapeutic probe can promote the efficient degradation of MCF-7 breast cancer cells.

[0037] When used in vivo, the near-infrared fluorescent diagnostic and therapeutic probe can achieve real-time visualization of ERα protein degradation in MCF-7 cells.

[0038] The near-infrared fluorescent diagnostic and therapeutic probe not only efficiently degrades ERα protein but also has near-infrared visualization performance.

[0039] The application prospect of the near-infrared fluorescence diagnostic and therapeutic probe in the treatment of breast cancer shows its superiority in improving treatment effects and reducing treatment toxicity.

[0040] The structure and components of the near-infrared fluorescence diagnostic and therapeutic probe can be appropriately adjusted according to actual needs to achieve precise treatment of different breast cancer subtypes.

[0041] The present invention found that the above compounds 6a-6d can be used to prepare anti-breast cancer drugs through in vitro breast cancer activity experiments. In particular, compound 6c has the best MCF-7 cell inhibitory activity and the best ERα protein degradation ability (IC 50 =0.051μM, DC 50 =0.12μM), and also has good fluorescence imaging performance, with an emission wavelength of up to 582nm and a Stokes shift of 116nm.

[0042] This invention uses the highly ERα-targeting fluorophore 2-(6-hydroxy-2-(4-hydroxyphenylvinyl)-4H-benzopyran-4-ylidene) malononitrile as a warhead that specifically binds to the target protein, employs methyl VHL as a ligand to recruit the E3 ligase, and utilizes carbon chains of varying lengths as linkers to design and synthesize a series of near-infrared fluorescent diagnostic and therapeutic probes for real-time monitoring of ERα protein degradation. Compared to existing technologies, this invention offers significant advantages and features, primarily in the following aspects:

[0043] Successfully combining PROTAC technology with a fluorescent probe targeting ERα enabled real-time visualization of ERα protein degradation in MCF-7 cells. This innovation provides a new strategy for monitoring and regulating ERα protein degradation, making the treatment process more precise and controllable.

[0044] Excellent anti-proliferative activity: The therapeutic NIR probe not only effectively degrades ERα protein but also exhibits excellent anti-proliferative activity. This provides strong support for the potential application of similar compounds in breast cancer treatment. These results provide strong support for the potential application of this class of compounds in breast cancer treatment and expand the application of PROTAC technology.

[0045] These characteristics make the present invention have broad application prospects in breast cancer treatment and related fields, and provide a more innovative and efficient treatment method for targeted visualization treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0047] Figure 1 This is a design diagram of a near-infrared fluorescent diagnostic and therapeutic probe that can monitor ERα protein degradation in real time.

[0048] Figure 2 Evaluation of the biological activity of 6a-6c. (A) Western blotting analysis of ERα protein levels in MCF-7 cells following treatment with 6a-6c and the control drug fulvestrant. (B) Evaluation of ERα levels in MCF-7 cells following treatment with different concentrations of 6c for 24 hours.

[0049] Figure 3 The optical properties of the system. (A) Absorption and (B) emission spectra at a concentration of 10 μM. (C) Fluorescence intensity versus concentration curve. (D) The linear relationship (R) obtained from Figure C. 2 =0.9937).

[0050] Figure 4 Confocal laser scanning microscopy images of MCF-7 cells treated with 6c, DAPI, and 6c + E2. Scale bar: 50 μm. Excitation wavelength: 488 nm.

[0051] Figure 5 Confocal laser scanning microscopy imaging analysis of 6c at 10 μM for different time periods. DAPI was used for intracellular localization (blue), ERα protein immunofluorescence staining (green), and A3 (10 μM, red). Scale bar: 50 μm. DETAILED DESCRIPTION

[0052] The outstanding features of the present invention are further illustrated by the following examples, which are intended only to illustrate the present invention but in no way to limit the present invention.

[0053] Example 1: Preparation of (2S,4R)-1-((S)-2-(6-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6a):

[0054] Under N2 protection, 2-(6-hydroxy-2-(4-hydroxystyryl)-4H-chromen-4-ylidene)malononitrile (1.00 g, 4.46 mmol) and tert-butyl 6-(2-formyl-5-(methoxymethoxy)phenoxy)hexanoate (2a) (1.73 g, 4.91 mmol) were added to 15 ml of toluene solution, followed by 0.15 ml of piperidine and 0.15 ml of acetic acid. The reaction mixture was heated to 120°C and refluxed for 12 hours. After completion of the reaction, the reaction solution was concentrated and extracted with dichloromethane. The combined organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained crude product was purified by column chromatography (dichloromethane / methanol = 100:1) to obtain tert-butyl (E)-6-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-(methoxymethoxy)phenoxy)hexanoate (3a).

