High-specificity near-infrared fluorescent probe for imaging melanoma as well as preparation method and application of high-specificity near-infrared fluorescent probe
By designing a near-infrared fluorescence probe activated by cascade glutathione and tyrosinase, the problem of insufficient specificity of existing fluorescence probes in detecting melanoma is solved, and high specific imaging of melanoma is achieved , avoid interference from false positive signals.
Patent Information
- Application Number
- CN202411753968.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-02-25
AI Technical Summary
The existing fluorescent probes are insufficient in detecting melanoma, which is prone to false positive signals and cannot be effectively improved.
A near-infrared fluorescence probe was designed, which activated the near-infrared fluorescence signal through the cascade activation of glutathione and tyrosinase, and realized the simultaneous detection of two related biomarkers in melanoma. .
This probe can effectively avoid interference from false positive signals and achieve high specific imaging of melanoma, with the advantages of novel structure, high targeting, strong sensitivity and good imaging performance.
Smart Images
Figure CN119504716A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a highly specific melanoma imaging near-infrared fluorescent probe and a preparation method and application thereof. Background Art
[0002] Malignant melanoma is characterized by early metastasis, rapid progression, poor prognosis, and high mortality, and its clinical diagnosis and treatment still face huge challenges. Near-infrared fluorescent probes provide a powerful means for the diagnosis of melanoma. Specific near-infrared fluorescent probes designed for biomarkers overexpressed in the melanoma microenvironment have the potential to visualize tumor boundaries and provide navigation for accurate diagnosis and surgical resection of melanoma. However, the fluorescent probes currently developed usually only respond to a single characteristic factor in melanoma, which is prone to interference from "false positive" signals due to insufficient specificity (Wu, L. et al., Nat. Rev. Chem. 2021, 5, 406-421). By detecting two biomarkers that are interrelated in the occurrence and development of melanoma, the false positive signal problem of most current fluorescent probes will be effectively improved.
[0003] Tyrosinase (TRY) is a multifunctional copper-containing metalloenzyme containing binuclear copper ions. It is a key rate-limiting enzyme in catalyzing the production of melanin. A large number of studies have shown that overexpression of tyrosinase is associated with the malignancy of melanoma and is an important biomarker for detecting melanoma. In addition, melanoma cells overproduce reactive oxygen species due to the Warburg effect. Compensatory, melanoma cells increase the production of glutathione (GSH) to eliminate elevated oxidative stress, thereby maintaining high levels of glutathione in cells. Therefore, high levels of glutathione are also an important microenvironmental feature of melanoma. More importantly, intracellular glutathione derivatives can participate in the regulation of melanin by interacting with the active site of tyrosinase. The development of near-infrared fluorescent probes activated by the tyrosinase and glutathione cascades associated with melanin is expected to achieve highly specific imaging of melanoma reactions to meet the needs of precise diagnosis and treatment of melanoma. Summary of the invention
[0004] In response to the above technical problems, the present invention provides a highly specific near-infrared fluorescent probe for melanoma imaging and its preparation method and application. The fluorescent probe can detect two biomarkers that are interrelated in the occurrence and development of melanoma, activate the near-infrared fluorescent signal through the cascade activation of glutathione and tyrosinase, and has the advantages of highly specific imaging of melanoma.
[0005] The present invention adopts the following technical scheme:
[0006] The first technical purpose of the present invention is to provide a highly specific near-infrared fluorescent probe for melanoma imaging, the structural formula of the near-infrared fluorescent probe is:
[0007] ;
[0008] Wherein: the substituent R is methyl or ethyl; X - It is iodide ion or bromide ion.
[0009] The second technical purpose of the present invention is to provide a method for preparing the highly specific near-infrared fluorescent probe for melanoma imaging as described above, which specifically comprises the following steps:
[0010] (1) adding the compound of formula II, base A and triphosgene to an organic solvent A to react to obtain a first reaction mixture;
[0011] (2) Adding the compound of formula III to the first reaction mixture, reacting, and separating to obtain the highly specific melanoma imaging near-infrared fluorescent probe; wherein the compound of formula II has the following structure:
[0012] ;
[0013] Wherein: the substituent R is methyl or ethyl; X - is iodide ion or bromide ion;
[0014] The structure of the compound of formula III is as follows:
[0015] .
[0016] Furthermore, the molar ratio of the compound of formula II in step (1) to the compound of formula III in step (2) is 1:0.5 to 1:5, for example, the molar ratio of the compound of formula II to the compound of formula III is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5; preferably 1:1.5.
