An ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe, its preparation method and application

Abstract

This invention discloses an ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe, its preparation method, and its applications, belonging to the field of medical technology. The fluorescent probes of this invention are dicyanomethylene-4H-benzodihydropyran compounds P1 and P2 containing borate ester groups, with the following structural formula. These fluorescent probes use DCM-OH as a backbone, acting as both a fluorophore and an ERβ ligand. The borate ester group is the H2O2 responsive group. The introduction of the borate ester group disrupts the push-pull effect of the DCM-OH fluorophore, preventing fluorescence emission. However, in a high-concentration H2O2 environment, the borate ester group is specifically cleaved by H2O2, subsequently exhibiting strong initiation fluorescence and enabling tumor imaging in vitro and in vivo. This invention brings new opportunities for the diagnosis and research of prostate cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to an ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe, its preparation method, and its application. Background Technology

[0002] In recent years, the therapeutic potential of ERβ in breast cancer, prostate cancer, lung cancer, nervous system diseases, and bone tissue has been increasingly revealed, drawing growing attention to this target. However, research on ERβ is still insufficient, and its subcellular distribution, isoform expression, and roles in different diseases require further confirmation. 1 ERβ is an important target for the treatment of prostate cancer, and its expression level varies in normal prostate tissue, prostate hyperplasia tissue, benign prostate cancer tissue, and high-grade prostate cancer tissue. 2 Prostate cancer (PCa) is the second leading cause of cancer-related deaths and the fifth leading cause of cancer death among men worldwide. 3 Prostate cancer is closely related to age, and some signaling pathways involving reactive oxygen species (ROS) play an important role in the occurrence and progression of cancer with increasing age. 4 Therefore, ERβ probes can be developed to monitor prostate lesions and cancerous changes, thereby advancing the early diagnosis of prostate cancer. To enhance the targeting specificity of probes to prostate cancer, environmentally responsive ERβ probes can also be designed by leveraging the unique microenvironment information of the tumor.

[0003] Increased ROS in the tumor microenvironment can promote tumor growth directly through cell damage, or indirectly through the inhibition of cytotoxic lymphocytes. 5 A prominent characteristic of tumor cells is a sharp increase in H2O2 content. As one of the most important reactive oxygen species, H2O2 plays a crucial role in maintaining protein folding, cell signaling, body defense, and cellular respiration. 6 However, increasing evidence suggests that excessive hydrogen peroxide production in the body not only easily causes oxidative damage to cell structures or biomolecules, but is also associated with serious diseases such as cardiovascular disease, neurodegenerative diseases, Alzheimer's disease, and cancer. 7 In diseases with high H2O2 expression, such as cancer and inflammation, H2O2 has become an important target, and many diagnostic reagents, prodrugs, drug delivery systems and fluorescent probes targeting H2O2 have been developed. 8-10 Groups that respond to H2O2 include ethers (thioethers, selenides, tellurides), ferrocene, phenylboronic acid esters / phenylboronic acid (PBE / PBA), aryl oxalates, and proline. Among these, phenylboronic acid esters / phenylboronic acid are the most common because PBE / PBA exhibits high selectivity and responsiveness to H2O2. 11

[0004] Small molecule fluorescent probes are an important component of optical imaging. Due to their advantages such as simple operation, low detection limit, good selectivity, high sensitivity, and high spatiotemporal resolution, they have become one of the most powerful tools for studying biological targets. 12 Compared with traditional fluorescent probes, near-infrared (NIR, 650nm-900nm) fluorescent probes have advantages such as deeper penetration into tissues and elimination of background autofluorescence interference, which greatly promotes the imaging of molecular processes in vivo. 13 Furthermore, its larger Stokes shift is beneficial for bioimaging, avoiding the adverse effects of excitation source and self-absorption. Dicyandimethyl-4H-benzodihydropyran and its derivatives (DCM dyes) possess unique spectral characteristics, such as near-infrared emission wavelengths and a large Stokes shift. Compared to traditional single-photon probes, DCM derivatives have a two-photon absorption cross-section, offering advantages such as higher spatial resolution, lower self-absorption, and deeper tissue penetration. 14 Previously, the first reports of ERβ-targeting near-infrared fluorescent probes and hypoxia-responsive fluorescent probes with DCM-OH as the backbone demonstrated that these small molecule ERβ probes, which are both ERβ ligands and fluorophores, showed good imaging capabilities of ERβ in vitro and in vivo. 15-16

[0005] References:

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[0015] 10.Morgan,B.;Van Laer,K.;Owusu,T.N.E.;Ezerina,D.;Pastor-Flores,D.;Amponsah,P.S.;Tursch,A.;Dick,T.P.,Real-time monitoring of basal H2O2 levelswith peroxiredoxin-based probes.Nat Chem Biol 2016,12(6),437-443.

