Fluorescent dye targeting human epidermal growth factor receptor 2 as well as preparation method and application of fluorescent dye

Abstract

The invention discloses a fluorescent dye targeting a human epidermal growth factor receptor 2 (HER2) as well as a preparation method and application of the fluorescent dye. The dye is composed of a fluorescent parent nucleus, a linker and a targeting group, and has near-infrared excitation and emission characteristics. The invention also provides an efficient preparation method of the dye. The dye has the innovativeness that fluorescence is quenched when the dye is in folded conformation in an aqueous solution, and the conformation is expanded and a strong fluorescence signal is released after the dye is specifically combined with HER2 protein, so that high signal-to-noise ratio detection is realized. The dye has the characteristics of quick response, high specificity, good biocompatibility and the like, and can be used in the fields of HER2 protein marking, molecular interaction research, drug screening, cell imaging and the like; and a novel fluorescence detection tool is provided for related research of HER2.

Description

Technical Field

[0001] This invention relates to the fields of life and health and high-end functional dyes, and particularly to a fluorescent dye that targets human epidermal growth factor receptor 2 (HER2), its preparation method and application. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2) is a key member of the type I RTK family, encoded by the ERBB2 gene, located in the 17q12 region of chromosome 17, and is a proto-oncogene. The 185 kDa transmembrane glycoprotein encoded by this gene includes three main functional domains: an extracellular ligand-binding domain, a transmembrane structural domain, and an intracellular tyrosine kinase active domain. The extracellular domain of HER2 lacks ligand-binding capacity; instead, it regulates cell proliferation, migration, and anti-apoptosis biological functions by activating downstream signaling pathways, ultimately driving tumorigenesis and metastasis. Aberrant HER2 expression (such as gene upregulation, protein overexpression, and mutation) has been shown to be closely associated with the malignant phenotypes of various solid tumors, including breast cancer, gastric cancer, and colorectal cancer. In breast cancer patients, HER2-positive tumors account for approximately 20%-25%, and this subtype of tumor has stronger invasiveness, a higher risk of recurrence, and poorer response to conventional chemotherapy. Accurate detection of HER2 protein expression levels is of great significance for tumor diagnosis, treatment planning, and prognostic assessment.

[0003] Clinically, HER2 status detection mainly relies on immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, traditional biopsies have limitations such as being invasive, involving tumor heterogeneity, and making dynamic monitoring difficult. Fluorescence imaging technology has the characteristics of being non-invasive, real-time, highly sensitive, and high-resolution, giving it a significant advantage in the field of biomedical imaging. In particular, fluorescent probes with excitation and emission wavelengths located in the near-infrared window have lower phototoxicity, deeper tissue penetration, significantly reduced autofluorescence interference from biological tissues, exhibit higher imaging signal-to-noise ratio, less tissue scattering, and higher imaging spatial resolution, making them one of the research hotspots in the field of high-end functional dyes. Currently, there are few reports on fluorescent probes that specifically target HER2-positive tumors, and the reported fluorescent probes have significant shortcomings in performance. For example, HJ Lee reported a responsive HER2 fluorescent probe that achieves fluorescence "on" through donor-receptor interaction binding, specifically recognizing HER2-positive breast cancer cells; however, the excitation and fluorescence emission wavelengths of this probe are relatively short (Anal. Chem. 2016, 88, 23, 11310). CS Li et al. modified lapatinib onto near-infrared fluorescent dye molecules to develop a series of normally bright HER2-targeting probes, but the signal-to-noise ratio at the tumor margin was low (Biosens. Bioelectron. 2022, 214, 114503). Therefore, there is an urgent need to create near-infrared fluorescent probes with high signal-to-noise ratio and specific fluorescence recognition of HER2-positive tumors. Summary of the Invention