[0017] Furthermore, in step (1), the organic solvent A is at least one of tetrahydrofuran, dichloromethane, toluene and chloroform, preferably dichloromethane.
[0018] Furthermore, in step (1), the base A is triethylamine, pyridine or 4-dimethylaminopyridine; preferably 4-dimethylaminopyridine.
[0019] Furthermore, in step (1), the ratio of the compound of formula II to the organic solvent A is 1 mol: 10-40 mL.
[0020] Further, in step (1), the reaction temperature is 0-5°C, and the reaction time is 10-120 minutes. For example, in step (1), the reaction temperature is 0°C, 1°C, 2°C, 3°C, 4°C or 5°C, and the reaction time is 10 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes or 120 minutes.
[0021] Further, in step (2), the reaction temperature is 20-30°C, and the reaction time is 4-16 hours. For example, in step (2), the reaction temperature is 20°C, 22°C, 24°C, 25°C, 26°C, 28°C or 30°C, and the reaction time is 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours or 16 hours.
[0022] Furthermore, in step (2), the compound of formula III is prepared by the following steps: 3-hydroxybenzyl alcohol, an organic solvent B and a base B are mixed to obtain a mixture, 2, 4-dinitrobenzenesulfonyl chloride is added to the mixture, the reaction time is 4 to 10 hours at 20 to 30° C., and separation is performed; wherein the organic solvent B is at least one of tetrahydrofuran, dichloromethane, toluene and chloroform, and the base B is triethylamine, pyridine or 4-dimethylaminopyridine; and the molar ratio of 3-hydroxybenzyl alcohol, base B and 2, 4-dinitrobenzenesulfonyl chloride is 0.6 to 2:1 to 2:0.5 to 1.5. For example, the molar ratio of 3-hydroxybenzyl alcohol, base B and 2, 4-dinitrobenzenesulfonyl chloride is 0.6-2:1-2:0.5-1.5, 0.8-2:1.2-2:0.7-1.5, 1-2:1.4-2:0.9-1.5, 1.2-2:1.6-2:1.1-1.5, 1.5-2:1.8-2:1.3-1.5 or 1.8-2:1.9-2:1.4-1.5. Preferably, the molar ratio of 3-hydroxybenzyl alcohol, base B and 2, 4-dinitrobenzenesulfonyl chloride is 1.2:1.5:1.
[0023] Furthermore, in step (2), the reaction temperature is room temperature and the reaction time is 5 hours.
[0024] Furthermore, in step (2), the ratio of 3-hydroxybenzyl alcohol to organic solvent B is 1.2 mol: 5-30 mL.
[0025] The third technical objective of the present invention is to provide the use of the highly specific melanoma imaging near-infrared fluorescent probe as described above in the detection of glutathione and tyrosinase.
[0026] The fourth technical objective of the present invention is to provide the use of the highly specific melanoma imaging near-infrared fluorescent probe as described above in the detection of melanoma.
[0027] The near-infrared fluorescent probe in the present invention can activate the cascade of glutathione and tyrosinase that are highly expressed in the characteristic microenvironment of melanoma, turn on the near-infrared fluorescent signal, and achieve highly specific imaging of melanoma.
[0028] The present invention connects the fluorescent group hemicyanine compound with the quenching group, so that the hemicyanine compound has no near-infrared fluorescence emission performance. After the 2,4-dinitrobenzenesulfonyl group is activated and broken by the high concentration of glutathione (GSH) in the tumor, the intermediate product 1 is released, and the intermediate product 1 is further activated by tyrosinase to release the fluorescent group (such as Figure 1 as shown).
[0029] Through this cascade activation, the near-infrared fluorescence signal of the fluorescent group is turned on, realizing the simultaneous detection of GSH and tyrosinase. Compared with the currently developed fluorescent probes that only activate a single characteristic factor in the tumor (tyrosinase, H2O2, microacid and GSH, etc.), the near-infrared fluorescent probe provided by the present invention has the characteristics of cascade activation by glutathione and tyrosinase highly expressed in the characteristic microenvironment of melanoma, and has high selectivity and strong sensitivity for melanoma, which is of great significance in the accurate detection of melanoma.
[0030] The beneficial effects of this application are as follows:
[0031] 1. The highly specific near-infrared fluorescent probe for melanoma imaging provided in the present application can detect two biomarkers that are interrelated in the occurrence and development of melanoma. By cascade activation of glutathione and tyrosinase, the near-infrared fluorescent signal is turned on to achieve highly specific imaging of melanoma, thereby avoiding the interference of "false positive" signals. At the same time, the probe has the advantages of novel structure, high targeting, strong sensitivity and good imaging performance.