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[0020] 15. Meng, QY; Xie, BH; Yu, HG; Shen, K.; Deng,

[0021] 16. Summary of the Invention

[0022] The purpose of this invention is to provide an ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe, and also to provide a method for preparing the probe and its applications.

[0023] The objective of this invention is achieved through the following technical solution:

[0024] An ERβ-targeting hydrogen peroxide-responsive near-infrared fluorescent probe is a dicyanomethylene-4H-benzodihydropyran compound containing a borate ester group. This borate ester group can be specifically cleaved by H2O2, undergoing an oxidation reaction to generate the corresponding phenolic hydroxyl group. This probe is designated as probe P1 or P2, and the structural formulas of P1 and P2 are shown below:

[0025]

[0026] The ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe uses DCM-OH as its backbone and borate ester groups as H2O2-responsive groups. Its luminescence principle is as follows: Figure 1 As shown, DCM-OH is both a fluorophore and an ERβ ligand. The introduction of the borate ester group disrupts the push-pull effect of the DCM-OH fluorophore, preventing fluorescence. However, in a high-concentration H₂O₂ environment, the borate ester group is specifically cleaved by H₂O₂ and removed, subsequently exhibiting strong initiation fluorescence and enabling tumor imaging in vitro and in vivo.

[0027] The preparation method of the ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe includes the following steps:

[0028] (1) Using 2'-hydroxy-5-methoxyacetophenone as a substrate, a condensation reaction was carried out with ethyl acetate to prepare compound 1-(2-hydroxy-5-methoxyphenyl)butane-1,3-dione. The cyclization reaction was carried out under the catalysis of concentrated sulfuric acid and acetic acid to give 6-methoxy-2-methyl-4H-benzodihydropyran-4-one. The acid-catalyzed reaction of 6-methoxy-2-methyl-4H-benzodihydropyran-4-one with malononitrile yielded 2-(6-methoxy-2-methyl-4H-benzodihydropyran-4-alkylene)malononitrile 1c, an important intermediate of the probe.

[0029] (2) The reaction of bromobenzaldehyde and pinacol diboronic acid ester yields 4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)benzaldehyde 2a.

[0030] (3) 2a and 1c underwent a Knoevenagel condensation reaction in the presence of piperidine and acetic acid to prepare (E)-2-(6-methoxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)styryl)-4H-benzodihydropyran-4-alkylene)malonitrile 1d; demethylation with BBr3 gave probe P1(E)-2-(6-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)styryl)-4H-benzodihydropyran-4-alkylene)malonitrile.

[0031] (4) Probe P1 reacts with 2-chloroethanesulfonyl chloride to obtain probe P2(E)-4-(diacenicomethyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)styryl)-4H-benzodihydropyran-6-yl vinyl sulfonate containing unsaturated olefin double bonds.

[0032] The ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe has different ultraviolet absorption and fluorescence emission spectra with H2O2 oxidation products of different concentrations, and can be used for probe-based detection of ultraviolet absorption and fluorescence intensity based on H2O2 concentration gradient.

[0033] The ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe described herein can be used for in vitro and in vivo imaging.

[0034] The ERβ-targeting hydrogen peroxide-responsive near-infrared fluorescent probe exhibits strong fluorescence imaging after being added to tumor cells for culture, and can be used for tumor cell imaging. The specific measurement method and conditions are as follows:

[0035] A. Add the probe to the phosphate buffer system at a concentration of 10 μM;

[0036] B. The reaction temperature was 37℃, and the pH of the incubation system was 7.4;

[0037] C. The reaction time is 15–30 minutes;

[0038] D. Detection conditions: excitation wavelength 488nm, emission wavelength range 600nm-700nm.