[0004] To address the aforementioned shortcomings, this invention proposes a near-infrared fluorescent dye based on the photoinduced electron transfer quenching effect of folded state. The fluorescent dye consists of a fluorescent core, a linker, and a targeting group. Its excitation and emission wavelengths are 630±20 nm and 650-750 nm, respectively, both located in the near-infrared region. It exhibits a folded conformation in aqueous solution. The fluorescence signal of the fluorescent core is quenched by the photoinduced electron transfer effect. After specifically binding to the HER2 protein, it can re-release the fluorescence signal, thereby achieving rapid and specific targeting of the HER2 protein. The fluorescence intensity ratio of targeted cells to non-targeted cells is ≥3:1, enabling high signal-to-noise ratio imaging of HER2-positive breast cancer. This is expected to provide an efficient tool for the rapid diagnosis of HER2-positive tumors, the study of their molecular mechanisms of development, and intraoperative fluorescence navigation.

[0005] Specifically, the fluorescent dye targeting human epidermal growth factor receptor 2 described in this invention has the following general formula 1 structure:

[0006] In general formula 1: X is selected from one of the following: oxygen atom, sulfur atom, and selenium atom; oxygen atom is the most preferred. L is selected from one of the structures described in formulas L1 to L3 below:

[0007] In formula L1, n is 2, 4, 6, 8, 10 or 12; more preferably 4. In equation L2, n is 1, 2, 3, or 4; In equation L3, n is 1, 2, 3 or 4.

[0008] T can be selected from HER2 inhibitor small molecules, preferably one of estrogen receptor small molecule inhibitors or antagonists; more preferably, a quinazoline tyrosine kinase inhibitor with a QL structure.

[0009] P can be selected from H, C1-C3 alkyl groups, such as CH3, CH2CH3; more preferably H.

[0010] In another aspect, the present invention protects a method for preparing a type of fluorescent dye targeting human epidermal growth factor receptor 2 as described above, comprising the following steps: (1) Mix 1-naphthylamine with the compound shown in general formula S-1 in a first organic solvent and reflux for 6-12 h. After the reaction is complete, cool to room temperature and remove the first organic solvent to obtain the intermediate shown in general formula 2. Dissolve the intermediate shown in general formula 2 in 1,4-dioxane, add a strong base aqueous solution, and stir at room temperature for 1-5 h to obtain the intermediate shown in general formula 3. (2) Dissolve the intermediate shown in general formula 3 in a second organic solvent, then add the compound shown in general formula S-2, add concentrated hydrochloric acid to the above reaction system under ice bath, allow to react fully, raise the temperature to 80-90℃ and continue the reaction for 20-24 h to obtain the compound shown in general formula 4. (3) Under alkaline conditions, the compound represented by general formula 4 and the compound represented by general formula 5 are subjected to an amidation reaction at room temperature to obtain the target compound;

[0011] For the technical solution described above, it is further preferred that, in step (1), the first organic solvent is selected from any one or a combination of several of water, methanol, ethanol, dichloromethane, ethyl acetate, isopropanol and acetone; For the technical solution described above, it is further preferred that, in step (1), the strong alkali is selected from any one of sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonia water; For the technical solution described above, more preferably, in step (1), the molar ratio of 1-naphthylamine to the compound represented by general formula S-1 is 1:(0.8-1.3), and more preferably, the ratio is 1:1.1.

[0012] For the technical solution described above, more preferably, in step (2), the second organic solvent is selected from any one or a combination of several of water, methanol, ethanol, acetonitrile, ethyl acetate, isopropanol and acetone; For the technical solution described above, it is further preferred that, in step (2), the molar ratio of the intermediate shown in general formula 3 to the compound shown in general formula S-2 is 1:(0.9-2.0).

[0013] For the technical solution described above, it is further preferred that, in step (3), the solvent used for the amidation reaction is selected from any one of anhydrous DMF, anhydrous DMSO, anhydrous acetonitrile, and anhydrous dichloromethane; For the technical solution described above, it is further preferred that the acid-binding agent used is selected from any one of K2CO3, KOH, Na2CO3, NaOH, DIEA, and triethylamine; For the technical solution described above, more preferably, the molar ratio of the compound represented by general formula 4 to the compound represented by general formula 5 is 1:(0.9-1.5), and more preferably, the ratio is 1:1.2.