[0032] 2. The present application provides a highly specific near-infrared fluorescent probe for melanoma imaging. It can be seen from the examples that the synthetic route of this scheme is simple and the fluorescent probe can be obtained with high yield and high purity.
[0033] 3. In view of the good imaging performance, the present application provides a highly specific melanoma imaging near-infrared fluorescent probe, whose fluorescence emission wavelength is in the near-infrared region, the normal living tissue signal is weak, and it can specifically and simultaneously respond to two melanoma-related biomarkers for detection, which is expected to provide a new method for early diagnosis and surgical navigation of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is a schematic diagram of the near-infrared fluorescent probe provided by the present invention in the cascade activation of GSH and tyrosinase;
[0035] Figure 2is the high-resolution mass spectrum of fluorescent probe 1;
[0036] Figure 3 is the fluorescence spectrum of fluorescent probe 1 in GSH and tyrosinase detection;
[0037] Figure 4 This is the selective imaging of GSH and tyrosinase by fluorescent probe 1 in cells;
[0038] Figure 5 This is the imaging picture of fluorescent probe 1 in different cells;
[0039] Figure 6 This is the imaging image of fluorescent probe 1 in melanoma-bearing mice. DETAILED DESCRIPTION
[0040] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
[0041] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.
[0042] Example 1 Synthesis of fluorescent probe 1
[0043] (1) Preparation of compound 1;
[0044] ;
[0045] In a round-bottom flask, add compound 3-hydroxybenzyl alcohol (1.2 mmol), then add dichloromethane (20 mL), add triethylamine (1.5 mmol), cool the solution in an ice bath, add 2, 4-dinitrobenzenesulfonyl chloride (1.0 mmol), react at room temperature for 5 hours, filter, extract, and separate by column chromatography to obtain compound 1. Compound 1 1 H NMR and 13 C NMR data are as follows: 1 H NMR (400 MHz, CDCl3): 1H NMR (400 MHz, CDCl3) δ 8.66 (d, J= 2.2 Hz, 1H), 8.49 (dd, J = 8.6, 2.2 Hz, 1H), 8.21 (d, J = 8.6 Hz, 1H), 7.38– 7.31 (m, 2H), 7.24 (s, 1H), 7.10 (d, J = 7.6 Hz, 1H), 4.69 (s, 2H), 1.85(s, 1H). 13 C NMR (101 MHz, CDCl3): δ 150.9, 149.0, 149.0, 143.9, 133.8, 133.7,130.3, 126.4, 126.3, 120.8, 120.4, 120.1, 64.1.
[0046] (2) Preparation of fluorescent probe 1;
[0047] ;
[0048] In a round-bottom flask, compound 2 (1 mmol) and dry dichloromethane (25 mL) were added with p-dimethylaminopyridine (1.5 mmol), and triphosgene (0.4 mmol) was added at 0°C. After stirring at 0°C for 30 minutes, compound 1 (2 mmol) was added, and the reaction was continued at room temperature for 8 hours, and then concentrated under reduced pressure and separated by column chromatography to obtain fluorescent probe 1. The high-resolution mass spectrum of the fluorescent probe 1 prepared in this example is shown in FIG. Figure 2 As shown, 1 H NMR and 13 C NMR data are as follows: 1 H NMR (400 MHz, DMSO-d6): δ10.44 (s, 1H), 8.54 (d, J=15.2 Hz, 1H), 7.79-7.80 (m, 2H), 7.67-7.69 (m, 2H), 7.44-7.57 (m, 6H), 7.31-7.33 (d, J=8.0 Hz, 2H), 7.11 (s, 1H), 6.99-7.03 (m, 2H), 6.55 (d, J=15.2 Hz, 1H), 5.17 (d, J=5.6 Hz, 4H), 3.88 (s,3H), 2.67-2.72 (m, 4H), 1.83 (m, 2H), 1.75 (s, 6H), 1.258 (s, 12H). 13C NMR(101 MHz, DMSO-d6): δ 178.2, 160.5, 158.8, 153.6, 142.8, 142.4, 140.8, 138.3,135.0, 130.2, 128.1, 127.6, 127.3, 120.7, 117.0, 114.9, 114.7, 114.2, 104.3, 84.1, 69.4, 50.8, 33.1, 30.1, 27.6, 25.1.