[0039] The ERβ-targeting hydrogen peroxide-responsive near-infrared fluorescent probe P2, when added to a mouse model of DU-145 prostate cancer cells, accumulates at the tumor site over time, and the tumor site shows stronger imaging than the background and other organs, making it suitable for in vivo diagnosis and monitoring of prostate cancer.

[0040] The aforementioned ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe, as an H2O2-responsive fluorescent probe substrate, can monitor ERβ-targeted prostate lesions and carcinogenesis based on tumor cell imaging. It can be used to prepare products for early diagnosis of prostate cancer and monitoring of prostate cancer progression.

[0041] A product for early diagnosis of prostate cancer or monitoring of prostate cancer progression, comprising the aforementioned ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe.

[0042] The ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe of this invention for detecting prostate cancer cells brings new opportunities for the diagnosis and research of prostate cancer, has practical application value, and can promote the development and application of early diagnosis of prostate cancer. This invention has the following advantages and beneficial effects:

[0043] (1) High specificity: When the probe enters the tumor cell, it can be oxidized and broken by H2O2 with high specificity, resulting in the release of borate ester groups and strong fluorescence.

[0044] (2) Inexpensive and readily available: The probe can be obtained through chemical synthesis, and the synthesis process is simple and easy to carry out.

[0045] (3) Anti-interference: Since detection in the near-infrared region (NIR) can avoid background noise generated by the environment and biological samples, it can effectively avoid interference from the self-absorption and autofluorescence of biological samples.

[0046] (4) High selectivity and good targeting: The probe enters the transplanted tumor model mouse and accumulates in the tumor site over time, enabling precise visualization. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the luminescence principle of the ERβ-targeted H2O2-responsive fluorescent probe.

[0048] Figure 2 These are the UV and fluorescence spectra of probes P1 and P2 before and after treatment with H2O2. (a) UV absorption spectrum of P1 before and after treatment with H2O2; (b) Emission spectrum of P1 before and after treatment with different concentrations of H2O2 (0-100 μM); (c) After treatment with H2O2, the fluorescence intensity of the solution showed a linear relationship in the range of 0-100 μM (R0). 2 =0.9609); (d) UV absorption spectra of P2 before and after treatment with H2O2; (e) Emission spectra of P2 before and after treatment with different concentrations of H2O2 (0-100μM); (f) After treatment with H2O2, the fluorescence intensity of the solution of P1 showed a linear relationship in the range of 0-100μM (R = 0.9609); 2 =0.9897).

[0049] Figure 3 This represents the fluorescence response of probe P2 to different analytes (a) and different pH conditions (b). The analytes are grouped as follows: 1. blank; 2. NaCl (100mM); 3. MgCl2 (100mM); 4. KCl (2.5mM); 5. Tyr (1mM); 6. GSH (1mM); 7. His (1mM); 8. Cys (1mM); 9. Glu (1mM); 10. Gly (1mM); 11. Arg (1mM); 12. NaOH (100μM); 13. Na2CO3 (100μM); 14. Na2SO4 (100μM); 15. NaNO2 (100μM); 16. NaClO (100μM); 17. NaCl (100μM); 18. H2O2 (100μM).

[0050] Figure 4 This describes the response of probes P1 and P2 to H2O2 during staining of MCF-7 and DU-145 cells. PMA: H2O2 inducer; DPI: H2O2 inhibitor. Excitation: 488nm, Emission: 600-700nm.

[0051] Figure 5 This is a colocalization map of probes P1 and P2 in DU-145 cells. The probes colocalize with the nuclear dye DAPI (blue), the mitochondrial dye MitoTracke (green), and the ERβ secondary antibody dye (purple), respectively.

[0052] Figure 6 This section describes in vivo and in vitro imaging in animals. (A) In vivo fluorescence imaging of P2 in DU-145 xenograft mice at different time points (0, 3, 6, 12, 15, 24, and 36 h). (B) In vitro fluorescence imaging of tumors and major organs 15 h after probe injection. (C) Comparative analysis of fluorescence intensity and background of tumors and major organs (heart, liver, lung, kidney, spleen). Detailed Implementation

[0053] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0054] Example 1: Preparation of an ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe

[0055] The ERβ-targeting hydrogen peroxide-responsive near-infrared fluorescent probe provided by this invention is a dicyanomethylene-4H-benzodihydropyran compound containing a borate ester group, which includes probes P1 and P2 with the structures shown below.