[0014] A third aspect of the present invention discloses the application of a class of fluorescent dyes targeting human epidermal growth factor receptor 2 as described above in the fields of biology, medicine, and chemistry.

[0015] With regard to the technical solution described above, the application further preferably includes using the fluorescent dye for protein labeling, protein molecule docking and protein inhibitor molecule screening, cell imaging, fluorescent dye probes, and for preparing adjuvants for tumor phototherapy and tumor fluorescence-guided surgery.

[0016] Compared with existing technologies, the advantages of the dye proposed in this invention are as follows: (1) High specificity: The fluorescent dye disclosed in this invention can specifically bind to the HER2 protein overexpressed by tumors, thereby specifically illuminating HER2-positive tumors; (2) High signal-to-noise ratio: The fluorescent dye disclosed in this invention increases the fluorescence signal intensity by about 5.2 times after specifically binding to the HER2 protein. Compared with the normal fluorescent dye, this responsive fluorescent dye helps to improve the imaging signal-to-noise ratio. (3) Near-infrared fluorescence excitation and emission bands: The excitation wavelength and emission wavelength of the fluorescent dye disclosed in this invention are approximately 630 nm and 680 nm, respectively, both located in the near-infrared region. It has lower phototoxicity, deeper tissue penetration depth, and can significantly reduce the interference of biological tissue autofluorescence, with less tissue scattering and higher imaging spatial resolution. (4) Rapid dynamic response: Cell uptake and fluorescence activation are completed within 15 minutes (Example 8, Figure 11 This represents a 16-fold improvement in response speed compared to Comparative Example 2 (4-hour response time), making it suitable for real-time intraoperative navigation. (5) Dual function: For the first time, the HER2 antagonist (QL) and the near-infrared dye (NB) are coupled through a reversible folding linker to avoid the metabolic off-target risk of multi-component probes; (6) Low cytotoxicity, high cell survival rate, wide pH range, and good water solubility.

[0017] In summary, the near-infrared fluorescent dye of the present invention can be used for high signal-to-noise ratio, high specificity fluorescence response to HER2 protein, and rapid dynamic response, providing an efficient tool for the diagnosis of HER2-positive tumors, intraoperative margin determination, and pathogenesis research. Attached Figure Description

[0018] Figure 1 NMR of HER2 recognition probe I 1 H (above) and 13 C-spectrum (Part 2); Figure 2 High-resolution mass spectrometry of HER2 recognition probe I; Figure 3 To determine the molecular conformation and frontier orbital energy levels of HER2 recognition probe I; Figure 4 The ultraviolet absorption spectrum of HER2 recognition probe I; Figure 5 The fluorescence emission spectrum of HER2 recognition probe I; Figure 6 The water solubility of HER2 recognition probe I; Figure 7 The fluorescence intensity of HER2 recognition probe I in aqueous solutions at different pH values ​​and in methanol; Figure 8 Fluorescence spectra of HER2 recognition probe I under different treatment methods; Figure 9 The fluorescence intensity at 680 nm for HER2 recognition probe I in the presence or absence of inhibitors; Figure 10 Cytotoxicity of HER2 recognition probe I; Figure 11 Time-fluorescence imaging intensity results for HER2 recognition probe I to recognize HER2 protein; Figure 12 Fluorescence imaging image of HER2-recognition probe I specifically recognizing (HER2+) BT474 cancer cells; Figure 13 Fluorescence imaging of HER2(-) MCF-7 cells incubated with HER2 recognition probe I; Figure 14 Fluorescence imaging of HER2(-) MCF-10A cells incubated with HER2 recognition probe I. Detailed Implementation

[0019] The following is in conjunction with the appendix Figure 1-14 The following specific examples further illustrate the present invention, demonstrating, in detail, a class of fluorescent dyes targeting human epidermal growth factor receptor 2, their preparation methods, and applications. However, the invention is not limited to these examples.