[0055] Compound 3a (2.00 g, 3.58 mmol) was added to 20 ml of anhydrous dichloromethane at 0-5°C. An appropriate amount of trifluoroacetic acid was then added dropwise and the reaction was stirred. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was then filtered and purified by column chromatography (dichloromethane / methanol = 50:1) to obtain (E)-6-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)hexanoic acid (4a).

[0056] Under nitrogen protection at 0-5°C, compound 4a (1.50 g, 3.27 mmol) was dissolved in 10 ml of DMF. EDCI (1.35 g, 6.54 mmol) and HOBt (0.88 g, 6.54 mmol) were added sequentially and stirred for 15 min. VHL ligand 5 (1.45 g, 3.27 mmol) and DIPEA (1.69 g, 13.09 mmol) were then slowly added. The reaction was stirred and allowed to warm to room temperature naturally, where it was stirred for 12 hours. After completion of the reaction, the mixture was poured into water and extracted with dichloromethane. The combined organic layers were washed sequentially with water and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 150:1) to yield compound 6a. 1 H NMR (400MHz, DMSO-d6) δ10.48(s,1H),10.28(s,1H),9.01(s,1H),8.39(d,J=7.8Hz,1H),8.06(d,J=2.6Hz,1H),7.89(m,2H),7.65(d,J=8.8 Hz,2H),7.42(d,J=8.0Hz,2H),7.39(m,3H),7.19(d,J=16.1Hz,1H),6.82(d,J=2.4Hz,1H),6.45(d,J=8.9Hz,2H),5.16(s,1H),4.89(t,J=7. 2Hz,1H),4.50(d,J=9.4Hz,1H),4.40(t,J=8.1Hz,1H),4.28(s,1H),4.04(s,2H),3.57(s,2H),2.47(s,3H),2.35–2.26(m,1H),2.22–2.14( m,1H),1.99(d,J=6.8Hz,1H),1.84–1.79(m,2H),1.64–1.59(m,1H),1.46(d,J=7.3Hz,1H),1.34(d,J=7.0Hz,3H),1.24(s,3H),0.91(s,9H). 13C NMR(101MHz,DMSO)δ172.0,170.6,169.6,161.8,159.1,155.0,152.5,151.5,147.8,1 45.7,144.7,133.6,131.1,129.7,129.6,128.8,128.8,126.4,124.2,120.2,118.0,11 7.6,116.6,115.3,114.9,108.6,108.1,104.8,99.9,68.8,68.0,58.6,57.3,56.4,56. 3,47.7,37.8,35.2,34.9,28.3,26.4,25.4,25.2,22.4,16.0.HRMS(ESI,positive)m / z calcd for[M+H] + C 49 H 52 N6O8S:885.3567,found:885.3645.

[0057] Example 2: Preparation of (2S,4R)-1-((S)-2-(8-(2-((E)-2-(4-(dicyanomethylene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6b):

[0058] Under N2 protection, 2-(6-hydroxy-2-(4-hydroxystyryl)-4H-chromen-4-ylidene)malononitrile (1.00 g, 4.46 mmol) and 8-(2-formyl-5-(methoxymethoxy)phenoxy)octanoic acid tert-butyl ester (2b) (1.87 g, 4.91 mmol) were added to 15 ml of toluene solution, followed by 0.15 ml of piperidine and 0.15 ml of acetic acid. The reaction mixture was heated to 120 ° C and refluxed for 12 hours. After the reaction was completed, the reaction solution was concentrated and extracted with dichloromethane. The combined organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography (dichloromethane / methanol = 100:1) to obtain tert-butyl (E)-8-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-(methoxymethoxy)phenoxy)octanoate (3b).

[0059] Compound 3b (2.00 g, 3.41 mmol) was added to 20 ml of anhydrous dichloromethane at 0-5°C. An appropriate amount of trifluoroacetic acid was then added dropwise and the reaction was stirred. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was then filtered and purified by column chromatography (dichloromethane / methanol = 50:1) to obtain (E)-8-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)octanoic acid (4b).