[0056]

[0057] The synthetic routes for probes P1 and P2 are as follows:

[0058]

[0059] Reagents and conditions: (a) NaH, EtOAc, 0℃, rt, 4h; (b) H2SO4, CH3COOH, 120℃, 30min; (c) Ac2O, 140℃, 14h; (d) Pd(dppf)Cl2, C H3COOK, DMF, 80℃, 3h; (e) Piperidine, CH3COOH, CH3CN, 85℃, 12h; (f) BBr3, DCM, 0℃, overnight; (g) Et3N, DCM, rt.

[0060] The specific steps include:

[0061] Synthesis of 1a: 2'-hydroxy-5-methoxyacetophenone (3.0 g, 18.0 mmol) was dissolved in 30 mL of ethyl acetate. After stirring to dissolve, 60% NaH (3.0 g, 75.0 mmol) was slowly added under ice bath conditions. The ice bath was removed, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the NaH was quenched with ice water, and the pH was adjusted to neutral with 3 M HCl. The mixture was filtered, and the solid was collected and dried to give a white crystalline solid 1a (3.2 g, 85% yield).

[0062] Synthesis of 1b: 1a (3.5 g, 16.8 mmol) was added to 40 mL of CH3COOH, followed by the slow addition of 3.6 mL of 95% concentrated sulfuric acid. After stirring to dissolve, the mixture was heated to 120 °C and refluxed for 30 min. After the reaction was stopped, the mixture was cooled and then slowly added to ice water. The pH was adjusted to neutral by slowly adding saturated Na2CO3 solution. The solution was extracted with dichloromethane (50 mL × 3), washed with saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, and the organic layer was concentrated to obtain needle-like gray solid 1b (2.4 g, yield 75%). 1 H NMR (400MHz, CDCl3) δ7.49(d,J=3.1Hz,1H),7.30(d,J=9.1Hz,1H),7.18(dd,J=9.1,3.1Hz,1H),6.11(s,1H),3.84(s,3H),2.34(s,3H).

[0063] Synthesis of 1c: 1b (2.8 g, 14.7 mmol) and malononitrile (1.2 g, 18.2 mol) were added to 20 mL of acetic anhydride, stirred until dissolved, and then refluxed at 140 °C for 14 h. After natural cooling to room temperature, the reaction solution was concentrated under vacuum, and 30 mL of deionized water was added. The mixture was then heated to reflux and reacted for 30 min. After the reaction was completed, the mixture was cooled to room temperature, extracted with dichloromethane (50 mL × 3), washed with saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, and the organic phase was concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain an orange-red solid 1c (1.8 g, yield 51%). 1 H NMR (400MHz, CDCl3) δ8.35(d,J=2.8Hz,1H),7.42(d,J=9.2Hz,1H),7.33(dd,J=9.2,2.8Hz,1H),6.72(s,1H),3.92(s,3H),2.46(s,2H).

[0064] Synthesis of 2a: Under argon purging, 4-bromobenzaldehyde (500.0 mg, 2.7 mmol), pinacol diborate (1.4 g, 5.4 mmol), Pd(dppf)Cl2 (200.0 mg, 0.27 mmol), and CH3COOK (800.0 mg, 8.1 mmol) were sequentially added to a reaction flask containing 10 mL of anhydrous DMF. After dissolution and stirring, the mixture was heated to 80 °C and reacted for 3 h. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL × 3), washed with saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, and the organic phase was concentrated. The solution was then subjected to column chromatography (petroleum ether / ethyl acetate = 5:1) to give a white solid (0.5 g, 80%). 1H NMR (400MHz, CDCl3) δ10.03(s,1H),7.95(d,J=6.8Hz,2H),7.85(d,J=6.6Hz,2H),1.35(s,12H).