[0020] Example 1 In the preparation, X is an oxygen atom; L can be selected from an alkane chain with 4 methylene groups; T is a small estrogen receptor molecule; and P is H. The reaction process is as follows:

[0021] Its preparation method includes the following steps: (1) Synthesis of dye NB-COOH First, ethyl 6-bromohexanoate (38.41 mmol, 8.57 g) was weighed into a 50 mL three-necked flask equipped with a thermometer and dissolved in anhydrous ethanol (17.5 mL). Then, 1-naphthylamine (34.92 mmol, 5 g) was added while stirring at room temperature. The mixture was refluxed under N2 protection for 12 h. After the reaction was complete, the mixture was allowed to return to room temperature, and the solvent was removed to give a brown oily crude product (11.5 g).

[0022] The crude, brown, oily product was then completely dissolved in 1,4-dioxane (87.3 mL), followed by the addition of a 2 M / L NaOH aqueous solution (0.175 mol, 6.98 g). The mixture was stirred at room temperature for 3 h. After the reaction was complete, the solvent was removed, the pH was adjusted to 2-3, and the product was extracted with ethyl acetate. The organic phase was separated, washed twice with saturated brine, and dried over anhydrous sodium sulfate. The product was then purified by column chromatography (DCM: MeOH = 20:1, v / v) to obtain a brown solid compound (7.75 g, 86%) with the structure shown in Figure I.

[0023] Compound I (3.89 mmol, 1 g) was weighed into a 50 mL flask under ice bath conditions. Acetonitrile (10 mL) was added and stirred until completely dissolved. Then, 2-nitroso-3-(dimethylamino)phenol (6.61 mmol, 1.1 g) was added. Concentrated hydrochloric acid (0.6 mL) was slowly added dropwise to the reaction system. After reacting for 10 min, the reaction temperature was raised to 85 °C and the reaction was continued for 24 h. After the reaction was completed, the mixture was concentrated, and the crude product was purified by silica gel column chromatography (DCM: MeOH = 10 / 1, v / v) to obtain a deep blue powder IINB-COOH (1.13 g, 72%).

[0024] The structure was verified to be correct: ¹H NMR (400 MHz, MeOD) δ 9.00 (d, J = 7.7 Hz, ¹H), 8.45 (bs, ¹H), 8.06 – 7.84 (m, 3H), 7.31 (d, J = 6.9 Hz, ¹H), 7.19 – 6.75 (m, 2H), 3.78 (bs, 2H), 3.39 (s, 6H), 2.44 (bs, 2H), 1.99 (bs, 2H), 1.81 (bs, 2H), 1.64 (bs, 2H). MS: m / z calcd for [M]+: 404.1969, found 404.1968. (2) Synthesis of the inhibitor QL Weigh 4-chloro-6-iodoquinazoline (17.2 mmol, 5.0 g) and dissolve it in 30 mL of isopropanol solution. Then add 3-chloro-4-fluoroaniline (18.9 mmol, 2.8 g). Heat the reaction mixture to 80 °C and maintain the temperature for 2.5 hours. After the reaction is complete, cool the mixture and filter it through diatomaceous earth. Wash the filter cake with isopropanol and finally dry it to obtain the desired compound III (6.5 g, 94%).

[0025] First, compound III (2 mmol, 800 mg) was weighed and added to a 25 mL three-necked flask containing 1.0 mL of N,N-dimethylformamide. After stirring thoroughly, 2.0 mL of N,N-dimethylformamide containing (4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)boronic acid (3 mmol, 660 mg), triphenylphosphine (0.4 mmol, 110 mg), and triethylamine (1.5 mL) was added. After stirring for five minutes, palladium acetate (0.4 mmol, 100 mg) was finally added. The mixture was heated at 80 °C for 24 hours under N2 protection. After the reaction was completed, the mixture was cooled to room temperature, 200 mL of water was added, and the mixture was extracted with ethyl acetate (4 × 200 mL). After separation, the mixture was dried over anhydrous sodium sulfate and rotary evaporated to obtain the crude product. First, the compound was purified by column chromatography (DCM: MeOH = 20:1, v / v) to obtain a solid powder (640 mg) mainly composed of 6-(4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)-N-(3-chloro-4-fluorophenyl)-quinazolin-4-amine. Then, all of the purified solid powder was added to a 5 mL single-necked flask containing DCM / TFA = 1:1, v / v, and stirred at room temperature for 1.5 hours. Dichloromethane and most of the trifluoroacetic acid were removed by rotary evaporation under reduced pressure to obtain compound IV, a yellow solid powder (329 mg, 38%).