[0060] Under nitrogen protection at 0-5°C, compound 4b (1.50 g, 3.08 mmol) was dissolved in 10 ml of DMF. EDCI (1.27 g, 6.17 mmol) and HOBt (0.83 g, 6.17 mmol) were added sequentially and stirred for 15 min. VHL ligand 5 (1.37 g, 3.08 mmol) and DIPEA (1.59 g, 12.33 mmol) were then slowly added. The reaction was stirred and allowed to warm to room temperature naturally, where it was stirred for 12 hours. After completion of the reaction, the mixture was poured into water and extracted with dichloromethane. The combined organic layers were washed sequentially with water and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 150:1) to yield compound 6b. 1 H NMR (400MHz, DMSO-d6) δ10.49(s,1H),10.34(s,1H),9.01(s,1H),8.41(d,J=7.8Hz,1H),8.05(d,J=2.7Hz,1H),7.89–7.78(m,2H),7.62(d,J=8.3Hz ,1H),7.55(d,J=9.1Hz,1H),7.43(d,J=8.3Hz,2H),7.39(m,3H),7.18(d, J=16.1Hz,1H),6.81(s,1H),6.45(d,J=8.6Hz,2H),5.18(m,1H),4.95–4.8 5(m,1H),4.49(d,J=9.3Hz,1H),4.40(t,J=8.1Hz,1H),4.28(s,1H),4.06 –3.98(m,2H),3.57(d,J=3.0Hz,2H),2.47(s,3H),2.31–2.19(m,1H),2.13 –2.04(m,1H),2.00(d,J=5.9Hz,1H),1.84–1.72(m,3H),1.51–1.45(m,3H ),1.35(d,J=7.0Hz,3H),1.29(d,J=7.1Hz,2H),1.23(s,3H),0.91(s,9H). 13C NMR (101MHz, DMSO) δ172.1,170.6,169.6,161.8,159.2,159.1,155.0,152.5,151.5,147.8,145.7, 144.7,133.8,131.1,129.7,128.8,126.4,124.2,119.9,118.0,117.5,116.5,115.3,114.9,108.6, 108.2,104.7,99.9,68.8,68.0,58.6,57.4,56.3,56.3,47.7,40.1,39.9,39.7,39.5,39.3,39.2,39 .1,38.9,37.7,35.2,34.9,28.7,28.6,28.5,26.4,25.6,25.4,22.4,16.0.HRMS(ESI,positive)m / z calcd for[M+H] + C 51 H 56 N6O8S:913.3880,found:913.3953.

[0061] Example 3: Preparation of (2S,4R)-1-((S)-2-(10-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)decylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6c):

[0062] Under N2 protection, 2-(6-hydroxy-2-(4-hydroxystyryl)-4H-chromen-4-ylidene)malononitrile (1.00 g, 4.46 mmol) and tert-butyl 10-(2-formyl-5-(methoxymethoxy)phenoxy)decanoate (2c) (2.00 g, 4.91 mmol) were added to 15 ml of toluene solution, followed by 0.15 ml of piperidine and 0.15 ml of acetic acid. The reaction mixture was heated to 120°C and refluxed for 12 hours. After completion of the reaction, the reaction solution was concentrated and extracted with dichloromethane. The combined organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained crude product was purified by column chromatography (dichloromethane / methanol = 100:1) to obtain tert-butyl (E)-10-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-(methoxymethoxy)phenoxy)decanoate (3c).

[0063] Compound 3c (2.00 g, 3.25 mmol) was added to 20 ml of anhydrous dichloromethane at 0-5°C. An appropriate amount of trifluoroacetic acid was then added dropwise and the reaction was stirred. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was then filtered and purified by column chromatography (dichloromethane / methanol = 50:1) to obtain (E)-10-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)decanoic acid 4c.