[0065] Synthesis of 1d: Under argon purging, 1c (500.0 mg, 2.1 mmol) and 2a (580 mg, 2.5 mmol) were added to 10 mL of acetonitrile solution, dissolved and stirred, followed by the sequential addition of piperidine and acetic acid (piperidine:acetic acid = 1 mL: 0.5 mL), and the mixture was heated to 85 °C and refluxed for 12 h. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL × 3), washed with saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, and the organic phase was concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 9:1) to give a yellow solid (0.3 g, 32%). 1 H NMR (400MHz, Acetone) δ8.38(d,J=2.8Hz,1H),7.76(dd,J=14.3,12.7Hz,2H),7.68(d,J=8.6Hz,2H),7.52(d d,J=9.2,2.9Hz,1H),7.21(d,J=16.0Hz,1H),6.95(d,J=8.6Hz,2H),6.91(s,1H),3.93(s,3H),1.30(s,12H).

[0066] Synthesis of P1: Under argon purging, 1d (300 mg, 0.66 mmol) was added to 10 mL of anhydrous DCM solution in an ice bath, dissolved and stirred, followed by rapid addition of BBr3 (500.0 mg, 2.0 mmol), and stirred overnight. After the reaction was complete, water was slowly added to quench the reaction, followed by extraction with dichloromethane (30 mL × 3), washing with saturated sodium chloride solution (20 mL × 1), drying with anhydrous sodium sulfate, concentrating the organic phase, and the crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 2:1) to give an orange-red solid P1 (0.2 g, 70%), (E)-2-(6-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)styryl)-4H-benzodihydropyran-4-alkylene)malonitrile. 1 H NMR(400MHz, DMSO)δ8.48(s,1H),8.00(d,J=2.6Hz,1H),7.68–7.60(m,2H),7.58(d,J=8.7Hz,2H), 7.43(d,J=11.8Hz,1H),7.15(d,J=15.9Hz,1H),6.85(s,1H),6.81(d,J=8.7Hz,2H),1.70(s,12H). 13C NMR (126MHz, DMSO) δ176.68(s),169.35(s),161.00(s),154.98(s),148.08(s),132.63(s),132.01(s),122.53( s),120.29(s),119.84(s),118.86(s),118.43(s),110.42(s),107.46(s),60.11(s),24.46(s).HRMS(ESI)calcd for C 26 H 23 BN₂O₄[M+K] + ,478.1424; found 478.1416.

[0067] Synthesis of P2: Under argon purging, P1 (200 mg, 0.46 mmol) was added to 10 mL of anhydrous DCM solution, dissolved and stirred. Then, 2-chloroethanesulfonyl chloride (90.0 mg, 0.55 mmol) and triethylamine (140.0 mg, 1.38 mmol) were added sequentially, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane (30 mL × 3), washed with saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, and the organic phase was concentrated. The crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 2:1) to give a red solid P2 (0.1 g, 42%), (E)-4-(diacenic)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)styryl)-4H-benzodihydropyran-6-yl vinyl sulfonate. 1 H NMR (400MHz, Acetone) δ8.24(d,J=2.5Hz,1H),7.76(d,J=16.0Hz,1H),7.69–7.65(m,3H),7.58(d,J=8.6Hz,1H),7.48(t,J=2.1Hz,1H ),7.44(dd,J=8.8,2.8Hz,1H),7.28(dd,J=8.6,2.5Hz,1H),7.20(d,J=16.0Hz,1H),6.95(d,J=8.5Hz,2H),6.88(s,1H),1.38(s,12H). 13C NMR(126MHz,CD3OD)δ177.97(s),158.99(s),153.22(s),147.73(s),146. 30(s),139.14(s),129.84(s),129.46(s),126.49(s),124.52(s),123.82( s),119.64(s),118.22(s),117.08(s),116.10(s),115.74(s),115.25(s) ,113.32(s),105.72(s),104.68(s),61.62(s),25.53(s).HRMS(ESI)calcd for C 28 H 25 BN2O6S[M+H] + ,528.1643; found 528.1635.