[0026] The structure was verified to be correct: ¹H NMR (400 MHz, DMSO) δ 11.01, 8.94, 8.88, 8.85, 8.38, 8.35, 8.11, 8.11, 8.10, 8.09, 7.92, 7.90, 7.86, 7.84, 7.81, 7.80, 7.80, 7.79, 7.79, 7.78, 7.77, 7.77, 7.59, 7.56, 7.54, 7.20, 7.18, 3.48, 3.29. 13C NMR (101 MHz, DMSO) δ 158.97, 158.68, 158.35, 158.03, 157.70,155.90, 153.45, 151.90, 150.08, 139.28, 134.86, 133.21, 129.15, 127.84,125.73, 124.45, 124.38, 119.54, 119.31, 119.12, 118.22, 117.06, 116.84,116.08, 115.27, 114.50, 45.05, 42.61. HRMS: m / z calcd for [M+H]+: 434.1470, found 434.1541. (3) Synthesis of probe NB-QL (HER2 recognition probe I) Finally, compound NB-COOH (0.5 mmol, 200 mg) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.7 mmol, 267 mg) were thoroughly dissolved in 3 mL of N,N-dimethylformamide, and then 320 μL of N,N-diisopropylethylamine was injected. After stirring for 20 min, 2 mL of N,N-dimethylformamide containing compound QL (0.6 mmol, 260 mg) was slowly injected into the system. As the reaction proceeded, the reaction solution turned blue. After the reaction was complete, the reaction solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH = 6 / 1, v / v) to obtain the fluorescent probe NB-QL (HER2 recognition probe I), which was a dark blue solid powder (371 mg, 90%).

[0027] The structure was verified to be correct: ¹H NMR (400 MHz, DMSO) δ 10.43 (s, ¹H), 10.14 (s, ¹H), 8.78 (s, ¹H), 8.74 (d, J = 8.1 Hz, ¹H), 8.68 (d, J = 8.2 Hz, ¹H), 8.60 (s, ¹H), 8.23 ​​(d, J = 6.6 Hz, ¹H), 8.14 (d, J = 8.7 Hz, ¹H), 7.97–7.88 (m, 2H), 7.85 (t, J = 7.4 Hz, 1H), 7.82–7.74 (m, 4H), 7.46 (t, J = 9.1 Hz, 1H), 7.16 (d, J = 9.2 Hz, 1H). Hz, 1H), 7.07-6.98 (m, 3H), 6.78 (s, 1H), 3.74 (s, 2H), 3.62 (s,4H), 3.24-3.14 (m, 10H), 2.42 (t, J = 7.0 Hz, 2H), 1.85-1.75 (m, 2H), 1.68-1.58 (m, 2H), 1.53-1.43 (m, 2H). 13C NMR (101 MHz, DMSO) δ 171.06, 157.97, 157.89, 155.32, 154.99,154.04, 152.57, 151.86, 150.82, 148.67, 147.59, 138.35, 137.01, 134.31,132.37, 131.66, 131.06, 130.11, 129.61, 129.46, 128.53, 128.10, 124.39,124.35, 124.19, 123.79, 123.09, 123.02, 119.27, 119.09, 117.03, 116.81,116.08, 115.76, 115.30, 96.16, 94.09, 48.70, 48.28, 45.10, 44.64, 41.20,40.95, 32.55, 28.59, 26.61, 24.96. HRMS: m / z calcd for [M]+: 819.3333, found819.3316. Example 2 Fluorescent probe fluorescence mechanism This example utilizes TDDFT (B3LYP / 6-31G) simulations to optimize the molecular structure and frontier molecular orbitals of NB-QL (HER2 recognition probe I) in water. The results show that NB-QL (HER2 recognition probe I) exhibits a folded conformation in water. The HOMO and LUMO orbital energy levels of the fluorophore NB are -5.899 eV and -3.276 eV, respectively, while the HOMO orbital energy level of the targeting group QL is -4.847 eV, falling between the HOMO and LUMO orbital energy levels of the fluorophore NB. When the fluorophore is excited, electrons from the HOMO orbital of the targeting group QL transition to the HOMO orbital of the fluorophore, preventing electrons from the LUMO energy level of QL from transitioning to the HOMO energy level of QL, thus quenching the fluorescence of the fluorophore. Upon binding to the HER2 protein, NB-QL (HER2 recognition probe I) becomes an unfolded conformation, increasing the distance between the fluorophore NB and the recognition group QL, weakening the PET effect, and causing the fluorophore NB to re-emit fluorescence, thereby achieving fluorescent recognition of the HER2 protein.