[0064] Under nitrogen protection at 0-5°C, compound 4c (1.50 g, 2.92 mmol) was dissolved in 10 ml of DMF. EDCI (1.20 g, 5.83 mmol) and HOBt (0.79 g, 5.83 mmol) were added sequentially, and the mixture was stirred for 15 min. VHL ligand 5 (1.30 g, 2.92 mmol) and DIPEA (1.51 g, 11.66 mmol) were then slowly added. The reaction was stirred and allowed to warm to room temperature naturally, where it was stirred for 12 hours. After completion of the reaction, the mixture was poured into water and extracted with dichloromethane. The combined organic layers were washed sequentially with water and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 150:1) to produce compound 6c. 1 H NMR(400MHz,DMSO-d6)δ10.47(s,1H),10.28(s,1H),9.01(s,1H),8.41(d,J =7.8Hz,1H),8.06(d,J=2.7Hz,1H),7.90–7.77(m,2H),7.62(d,J=8.4Hz,1H ),7.55(d,J=9.1Hz,1H),7.43(d,J=8.3Hz,2H),7.39–7.31(m,3H),7.18(d, J=16.1Hz,1H),6.82(s,1H),6.48(s,2H),5.12(d,J=3.4Hz,1H),4.94(s,1H) ,4.49(d,J=9.3Hz,1H),4.43(s,1H),4.28(s,1H),4.02(t,J=6.3Hz,2H),3. 60(s,2H),2.46(s,3H),2.28–2.17(m,1H),2.11–2.04(m,1H),2.02–1.95(m ,1H),1.85–1.74(m,3H),1.55–1.43(m,4H),1.42(s,2H),1.36(d,J=7.0Hz, 3H),1.33(d,J=2.6Hz,1H),1.29(s,2H),1.22(d,J=2.1Hz,3H),0.92(s,9H). 13C NMR (101MHz, DMSO) δ172.1,170.7,169.7,161.8,159.3,159.1,155.0,152.4,151.5,147.8,145.7,144. 7,133.9,131.2,130.0,129.7,128.9,126.4,124.1,119.8,118.0,117.6,116.5,115.3,114.9,108.6,10 8.2,104.7,99.9,68.8,68.1,58.6,57.5,56.4,56.3,47.8,40.1,39.9,39.7,39.5,39.3,39.1,38.9,37. 8,35.2,34.9,29.1,28.9,28.8,28.7,28.6,26.4,25.8,25.5,22.5,16.0.HRMS(ESI,positive)m / zcalcd for[M+H] + C 53 H 60 N6O8S:941.4193,found:941.4257.

[0065] Example 4: Preparation of (2S,4R)-1-((S)-2-(12-(2-((E)-2-(4-(dicyanomethylene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)dodecanoyl)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (6d):

[0066] Under N2 protection, 2-(6-hydroxy-2-(4-hydroxystyryl)-4H-chromen-4-ylidene)malononitrile (1.00 g, 4.46 mmol) and 12-(2-formyl-5-(methoxymethoxy)phenoxy)t-butyl dodecanoate (2d) (2.14 g, 4.91 mmol) were added to 15 ml of toluene solution, followed by 0.15 ml of piperidine and 0.15 ml of acetic acid. The reaction mixture was heated to 120 ° C and refluxed for 12 hours. After completion of the reaction, the reaction solution was concentrated and extracted with dichloromethane. The combined organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography (dichloromethane / methanol = 100:1) to give tert-butyl (E)-12-(2-(2-(4-(dicyanomethylene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-(methoxymethoxy)phenoxy)dodecanoate (3d).

[0067] Compound 3d (2.00 g, 3.11 mmol) was added to 20 ml of anhydrous dichloromethane at 0-5°C. An appropriate amount of trifluoroacetic acid was then added dropwise and the reaction was stirred. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was then filtered and purified by column chromatography (dichloromethane / methanol = 50:1) to obtain (E)-12-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)dodecanoic acid 4d.

[0068] Under nitrogen protection at 0-5°C, compound 4d (1.50 g, 2.76 mmol) was dissolved in 10 ml of DMF. EDCI (1.14 g, 5.53 mmol) and HOBt (0.75 g, 5.53 mmol) were added sequentially, and the mixture was stirred for 15 min. VHL ligand 5 (1.23 g, 2.76 mmol) and DIPEA (1.43 g, 11.06 mmol) were then slowly added. The reaction was stirred and allowed to warm to room temperature naturally, where it was stirred for 12 hours. After completion of the reaction, the mixture was poured into water and extracted with dichloromethane. The combined organic layers were washed sequentially with water and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 150:1) to yield compound 6d. 1 H NMR(400MHz, DMSO-d6)δ9.00(s,1H),8.42(d,J=7.7Hz,1H),8.10–8.01(m,1H),7.83–7.72(m,2H),7.51(d,J=9.1Hz,1H) ,7.48–7.32(m,6H),7.32–7.27(m,1H),7.06(d,J=16.0Hz,1H),6.77(s,1H),6.51(s,1H),5.16(s,1H),4.90(t,J=7.2Hz ,1H),4.49(d,J=9.4Hz,1H),4.40(t,J=8.1Hz,1H),4.28(s,1H),4.01(s,2H),3.58(d,J=3.4Hz,2H),2.46(s,3H),2.24– 2.14(m,1H),2.08–1.93(m,3H),1.83(m,3H),1.49(m,4H),1.35(d,J=6.9Hz,3H),1.29(s,7H),1.22(s,4H),0.92(s,9H). 13C NMR(101MHz,DMSO)δ171.1,170.1,169.8,161.9,158.5,155.3,152.7,151.9,148.2,14 6.8,146.1,133.8,131.3,129.3,126.8,124.1,118.4,114.6,112.1,104.9,101.0,69.2 ,59.0,56.8,48.2,42.6,40.5,40.3,40.1,39.9,39.7,39.5,39.3,38.1,38.0,35.6,31 .6,29.6,29.5,29.3,29.2,26.8,26.3,25.9,22.8,22.5,16.4.HRMS(ESI,negative)m / z calcd for[MH] - C 55 H 64 N6O8S:967.4506,found:967.4495.