[0068] Example 2: Determination of the relative affinity of the probe

[0069] The affinity of the target compound for ERα and ERβ was determined by fluorescence polarization. The compound affinity was a relative value of the endogenous E2 affinity, with RBA set at 100%. In a 384-well plate, 20 μL of potassium phosphate buffer containing 0.8 μM ERα or ERβ protein, 150 nM estradiol (CS), and 2.4 μg bovine immunoglobulin was added, followed by 20 μL of the target compound solution. The compound concentration gradient was 3.16 × 10⁻⁶. -4 M, 1×10 -4 M, 3.16×10 -5 M, 1×10 -5 M, 3.16×10 -6 M, 1×10 -6 M, 3.16×10 -7 M, 1×10 -7 M, 3.16×10 -8 M, 1×10 -8 M, 3.16×10 -9 M. After incubating at room temperature in the dark for 2 hours, read the plate using a microplate reader, selecting 485nm as the primary wavelength and 528nm as the reference wavelength. Analyze the experimental results and apply the formula Receptor Affinity RBA = Test Substance K i / Estradiol K iThe RBA value for each compound was calculated by multiplying by 100, and the results are shown in Table 1. Both probes exhibited similar isotype selectivity. Due to the inclusion of a large borate ester group, they showed almost no affinity for ERα, but some affinity for ERβ. Probe P1 showed lower binding affinity for ERβ, and its ERβ selectivity was three times that of ERα. Introducing an unsaturated olefin double bond to the left side of probe P1 increased the affinity of P2 for ERβ, resulting in higher isotype selectivity; its ERβ selectivity was 7.75 times that of ERα. This indicates that probe P2 is a very promising ERβ-targeting fluorescent probe.

[0070] Table 1. Relative binding affinity (RBA) of probes P1 and P2 for estrogen receptor α (ERα) and estrogen receptor β (ERβ). a

[0071]

[0072] a Relative binding affinity is measured by fluorescence polarization and expressed as IC50. 50 estradiol / IC 50 Compound × 100 ± standard deviation (binding affinity of estradiol is set at 100%).

[0073] Example 3 Cell Viability Test

[0074] Normal breast cancer cells MCF-10A, breast cancer cells MCF-7, and prostate cancer cells DU-145 were cultured in DMEM liquid medium containing 10% fetal bovine serum and phenol red. When the cell density reached 80%–90%, the cells were digested, and the cell suspension was seeded into 96-well cell culture plates using DMEM medium without phenol red and containing 10% estradiol (CS). After complete cell attachment, the original culture medium was discarded, and 100 μL of a fresh compound solution prepared in DMEM medium containing 10% estradiol (CS) was added to each well. The compound concentration gradient was 1 × 10⁻⁶. -7.5 M, 1×10 -7 M, 1×10 -6.5 M, 1×10 -6 M, 1×10 -5.5 M, 1×10 -5 M, 1×10 -4.5 M, 1×10 -4 M. After 4 days of drug treatment and culture, remove the culture plate, aspirate the culture medium, add 100 μL of CCK8 working solution to each well, and incubate at 37℃ in a 5% CO2 incubator for 1.5–2 hours. Read the plate on a microplate reader, select 450 nm as the reference wavelength, analyze the experimental results, and calculate the IC50. 50The results are shown in Table 2. Probe P2 showed no cytotoxicity against normal MCF-10A and prostate cancer cells DU-145, but exhibited some inhibitory effect on cancer cells MCF-7, with an IC50 value of [missing information]. 50 The value was 16.34 μM; while P1 had no killing effect on these three cell lines. This indicates that when the probe of this invention is used for imaging, it will not damage normal cells and tissues, and can accurately reflect the expression level of ERβ in the lesion area.

[0075] Table 2 Cell viability assays of probes P1 and P2

[0076]

[0077] Example 4 Fluorescence Performance Test

[0078] The probe solution was prepared using 10 mM PBS (pH = 7.4), and its optical properties were determined using a SHIMADZU UV-2600 UV-Vis spectrophotometer and a HITACHI F-4600 fluorescence spectrophotometer. The fluorescein (Φ) was used as the analytical measure. f1 =0.85) was used as a control, and the fluorescence quantum yields of P1 and P2 were calculated using the following formula. The results are shown in Table 3. The excitation wavelength of P1 is 410 nm, and the emission wavelength reaches 653 nm, which is in the near-infrared region, indicating good potential for animal imaging. The excitation wavelength of P2 is also 413 nm, and the emission wavelength reaches 655 nm, which also meets the requirements for in vivo imaging. At the same time, the Stokes shifts of P1 and P2 both exceed 240 nm, indicating strong resistance to background interference.