[0049] Example 2: Application of fluorescent probe 1 in GSH and tyrosinase cascade activation
[0050] A near-infrared fluorescent probe provided in the present application can activate the cascade of glutathione and tyrosinase that are highly expressed in the characteristic microenvironment of melanoma, turn on the near-infrared fluorescent signal, and realize high-specific imaging of melanoma. This example studies the application of the fluorescent probe 1 prepared in Example 1 in the cascade activation of GSH and tyrosinase. The application of the fluorescent probe in the cascade activation of GSH and tyrosinase was determined by fluorescence emission spectroscopy. Specifically, (i) PBS (PBS concentration in the mixed solution is 10 mM, pH of the mixed solution = 7.4) was added to the fluorescent probe (the concentration of the fluorescent probe in each mixed solution was 5 μmol / L) solution at room temperature; (ii) GSH (GSH concentration in the mixed solution was 10 mmol / L); (iii) tyrosinase (tyrosinase concentration in the mixed solution was 400 U / mL); (iv) GSH and tyrosinase (GSH in the mixed solution was 10 mmol / L; tyrosinase in the mixed solution was 400 U / mL); and mixed solutions were obtained respectively. The test solution was at λ ex = Fluorescence spectrum at 680 nm (λ em = 700 – 800 nm). Figure 3 It can be seen that the fluorescent probe only produces a strong fluorescence signal (λ em = The fluorescence intensity at 705 was as high as 2.2E+06), indicating that the fluorescent probe can only emit fluorescent signals for detection when activated by GSH and tyrosinase cascade.
[0051] Example 3: Application of fluorescent probe 1 in selective imaging analysis of GSH and tyrosinase
[0052] This example studies the application of the fluorescent probe 1 prepared in Example 1 in the selective imaging analysis of GSH and tyrosinase. Human malignant melanoma cells A375 containing high concentrations of GSH and tyrosinase were selected as the object. First, A375 cells were cultured in a cell culture incubator at 37°C, 21% (volume percentage) oxygen, and 5% (volume percentage) carbon dioxide for 24 hours, and then the cells were digested with trypsin and transferred to a cell imaging dish (the number of cells was 1 10 6 (i) PBS (PBS concentration in the mixture was 10 mM, pH of the mixture was 7.4) was added and incubated for 4 hours; (ii) fluorescent probe 1 was added and incubated for 4 hours (the final concentration of fluorescent probe in the mixture was 5 μmol / L); (iii) A375 cells were pre-incubated with N-ethylmaleimide for 2 hours to eliminate intracellular GSH, and then fluorescent probe 1 was added and incubated for 4 hours (the final concentration of fluorescent probe 1 in the mixture was 5 μmol / L); (iv) A375 cells were pre-incubated with kojic acid for 2 hours to inhibit intracellular tyrosinase activity, and then fluorescent probe 1 was added and incubated for 4 hours (the final concentration of fluorescent probe 1 in the mixture was 5 μmol / L); the mixtures were obtained respectively. The culture medium in the imaging dish was washed away with PBS buffer, and the images were obtained under a laser scanning confocal microscope (λ ex = 633 nm, λ em =680-750 nm) for fluorescence imaging. Figure 4 It can be seen that a strong near-infrared fluorescence signal was detected only in the experimental group (ii) cells. In contrast, no obvious fluorescence signal was observed in the experimental groups (i), (iii) and (iv). This is because among the four selected groups of cells, only the experimental group (ii) cells contained high concentrations of GSH and tyrosine at the same time. This phenomenon shows that the fluorescent probe 1 can enter the cells and be selectively activated by tyrosinase and GSH to produce a significant fluorescence signal, achieving highly selective imaging detection of GSH and tyrosinase.
[0053] Example 4: Application of fluorescent probes in specific imaging analysis of melanoma cells
[0054] This example studies the application of the fluorescent probe 1 prepared in Example 1 in the specific imaging analysis of melanoma cells. Human malignant melanoma cells A375, human breast cancer cells HepG2, human cervical cancer cells HaLa, and human normal liver cells LO2 were selected as the research objects. First, A375, HepG2, HaLa, and LO2 cells were cultured in a cell culture incubator at 37°C, 21% oxygen (volume percentage), and 5% carbon dioxide (volume percentage) for 24 hours, and then the cells were digested with trypsin and transferred to a cell imaging dish (the number of cells was 1 106 The same concentration of fluorescent probe 1 was added to each mixture (the final concentration of fluorescent probe 1 in each mixture was 5 μmol / L) to obtain mixtures. The above four groups of cells were cultured in a cell culture incubator at 37°C and 5% carbon dioxide (volume percentage) for 4 hours, and the culture medium in the imaging dish was washed with PBS buffer, and fluorescence imaging was performed using a laser scanning confocal microscope (λ ex = 633 nm, λ em = 680-750 nm). Figure 5 As can be seen, strong near-infrared fluorescence signals were detected only in A375 cells. In contrast, no obvious fluorescence signals were observed in HepG2, HaLa, and LO2 cells. This is because among the four selected groups of cells, only human malignant melanoma cells A375 contain high concentrations of GSH and tyrosine at the same time. This phenomenon shows that the probe can achieve highly selective imaging detection of melanoma cells.