[0028] Example 3 Physicochemical properties of probe molecules Absorption and fluorescence spectra of the fluorescent probe NB-QL (HER2 recognition probe I) in different buffer solutions. The absorption peak positions of the probe are different in different solvents, and there are two absorption peaks in the 300-800 nm range. At the same time, the fluorescence emission spectrum shows that the probe has high fluorescence intensity in methanol and dichloromethane, while the fluorescence in culture medium and PBS is almost completely quenched.

[0029] Example 4 Investigation of the water solubility of fluorescent probes Different volumes of probes were added to cuvettes containing the same volume of PBS buffer solution, and the absorbance of the solutions at different concentrations was measured.

[0030] The absorbance of the solution increases with the increase of the probe concentration in the solution. In the range of 0-30 μM, the absorbance and the probe concentration have a good linear relationship, which indicates that the fluorescent probe obtained by the present invention has good water solubility.

[0031] Example 5 Study on fluorescence stability of fluorescent probes in aqueous solutions at different pH values ​​and in methanol The probe was tested using fluorescence emission spectroscopy. First, aqueous solutions with pH values ​​of 4, 5, 6, 7, 8, 9, and 10 were prepared. Then, the probe stock solution was measured using a micropipette and dissolved in quartz cuvettes containing 3 mL of aqueous solutions with different pH values. The fluorescence spectra of the probe NB-QL (HER2 recognition probe I) were measured.

[0032] The results showed that NB-QL (HER2 recognition probe I) in aqueous solution also exhibited very weak fluorescence emission over a wide pH range of 4-10, in stark contrast to its emission in methanol. Example 6 Fluorescent probe response to human recombinant protein HER2 The fluorescent probe was incubated with human recombinant protein HER2 at 37 ºC for 10 minutes. As a control, human recombinant protein HER2 was pretreated with the HER2 tyrosine kinase inhibitor lapatinib (LAP) for 30 minutes before the probe was added. Fluorescence emission spectra under the three conditions were recorded using a micro-ultraviolet-visible spectrophotometer with an excitation wavelength of 630 nm and an emission wavelength of 655–710 nm.

[0033] When the protein was pretreated with an excess of lapatinib, fluorescence enhancement was significantly suppressed, indicating that NB-QL (HER2 recognition probe I) can restore its fluorescence by specifically binding to HER2.

[0034] Example 7 Cytotoxicity assessment of fluorescent probes Through MTT (3 (4,5) dimethylthiazole ( 2 base) 3,5 (Diphenyltetraazole) to assess cell viability.

[0035] The experimental procedure was as follows: BT-474, MCF-7, and MCF-10A cells were seeded at a density of 1.5 × 10⁵ cells / mL in 96-well plates and cultured in 150 μL of medium containing 10% FBS. After 24 hours of cell attachment, the plates were washed with 150 μL / well of PBS. The cells were then incubated with DMEM containing different concentrations of probes. Afterward, the cells were further cultured in an incubator for 24 hours.

[0036] For the viability test, 10 μL MTT (5 mg / mL) prepared in PBS was added to each well, and the plate was incubated at 37°C for 4 h in a humidified incubator with 5% CO2. The culture medium was then carefully removed, and the purple crystals were dissolved in 150 μL LDMSO. The absorbance at 570 nm was measured using a Thermo Fisher Scientific microplate reader.