[0079] Φ (sample) =Φ (standard) ×(A (standard) / A (sample) )×(S (sample) / S (standard) )

[0080] The abbreviations mean: Φ: fluorescence quantum yield, A (standard) and A (sample) : Control and probe at λ em Absorption value at S (standard) and S (sample) Peak areas of emission from the control and probe. Slit width = 10 / 10nm.

[0081] Table 3. Fluorescence quantum yields of probes P1 and P2 in PBS (pH = 7.4)

[0082]

[0083] Probe solutions were prepared using 10 mM PBS (pH = 7.4), and different concentrations of H₂O₂ (0-100 μM) were added. The optical properties of the probes were measured using a SHIMADZUUV-2600 UV-Vis spectrophotometer and a HITACHI F-4600 fluorescence spectrophotometer. Results are shown below. Figure 2 Experimental results showed significant differences in the UV absorption and fluorescence emission spectra of the probe solutions before and after H2O2 treatment. After H2O2 treatment, the absorption spectra of both P1 and P2 exhibited a red shift, indicating that the properties of the probes changed before and after the H2O2 response. The fluorescence spectra of P1 and P2 initially showed low fluorescence intensity and no fluorescence in the 600-700 nm range. However, after H2O2 treatment, both P1 and P2 produced fluorescence emission peaks in the 600-700 nm range, suggesting that the oxidized products possess excellent potential for in vivo imaging. After P1 was hydrolyzed by H2O2, the fluorescence intensity of the solution showed a linear relationship with the probe concentration in the 0-100 μM range (R0). 2 =0.9609), while after P2 was treated with H2O2, the fluorescence intensity of the solution showed a linear relationship with the probe concentration in the range of 0-100 μM (R = 0.9609). 2 =0.9897), which indicates that the probe treated with H2O2 has a high sensitivity to concentration.

[0084] For the anti-interference experiment, cations, anions, various amino acids, and active substances were added to the probe P2 solution prepared in 10 mM PBS (pH = 7.4). For the fluorescence response experiment under different pH conditions, probe P2 solutions were prepared using 10 mM PBS at different pH values. The response was measured using a HITACHI F-4600 spectrophotometer (slit width = 10 / 10 nm), and the results are shown below. Figure 3 . Figure 3 a indicates that the fluorescence intensity of P2 in each analyte group is not significantly different and is lower than that in the positive control group treated with H2O2. This suggests that the probe is not interfered with by other substances when used for imaging of the physiological environment. Figure 3 b indicates that the fluorescence intensity of P2 changes very little in different pH environments, and these probes can be used for imaging of physiological environments and are not affected by pH.

[0085] Example 5 Cell Imaging

[0086] DMEM medium containing cells (MCF-7 or DU-145) was placed in a cell culture incubator at 37°C. After cell resuscitation, the cells were transferred to confocal microplates and cultured for 24 h. Probe (10 μM), probe + phorbol myristate acetate (PMA) (10 μM), and probe + diphenyl iodide chloride (DPI) (10 μM) were added to the microplates to stain the viable cells for 15 min. The cells were then carefully washed three times with PBS buffer. Results are shown in the figure. Figure 4 Experimental results showed that without PMA, only weak fluorescence was observed in MCF-7 and DU-145 cells, while with PMA, more obvious fluorescence was observed in both cell types. This indicates that P1 and P2 have a strong response to H2O2, specifically removing borate ester groups in the H2O2 environment to release fluorescence. Simultaneously, adding DPI to inhibit H2O2 production caused the complete disappearance of fluorescence signals in MCF-7 and DU-145 cells, and the cells shrank and deformed. This suggests that H2O2 consumption not only leads to a decrease in probe fluorescence intensity but also has a significant damaging effect on cancer cells, which has reference value for the future development of bifunctional probes.

