A fluorescent probe for high specificity tumor imaging, a preparation method and application thereof

Abstract

The application discloses a kind of high specificity tumor imaging fluorescent probe, preparation method and application thereof, belong to fluorescent probe technical field, the structural formula of the fluorescent probe is as shown in formula III, formula IV: compared with the specificity fluorescent probe that currently exists, high specificity tumor imaging fluorescent probe III and IV will tumor growth attack system and tumor defense system combination, without introducing bulky targeting group in molecule, also do not need to obtain specific site by a large number of screening, with better performance-price ratio.Compared with the fluorescent probe I and II without being improved, high specificity tumor imaging fluorescent probe can effectively realize the distinction of normal cells and tumor cells at cell level.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and relates to a fluorescent probe for high-specificity tumor imaging, a preparation method and application thereof, in particular to the application of realizing high-precision visualized imaging of a liver tumor boundary by utilizing the complementary relationship between tumor growth, invasion and migration related peptidases and biomarkers for maintaining the stubborn growth of liver cancer cells in anoxic and redox imbalance environment. BACKGROUND

[0002] Hepatocellular carcinoma, as the most important subtype of liver cancer, is of great significance to its research. The specific fluorescent probe designed for the biomarker overexpressed in the lesion has the potential to visualize the tumor boundary and assist in high-quality clinical tumor resection. Alanine aminopeptidase (APN / CD13) assists in hydrolyzing the basement membrane during tumor growth, thereby promoting the release of vascular endothelial growth factor (VEGF), leading to a higher density of blood vessels near the tumor, which is easier to uptake nutrients from the organism, providing extremely convenient conditions for tumor invasion and metastasis, and is considered as an attack of the tumor. In addition, in order to ensure the stubborn growth of the tumor under anoxic, immune system and external drug stimulation, the tumor-specific aerobic glycolysis metabolic mode will effectively reduce the generation of active oxygen with killing ability in cells, and overexpresses biomarkers such as quinone oxidoreductase (hNQO1) to provide a strong defense for the tumor. By utilizing the attack characteristics exhibited by tumor growth and the defense characteristics of tumor survival and development, a series of small-molecule fluorescent probes are designed for the multi-dimensional characteristics of the tumor. These fluorescent probes will improve the false positive signal problem existing in most current fluorescent probes, have the ability to visualize the tumor boundary and assist in high-quality clinical tumor resection.

[0003] In recent years, fluorescent analysis technology has been used in many fields such as biomolecule labeling, environmental monitoring, cell staining and clinical diagnosis. Most of the fluorescent probes reported for tumor diagnosis only utilize the single-sided markers of the tumor, which will lead to insufficient specificity of the probe and cause false positive signals. This is because some of the markers selected to design the probe still play an important role in the physiological environment in addition to playing an important role in the pathological environment. Therefore, understanding the relationship between the multi-aspect markers of the tumor and designing fluorescent probes with high specificity and no false positive signals will provide the greatest help for distinguishing the tumor boundary and realizing high-precision visualized imaging. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a fluorescent probe for high-specificity tumor imaging, a preparation method and application thereof, the fluorescent probe has the property of high-precision visualization of hepatocellular carcinoma tissue and cancer adjacent tissue boundary, the present application utilizes biomarkers of tumor attack and defense two processes, aims to design and apply a fluorescent probe with high specificity for visualization of near-infrared fluorescence imaging of liver tumor boundary, and provides a new choice for realizing high-precision surgery in clinic.

[0005] In order to achieve the above purpose, the present application provides the following technical solutions:

[0006] The present application is a small molecule near-infrared fluorescent probe, the probe takes peptidase as a recognition substrate, is used for detecting cancer cell growth, invasion and migration, the structural formula of the probe is shown in formula I and formula II:

[0007]

[0008] The present application is a fluorescent probe for high-specificity tumor imaging, the structural formula of the fluorescent probe is shown in formula III and formula IV:

[0009] The fluorescent probes III and IV have the characteristics of high sensitivity, high specificity, high spatial resolution and rapid imaging.

[0010] The present application provides a preparation method of a small molecule near-infrared fluorescent probe, comprising the following steps:

[0011] (1) potassium carbonate, resorcinol (compound 1) and a solvent are added to a first reactor for activation, compound 2 is added dropwise, then the temperature is raised to a predetermined temperature, and near-infrared hemicyanine dye (compound 3) is obtained after reaction;

[0012] (2) compound 3, potassium carbonate and 9H-fluoren-9-yl)methyl(1-((4- (bromomethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate (compound 7) are placed in a second reactor, and a solvent is added, and compound 8 is obtained by dissolving and refluxing at a set temperature;

[0013] (3) compound 8 is placed in a third reactor and dissolved with dichloromethane, piperidine is added dropwise at a predetermined temperature, and fluorescent probe I is obtained after reaction;

[0014] The chemical reaction formula is:

[0015]

[0016] The solvent is acetonitrile in the preparation of the small molecule near-infrared fluorescent probe.