[0037] Cell viability = (OD experimental group) OD blank control / (OD negative control) (OD blank control) × 100%, where the negative and blank control groups were the no-drug group and the blank culture medium group, respectively.

[0038] The test results showed that the survival rate of MCF-7 cells was very high, close to 100%, indicating that the fluorescent probe had low toxicity.

[0039] Example 8 Fluorescent probe fluorescence imaging of BT474 cells: time-fluorescence intensity HER2(+) BT474 cells were seeded onto 35 mm confocal dishes and incubated at 37°C with 5% CO2. Once the cell density reached 60%, the cells were incubated with culture medium containing 0.1 μM of a fluorescent probe. Cell fluorescence intensity was measured at different time points (0, 15, 30, and 60 min) using confocal fluorescence microscopy. The results showed that cell uptake was completed within 15 min, with BT-474 cells exhibiting a strong fluorescence signal, and the fluorescence intensity remained stable for the following 45 minutes. These results indicate that the probe NB-QL (HER2 recognition probe I) has good cell uptake capability.

[0040] Example 9 Investigation of the specificity of fluorescent probes for HER2+ Two cell lines were prepared. HER2(+) BT474 cells were seeded onto 35 mm confocal dishes and incubated at 37°C with 5% CO2. Once the cell density reached 60%, the first group was pre-cultured with 5 μM lapatinib (LAP), followed by incubation with medium containing 0.1 μM fluorescent probe. Imaging was performed using a confocal fluorescence microscope. The results showed that fluorescence was significantly suppressed in the LAP-pretreated group compared to the unpretreated group. This indicates that the fluorescent probe can specifically illuminate HER2(+) BT474 cells.

[0041] Example 10 Investigation of the specificity of fluorescent probes HER2(-) MCF-7 and MCF 10A cells were seeded onto 35 mm confocal dishes and incubated at 37°C and 5% CO2 for 24 hours. Cells were then incubated for 15 min with medium containing 0.1 μM fluorescent probe, after which the medium was aspirated and replaced with freshly prepared medium. Fluorescence intensity was measured using a confocal microscope. The results are shown below. Figure 13 As shown, the fluorescence intensity of MCF-7 cells was significantly lower than that of BT474 cells. HER2(-) MCF-10A cells also showed only weak fluorescence after incubation with the probe. Figure 14 This indicates that the fluorescent probe can only selectively illuminate HER2(+) BT474 cells.

[0042] Comparative Example 1 James et al. reported a fluorescent probe targeting the EGFR / ERBB family of kinases. This probe uses the quinazoline core structure of an EGFR / ERBB inhibitor as its backbone, extending the aromatic core conjugated system through chemical modification of the N-methylpiperazine group. A "shut-on" fluorescence response mechanism is constructed based on the conformational change before and after probe binding to the kinase. In solution, the probe undergoes fluorescence quenching due to intramolecular charge transfer state (TICT); however, when it specifically binds to the ATP-binding pocket of the ERBB2 kinase, conformational restriction triggers fluorescence emission. By introducing a Type II inhibitor pharmacophore, the probe's affinity is enhanced to a level comparable to the clinical drug lapatinib, demonstrating precise recognition of HER2-overexpressing cells in live cell models. Notably, the probe has an absorption wavelength of 317 nm, an emission wavelength of 487 nm, and a blue shift of 40 nm to 447 nm after binding to the kinase domain.

[0043] The shorter wavelength amplifies interference from biological autofluorescence, reducing the signal-to-noise ratio in imaging. Furthermore, it is easily scattered and absorbed by living biological tissues, resulting in limited penetration depth, which greatly restricts its application in the detection of living tumor tissues.

[0044]

[0045] Comparative Example 2 Gu et al. reported a small-molecule near-infrared fluorescent probe, YQ-H-06, targeting HER2+ tumors. The probe employs a ternary molecular design: the near-infrared dye MPA serves as the fluorescent reporter unit, amino acid chains form the flexible linker, and a lapatinib-derived structure acts as the target recognition unit. Spectroscopic characterization showed that YQ-H-06 has an absorption peak at 780 nm and an emission peak at 820 nm, classifying it as a near-infrared fluorescent probe.