[0087] In the colocalization experiment, cells in a confocal dish were first stained with a probe for 15 min, then washed three times with PBS buffer, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and washed with PBS after 10 min. DAPI was added to stain the nucleus or Mito-tracker Green to stain the mitochondria, and after 30 min, the free dye was washed away with PBS to observe the imaging results. In immunofluorescence staining, DU-145 cells were stained with a probe (10 μM) for 30 min, then fixed and permeabilized. Cells were washed three times with PBS and incubated at 37°C with monoclonal anti-ERβ antibody (1:200) for 12 h. Cells were washed with PBS, and ERβ was stained with the secondary antibody DyLight 488AffiniPure Goat Anti-Rabbit IgG (1:200) for 1 h. After washing away the free dye, imaging was performed. Results are shown below. Figure 5 P1, P2, and DAPI showed inconsistent locations in DU-145 cells with high ERβ expression, distributed in the extracellular region with minimal overlap with the nuclear dye DAPI. Both probes exhibited high co-localization with mitochondria, indicating their targeting of mitochondria. Immunofluorescence staining further confirmed the probes' ERβ labeling ability. P1 and P2 partially co-localized with ERβ, with fluorescence signals primarily present at the extracellular margin, similar to the mitochondrial co-localization results.

[0088] Example 5 Animal Imaging

[0089] In animal imaging experiments, a mouse model of DU-145 tumor transplantation was established. First, DU-145 prostate cancer cells were expanded and cultured, then subcutaneously injected into the lumbar region of 6-week-old male Balb / c nude mice. When the tumor grew to approximately 80-100 mm³, the probe was dissolved in PBS and injected into the mice via the tail vein. Thirty minutes later, the mice were anesthetized with 2% sodium pentobarbital, and imaging was performed using a live animal imaging system (Bruker Xtreme BI). Imaging results were observed every 3 hours. Results are shown below. Figure 6 Because P2 can target the highly expressed ERβ protein in DU-145 xenograft tumors, the probe gradually accumulates at the tumor site. After 15 hours, the fluorescence signal in the tumor area is stronger than the background, indicating good tumor targeting. A slight fluorescence signal is also visible in the liver area. Figure 6 A). Subsequently, in vitro animal imaging was performed on mice to further determine the exact tissue distribution of probe P2. The fluorescence signal in tumor tissue was significantly stronger than that in tissues such as the heart, liver, spleen, lung, and kidney, but weaker fluorescence was also observed in the liver and kidney. Figure 6 B and 6C) indicate that the probe may be metabolized by these two organs.

Claims

1. An ERβ-targeting hydrogen peroxide-responsive near-infrared fluorescent probe, characterized in that: The probe described is probe P2 with the structure shown below: P2。 2. The method for preparing the ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe according to claim 1, characterized in that, Includes the following steps: (1) Using 2'-hydroxy-5'-methoxyacetophenone as a substrate, a condensation reaction with ethyl acetate was carried out to prepare compound 1-(2-hydroxy-5-methoxyphenyl)butane-1,3-dione, which was then cyclized under the catalysis of concentrated sulfuric acid and acetic acid to give 6-methoxy-2-methyl-4-dione. H -Benzodihydropyran-4-one; acid-catalyzed 6-methoxy-2-methyl-4-one H Benzodihydropyran-4-one reacts with malononitrile to give intermediate 2-(6-methoxy-2-methyl-4-) H -Benzadihydropyran-4-alkylene)malonitrile; (2) The reaction of bromobenzaldehyde and pinacol diboronic acid ester yields 4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)benzaldehyde; (3) The products of steps (1) and (2) are prepared by Knoevenagel condensation in the presence of piperidine and acetic acid. E )-2-(6-methoxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)styryl)-4 H -benzodihydropyran-4-alkylene)malonitrile; demethylation with BBr3 gave probe P1( E )-2-(6-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)styryl)-4H-benzodihydropyran-4-alkylene)malonitrile; P1 (4) Probe P1 reacts with 2-chloroethanesulfonyl chloride to give probe P2 containing an unsaturated olefin double bond. E )-4-(diacetonylmethyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)styryl)-4 H 6-Benzodihydropyran-6-ylethylene sulfonate.

3. The application of the ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe of claim 1 in the preparation of products for prostate cancer diagnosis.

4. The application of the ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe of claim 1 in the preparation of products for monitoring prostate cancer progression.

5. A product for diagnosing or monitoring the progression of prostate cancer, characterized in that: It includes the ERβ-targeted hydrogen peroxide-responsive near-infrared fluorescent probe of claim 1.

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