[0017] The molar ratio of the potassium carbonate, the resorcinol and the compound 2 is (2-5):(2-5):1.

[0018] The molar volume ratio of the compound 2 and the acetonitrile is 1:(5-10) mmol / mL.

[0019] The molar ratio of the compound 3, the potassium carbonate and the compound 7 is 1:(1-3):(1-3).

[0020] The molar volume ratio of the compound 3 and the acetonitrile is 1:(5-10) mmol / mL.

[0021] The molar ratio of the compound 8 and the piperidine is 1:(10-20).

[0022] The molar volume ratio of the compound 8 and the dichloromethane is 1:(5-10) mmol / mL.

[0023] The application provides a preparation method of a fluorescent probe for high-specificity tumor imaging.

[0024] (1) N-(4-(hydroxymethyl)phenyl)-N,2,2-trimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-diene-1-yl)propanamide (compound 9) and triphosgene are placed in a reaction container filled with an inert atmosphere, and ultra-dry dichloromethane and DIPEA are injected into the reaction system, and the reaction is set for a time under ice bath;

[0025] (2) Then the system is dried, and the dichloromethane solution of the fluorescent probe I obtained in claim 3 and the dichloromethane solution of DIPEA are injected, and the fluorescent probe III (ANQ) is obtained after reaction;

[0026] The chemical reaction formula is as follows:

[0027]

[0028] The molar ratio of the compound 9, triphosgene and DIPEA is 1:(1-2):(1-2) in the preparation of the fluorescent probe for high-specificity tumor imaging.

[0029] The molar ratio of the fluorescent probe I and DIPEA is 1:(1-2).

[0030] The molar volume ratio of the fluorescent probe I and dichloromethane is 1:(2-5) mmol / mL.

[0031] The inventors respectively characterize by nuclear magnetic resonance hydrogen spectrum, mass spectrum, ultraviolet spectrum and the like, indicating that the high specificity imaging tumor boundary fluorescent probe ANQ is successfully synthesized.

[0032] As a general inventive concept, the application further provides a preparation method of a small molecule near-infrared fluorescent probe, comprising the following steps:

[0033] (1) potassium carbonate, 4-fluoro-resorcinol (compound 10), solvent are added to the first reactor for activation, compound 2 is added dropwise, then the temperature is raised to a predetermined temperature, and after reaction, a near-infrared hemicyanine dye (compound 11) is obtained;

[0034] (2) compound 11, potassium carbonate, 9H-fluoren-9-yl)methyl(1-((4- (bromomethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate (compound 7) are placed in the second reactor, and a solvent is added, dissolved and refluxed at a set temperature to obtain compound 12;

[0035] (3) compound 12 is placed in the third reactor and dissolved with dichloromethane, piperidine is added dropwise at a predetermined temperature, and after reaction, the fluorescent probe II is obtained;

[0036] The chemical reaction formula involved is:

[0037]

[0038] In the preparation process of the above-mentioned small molecule near-infrared fluorescent probe, acetonitrile is used as the solvent;

[0039] The molar ratio of potassium carbonate, 4-fluoro-resorcinol and compound 2 is (2-5):(2-5):1;

[0040] The molar volume ratio of compound 2 and acetonitrile is 1:(5-10) mmol / mL;

[0041] The molar ratio of compound 11, potassium carbonate and compound 7 is 1:(1-3):(1-3);

[0042] The molar volume ratio of compound 11 and acetonitrile is 1:(5-10) mmol / mL;

[0043] The molar ratio of compound 12 and piperidine is 1:(10-20);

[0044] The molar volume ratio of compound 12 and dichloromethane is 1:(5-10) mmol / mL.