[0055] Example 5: Application of fluorescent probes in in vivo fluorescence imaging analysis of melanoma
[0056] This example studies the application of the fluorescent probe 1 prepared in Example 1 in the in vivo fluorescence imaging analysis of melanoma. Mice with human malignant melanoma cell A375 transplanted tumors (tumor size of 600 mm) were selected. 3 ) was used as the experimental model and randomly divided into an experimental group and a control group. The mice in the experimental group were injected with fluorescent probe 1 (volume 100 μL, concentration 25 μmol / L), while the mice in the control group were injected with an equal amount of saline. After injection, fluorescence imaging was performed using a small animal in vivo fluorescence imaging instrument. The results are shown in Figure 6 shown. Figure 6 Mouse No. 1 is the control group mouse, and mouse No. 2 is the drug-treated group mouse. Figure 6 It can be seen that the fluorescent signal at the tumor site of the mice in the drug group is more significant, compared with no fluorescent signal at the tumor site of the mice in the control group. This is because the fluorescent probe 1 in the drug group mice interacts with tyrosinase and GSH, causing the near-infrared fluorescent signal to light up, realizing the imaging analysis of melanoma in animals.
Claims
1. A highly specific near-infrared fluorescent probe for melanoma imaging, characterized in that: The structural formula of the highly specific melanoma imaging near-infrared fluorescent probe is: ； Wherein: the substituent R is methyl or ethyl; X - It is iodide ion or bromide ion.
2. The method for preparing a highly specific near-infrared fluorescent probe for melanoma imaging according to claim 1, characterized in that: The following steps are involved: (1) adding the compound of formula II, base A and triphosgene to an organic solvent A to react to obtain a first reaction mixture; (2) adding the compound of formula III to the first reaction mixture, reacting, and separating to obtain the highly specific melanoma imaging near-infrared fluorescent probe; Wherein, the compound of formula II has the following structure: ； Wherein: the substituent R is methyl or ethyl; X - is iodide ion or bromide ion; The structure of the compound of formula III is as follows: 。 3. The method for preparing a highly specific near-infrared fluorescent probe for melanoma imaging according to claim 2, characterized in that: The molar ratio of the compound of formula II in step (1) to the compound of formula III in step (2) is 1:0.5 to 1:
5.
4. The method for preparing a highly specific near-infrared fluorescent probe for melanoma imaging according to claim 2, characterized in that: In step (1), the organic solvent A is at least one of tetrahydrofuran, dichloromethane, toluene and chloroform.
5. The method for preparing a highly specific near-infrared fluorescent probe for melanoma imaging according to claim 2, characterized in that: In step (1), the base A is triethylamine, pyridine or 4-dimethylaminopyridine.
6. The method for preparing a highly specific near-infrared fluorescent probe for melanoma imaging according to claim 2, characterized in that: In step (1), the reaction temperature is 0-5°C, and the reaction time is 10-120 minutes; in step (2), the reaction temperature is 20-30°C, and the reaction time is 4-16 hours.
7. The method for preparing a highly specific near-infrared fluorescent probe for melanoma imaging according to claim 2, characterized in that: In step (2), the compound of formula III is prepared by the following steps: 3-hydroxybenzyl alcohol, an organic solvent B and a base B are mixed to obtain a mixture, 2, 4-dinitrobenzenesulfonyl chloride is added to the mixture, the reaction time is 4 to 10 hours at 20 to 30° C., and separation is performed; wherein the organic solvent B is at least one of tetrahydrofuran, dichloromethane, toluene and chloroform, and the base B is triethylamine, pyridine or 4-dimethylaminopyridine; and the molar ratio of 3-hydroxybenzyl alcohol, base B and 2, 4-dinitrobenzenesulfonyl chloride is 0.6 to 2:1 to 2:0.5 to 1.
5.
8. Use of a highly specific near-infrared fluorescent probe for melanoma imaging as claimed in claim 1 in detecting glutathione and tyrosinase.
9. Use of a highly specific melanoma imaging near-infrared fluorescent probe as claimed in claim 1 in detecting melanoma.