[0046]

[0047] This probe exhibits excellent targeting performance: it has a significant affinity for HER2-overexpressing cells, and its binding can be effectively competitively inhibited by lapatinib. Simultaneously, the probe demonstrates outstanding photostability: it maintains fluorescence signal stability for over 6 hours under continuous irradiation with a 785 nm laser (0.8 mW / cm²). However, the probe's kinetic properties have significant limitations: its response speed is exceptionally slow, requiring 4 hours to reach maximum uptake in cells. The decline in cell viability due to prolonged incubation and severe probe photobleaching severely restrict its ability to monitor rapid signal transduction processes in real time. In in vivo tissue imaging, the fluorescence signal ratio between tumor tissue and normal tissue after probe injection is only about 2 times, far below the signal resolution threshold required for clinical diagnosis, making accurate identification of tumor boundaries difficult and failing to meet the needs of precise clinical diagnosis.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of fluorescent dyes targeting human epidermal growth factor receptor 2, characterized in that: It has the following general formula 1 structure: In general formula 1: X is selected from one of the following: oxygen atom, sulfur atom, and selenium atom; L is selected from one of the structures described in formulas L1 to L3 below: In formula L1, n is 2, 4, 6, 8, 10 or 12; In equation L2, n is 1, 2, 3, or 4; In equation L3, n is 1, 2, 3 or 4. T is selected from HER2 inhibitor small molecules; P is selected from H and C1-C3 alkyl groups.

2. The method for preparing the fluorescent dye as described in claim 1, characterized in that: Includes the following steps: (1) Mix 1-naphthylamine with the compound shown in general formula S-1 in a first organic solvent and reflux for 6-12 h. After the reaction is complete, cool to room temperature and remove the first organic solvent to obtain the intermediate shown in general formula 2. Dissolve the intermediate shown in general formula 2 in 1,4-dioxane, add a strong base aqueous solution, and stir at room temperature for 1-5 h to obtain the intermediate shown in general formula 3. (2) Dissolve the intermediate shown in general formula 3 in a second organic solvent, then add the compound shown in general formula S-2, add concentrated hydrochloric acid to the above reaction system under ice bath, allow to react fully, raise the temperature to 80-90℃ and continue the reaction for 20-24 h to obtain the compound shown in general formula 4. (3) Under alkaline conditions, the compound shown in general formula 4 and the compound shown in general formula 5 are subjected to an amidation reaction at room temperature to obtain the target compound.

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

4. The method according to claim 2, characterized in that: In step (1), the molar ratio of 1-naphthylamine to the compound represented by general formula S-1 is 1:(0.8-1.3).

5. The method according to claim 2, characterized in that: In step (2), the second organic solvent is selected from any one or a combination of several of water, methanol, ethanol, acetonitrile, ethyl acetate, isopropanol and acetone; in step (3), the solvent used for the amidation reaction is selected from any one of anhydrous DMF, anhydrous DMSO, anhydrous acetonitrile and anhydrous dichloromethane.

6. The method according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate shown in general formula 3 to the compound shown in general formula S-2 is 1:(0.9-2.0).

7. The method according to claim 2, characterized in that: The acid-binding agent used is selected from any one of K2CO3, KOH, Na2CO3, NaOH, DIEA, and triethylamine.

8. The method according to claim 2, characterized in that: The molar ratio of the compound shown in Formula 4 to the compound shown in Formula 5 is 1:(0.9-1.5).

9. The fluorescent dye according to claim 1, characterized in that: The applications of the fluorescent dyes described herein in the fields of biology, medicine, and chemistry.

10. The application according to claim 9, characterized in that: This includes using the fluorescent dye for protein labeling, protein docking and screening of protein inhibitor molecules, cell imaging, fluorescent dye probes, and as an adjuvant for preparing tumor phototherapy and tumor fluorescence-guided surgery.

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