[0069] Relative affinity determination of compounds

[0070] The affinity of compounds 6a-6d for ERα and ERβ was determined by fluorescence polarization, where the affinity of the compound was defined relative to the affinity for the endogenous estrogen E2. The affinity of E2 for the receptor was set to RBA = 100%. The relative binding affinity (RBA) was calculated as follows: RBA (%) = IC 50 E2 / IC 50 As shown in Table 1, compounds 6a-6d all had higher affinities for ERα than for ERβ, with 6c showing the highest affinity for ERα. The RBA value of 6c for ERα was 7.99 ± 0.12 and the α / β ratio was 2.03.

[0071] Table 1 Relative binding affinities of target compounds 6a-6c to ERα and ERβ a .

[0072]

[0073] a RBA values were determined by competitive radiometric bindingassays(RBA,estradiol=

[0074] 100%).

[0075] Cell viability test

[0076] CCK-8 (Cell Counting Kit-8) was used to determine the cell growth inhibitory activity of 6a-6c at different concentrations on MCF-7 and DU-145 cells. 3 Cells were plated and incubated for 24 hours. Subsequently, the cells were added with different concentrations of serial dilutions of 6a-6c containing 0.5% dimethyl sulfoxide (DMSO) and incubated for 48 hours. After that, the cell culture medium was replaced with 100 μL of fresh culture medium and 10 μL of CCK8 was added. After incubation at 37°C for 15 minutes, the plate was read on a microplate reader, and the wavelength of 450 nm was selected as the reference wavelength. The experimental results were analyzed and the IC 50 . Each drug concentration was tested with four technical replicates and three biological replicates. As shown in Table 2, compounds 6a-6d exhibited significant antiproliferative activity in MCF-7 cells. In contrast, these compounds had relatively weak inhibitory effects on DU-145 cells. It is worth noting that the length of the linker plays a key role in determining the inhibitory activity of ERα. As the length of the carbon chain increases, the inhibitory activity continues to increase. When the linker length is extended to 10 carbon atoms, the antiproliferative activity reaches the optimal level (IC 50 =0.051 μM). However, further extension of the linker resulted in a weakening of the inhibitory activity.

[0077] Table 2 Effects of target compounds 6a-6c on the viability of MCF-7 cells and DU-145 cells a .

[0078]

[0079] a Experimental values represent an average of at least three independent experiments±standard deviation(mean±SD).

[0080] ER degradation activity assay

[0081] The ability of compounds 6a-6c to induce ERα degradation in MCF-7 cells was evaluated by Western blotting (WB). MCF-7 cells were plated at 7×10 5Cells were cultured in 6-well plates at a density of 100 cells, and 6a-6c were added at different time intervals. After incubation, proteins were extracted using SDS lysis buffer. Subsequently, these protein samples were separated on 8% SDS-PAGE gels and transferred to PVDF membranes. They were incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-labeled secondary antibodies for 1 hour at room temperature, and finally the membranes were developed using ECL reagents. Figure 2 As shown in A, compounds 6a-6c all showed the ability to induce ERα protein degradation in MCF-7 cells at a concentration of 5 μM, and the degradation efficiency gradually increased with the increase of the linker chain length. Consistent with the ERα inhibitory activity, 6c with a linker chain length of 10 carbon atoms showed the most significant degradation efficacy, and its DC 50 The value is 0.12μM ( Figure 2 B).