[0045] The application further provides a preparation method of a high specificity tumor imaging fluorescent probe, comprising the following steps:

[0046] (1) N-(4-(hydroxymethyl)phenyl)-N,2,2-trimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-diene-1-yl)propanamide (compound 9), triphosgene are placed in a reaction vessel filled with an inert atmosphere, and ultradry dichloromethane and DIPEA are injected into the reaction system, and the reaction is set for a certain time under ice bath;

[0047] (2) Then the system is pumped dry, and a dichloromethane solution of fluorescent probe II and a dichloromethane solution of DIPEA are injected, and fluorescent probe IV (FANQ) is obtained after reaction;

[0048] The chemical reaction formula involved is:

[0049]

[0050] In the preparation process of the above-mentioned high-specificity tumor imaging fluorescent probe, the molar ratio of compound 9, triphosgene and DIPEA is 1: (1-2): (1-2);

[0051] The molar ratio of fluorescent probe II and DIPEA is 1: (1-2);

[0052] The molar volume ratio of fluorescent probe II and dichloromethane is 1: (2-5) mmol / mL.

[0053] The inventors characterized by nuclear magnetic resonance hydrogen spectrum, mass spectrometry, ultraviolet spectrum and other means, which indicated that the high-specificity tumor boundary imaging fluorescent probe FANQ was successfully synthesized.

[0054] The application provides application of the small molecule near-infrared fluorescent probe in a reference probe.

[0055] The application provides application of the high-specificity tumor imaging fluorescent probe in detection of tumor-specific markers.

[0056] The application provides the high-specificity tumor imaging fluorescent probe, a series of probes based on a high-efficiency tumor distinguishing strategy, which are obtained by comparison and screening in vitro and in cells.

[0057] The application of the high-specificity tumor imaging fluorescent probe in detection of tumor-specific markers adopts the above-mentioned probe to test tumor-specific substrates.

[0058] Compared with the prior art, the application has the following beneficial effects:

[0059] Compared with the existing specific fluorescent probes, the fluorescent probes II and IV with high specificity tumor imaging advantage of the present application combine the attack system of tumor growth and the defense system of tumor, do not need to introduce a large volume of targeting group in the molecule, and do not need to obtain specific sites through a large number of screening, and have better cost performance.

[0060] Compared with the unimproved fluorescent probes I and III, the fluorescent probes with high specificity tumor imaging can effectively realize the differentiation of normal cells and tumor cells at the cell level.

[0061] The mice with sufficient activity and healthy liver and kidney function cannot activate the fluorescent probes II and IV with high specificity tumor imaging, which will provide a reliable basis for realizing the visualization of the liver tumor boundary;

[0062] Human tissues have more complex components and structures, and the fluorescent probes designed based on a single aspect of tumor will be difficult to exhibit the high selectivity tested in vitro, and the fluorescent probes II and IV with high specificity tumor imaging can still exhibit excellent selectivity in complex organisms, and have excellent characteristics of visualizing the tumor boundary. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 is the high resolution mass spectrum of the fluorescent probe III (ANQ) 1 H NMR spectrum

[0064] Figure 2 is the H NMR spectrum of the fluorescent probe IV (FANQ) 1 H NMR spectrum

[0065] Figure 3 is the high resolution mass spectrum of the fluorescent probe III (ANQ)

[0066] Figure 4 is the high resolution mass spectrum of the fluorescent probe IV (FANQ)

[0067] Figure 5 is the confocal imaging diagram of the fluorescent probe I (AN) and the fluorescent probe III after incubation in different types of cells for the same time

[0068] Figure 6 is the fluorescent imaging diagram of the compound fluorescent probe II (FAN) and the fluorescent probe IV (FANQ) respectively observed in real time after being injected into healthy mice through the tail vein

[0069] Figure 7 is the fluorescent imaging diagram of the compound fluorescent probe II (FAN) and the fluorescent probe IV (FANQ) respectively observed after being sprayed and incubated in a clinical orthotopic hepatocellular carcinoma sample. DETAILED DESCRIPTION

[0070] The specific embodiments of the present invention will be further illustrated below, but the specific embodiments of the present invention are not limited to the following embodiments.

[0071] This invention discloses a small-molecule near-infrared fluorescent probe that uses peptidase as a recognition substrate to detect cancer cell growth, invasion, and migration. The structural formula of the probe is shown in Formula I and Formula II.

[0072]

[0073] This invention discloses a highly specific fluorescent probe for tumor imaging, the structural formula of which is shown in Formula III and Formula IV:

[0074]

[0075] The fluorescent probes described herein are named III (ANQ) and IV (FANQ), respectively, and they are characterized by high sensitivity, high specificity, high spatiotemporal resolution, and rapid imaging. The fluorescent probes III and IV described above are highly specific fluorescent probes for tumor imaging, constructed using quinone oxidoreductase (hNQO1), a biomarker that sustains the refractory growth of liver cancer cells in a hypoxic and redox-imbalanced environment, as the recognition site.