[0082] Optical properties

[0083] Absorption spectrum studies show that compounds 6a-6c exhibit their main absorption peak at 466 nm, accompanied by a relatively weak absorption peak at 648 nm ( Figure 3 A). Under 466nm excitation, its maximum emission wavelength (λem) is 582nm, with a large Stokes shift of 116nm, as shown in Figure 3 As shown in B. Considering the cytotoxicity and degradation efficiency, 6c was selected to study the relationship between photostability and fluorescence intensity and concentration. Figure 3 As shown in C-3D, within the concentration range of 0-50 μM, the fluorescence intensity increases directly proportional to the concentration and exhibits a good linear relationship with a high correlation coefficient (R2 = 0.9937). When the concentration exceeds 50 μM, the fluorescence intensity is quenched or saturated, resulting in the fluorescence intensity no longer showing a linear relationship.

[0084] Cell imaging

[0085] The inventors selected ERα + MCF-7 cells were cultured and incubated with 10 μM A3 in DMEM basal medium for 30 minutes. The results showed that MCF-7 cells showed strong red fluorescence. To further confirm that the fluorescence came from the binding of 6c to ERα protein, the inventors blocked the ERα-A3 interaction by adding excess estradiol (E2, 100 μM). After this treatment, no obvious fluorescence was observed ( Figure 4After immunofluorescence staining of ERα protein, it was found that the green fluorescence signal gradually weakened over time and almost completely disappeared after 24 hours of treatment with 6c (10μM). This result is consistent with the degradation effect of 6c on ERα. In the first 24 hours, the red fluorescence signal in MCF-7 cells gradually increased, which was due to the increased cellular uptake of compound 6c and its binding to ERα ( Figure 5 These imaging results confirm the ability of compound 6c to simultaneously detect and degrade endogenous ERα in MCF-7 cells, highlighting its efficacy and sensitivity as a real-time tool to track the degradation process.

[0086] The above description is only a specific embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or replacements within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A near-infrared fluorescent diagnostic and therapeutic probe for real-time monitoring of ERα protein degradation, with the following structural formula: ， in, n = 9。 2. A method for preparing the near-infrared fluorescence diagnostic and therapeutic probe according to claim 1, characterized in that: The specific steps include: Under N2 protection, 2-(6-hydroxy-2-(4-hydroxystyryl)-4H-chromene-4-ylidene) malononitrile and tert-butyl 10-(2-aldehyde-5-(methoxymethoxy)phenoxy)decanoate were added to a toluene solution, followed by dropwise addition of equimolar amounts of piperidine and acetic acid. The reaction mixture was heated to 120°C and refluxed for 12 hours. The mixture was then concentrated to an appropriate volume and extracted with dichloromethane. The combined organic layers were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product which was purified by column chromatography to obtain tert-butyl (E)-10-(2-(2-(4-(dicyanomethylidene)-6-hydroxy-4H-chromene-2-yl)vinyl)-5-(methoxymethoxy)phenoxy)decanoate. Tert-butyl (E)-10-(2-(2-(4-(dicyanomethylene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-(methoxymethoxy)phenoxy)decanoate was added to an appropriate amount of anhydrous dichloromethane at 0-5°C, followed by dropwise addition of an appropriate amount of trifluoroacetic acid with stirring. The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was then filtered and purified by column chromatography to obtain (E)-10-(2-(2-(4-(dicyanomethylene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)decanoic acid. Under nitrogen protection, at 0-5 ° C, (E)-10-(2-(2-(4-(dicyanomethylene)-6-hydroxy-4H-chromen-2-yl)vinyl)-5-hydroxyphenoxy)decanoic acid was dissolved in DMF, EDCI and HOBt were added, and then (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide and DIPEA were added in sequence. The reaction was stirred for 10 minutes, then the temperature was raised to room temperature and the reaction was stirred for 12 hours. After completion, the mixture was poured into water and extracted with dichloromethane, the organic layers were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by column chromatography to obtain (2S,4R)-1-((S)-2-(10-(2-((E)-2-(4-(dicyanomethylidene)-6-hydroxy-4H-benzopyran-2-yl)vinyl)-5-hydroxyphenoxy)decylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

3. Use of the near-infrared fluorescent diagnostic and therapeutic probe according to claim 1 in the preparation of anti-breast cancer drugs, characterized in that: It can effectively inhibit the growth of MCF-7 breast cancer cells.

4. A use of the near-infrared fluorescent diagnostic and therapeutic probe according to claim 1 in the preparation of an anti-breast cancer drug, characterized in that: The near-infrared fluorescent diagnostic and therapeutic probe can realize real-time visualization of ERα protein degradation in MCF-7 cells.

Images