[0076] Fluorescent probe III (ANQ) and fluorescent probe IV (FANQ) serve as specific substrate probes for tumor-invasive alanine aminopeptidase (APN) and tumor-reducing resistant biomolecules. They exhibit no fluorescence upon excitation at 660 nm. After sequentially reacting with tumor-reducing resistant biomolecules and tumor-invasive alanine aminopeptidase, they induce the recognition site to leave and then release fluorescence at 705 nm. The activity or content of various biomarkers can be detected by quantitatively measuring the fluorescence intensity of the reaction products per unit time.

[0077] The present invention provides a fluorescent probe for highly specific tumor imaging, which is tested in vitro and intracellularly to obtain a highly efficient tumor-specific probe that is best suited for in vivo and clinical applications.

[0078] The synthesis process of the ANQ probe is shown below:

[0079]

[0080] The fluorescent probe III of this invention exhibits a maximum absorption of 658 nm and extremely low background fluorescence at 705 nm when no recognition substrate is added. Even when only alanine aminopeptidase or quinone oxidoreductase is added, the fluorescent probe III still displays weak fluorescence. The absorption at 658 nm weakens only when alanine aminopeptidase, quinone oxidoreductase, and the coenzyme NADH are present simultaneously, with the maximum absorption and maximum emission red-shifted to 680 nm and 705 nm, respectively.

[0081] As a fluorescent probe with high specificity, the fluorescent probe III can distinguish normal cells and tumor cells by the expression amount and activity of APN and hNQO1.

[0082] The synthesis process of the FANQ probe is as follows:

[0083]

[0084] The fluorescent probe IV (FANQ) described in the application has a maximum absorption of 658 nm without adding a recognition substrate, and has an extremely low background fluorescence at 705 nm. When only alanine aminopeptidase or quinone oxidoreductase is added, the fluorescent probe IV still exhibits the characteristics of weak fluorescence. When and only when alanine aminopeptidase (APN) and quinone oxidoreductase (hNQO1) and coenzyme NADH exist simultaneously, the absorption at 658 is weakened, and the maximum absorption and the maximum emission are red-shifted to 680 nm and 705 nm, respectively.

[0085] As a fluorescent probe with high specificity, the fluorescent probe IV can distinguish normal cells and tumor cells by the expression amount and activity of APN and hNQO1.

[0086] The application of the high-specificity tumor imaging fluorescent probe in detecting tumor-specific markers, the above-mentioned probe fluorescent probe III and fluorescent probe IV are used as specific substrate probes of alanine aminopeptidase and quinone oxidoreductase in a tumor environment, which has no fluorescence under excitation at 660 nm. After the sequence reaction with quinone oxidoreductase and alanine aminopeptidase, the recognition site is induced to leave and release fluorescence at 705 nm. The activity or content of various biomarkers is detected by quantitatively detecting the fluorescence intensity of the reaction product per unit time.

[0087] The specific use method of the above-mentioned high-specificity tumor imaging fluorescent probe is as follows:

[0088] In the system, compound III and compound IV are used as probes, the reaction temperature is between 30℃ and 38℃ in a PBS buffer containing 20% EtOH, the incubation pH environment is between 3 and 9, and the reaction time is 0-240 min, so as to ensure that the corresponding recognition site of the above-mentioned probe leaves to reach the quantitative upper limit. The high-specificity detection of the probe and the fluorescent dye can be realized by using an ultraviolet spectrophotometer and a fluorescence detector at the same time. The fluorescence detection conditions are as follows: the excitation wavelength is 660 nm, and the maximum emission wavelength is 705 nm.

[0089] Further, the reaction temperature is preferably 37℃; and the incubation pH environment is preferably 7.

[0090] The application is different from the past fluorescent probes based on tumor unilateral design, and the tumor root factor is considered from multiple angles to achieve a breakthrough.

[0091] The biological molecules overexpressed by tumor cells in the growth and proliferation process exhibit important pathological properties, but also play an important role in normal physiological environment, alanine aminopeptidase can promote angiogenesis to accelerate tumor growth and proliferation, and alanine aminopeptidase can also promote the reabsorption of amino acids in metabolic organs such as liver and kidney, so the fluorescent probe only for alanine aminopeptidase will have a false positive problem when dealing with liver cancer or kidney cancer disease, and cannot realize the differentiation of tumor boundary. Therefore, the combination of tumor attack system and reduction environment defense system will effectively improve the signal accuracy and realize the precise resection in clinic.

[0092] The application will be further described below through specific examples and drawings.

[0093] Example 1

[0094] Synthesis of fluorescent probe III (ANQ):

[0095] (1) 3.5 mmol of potassium carbonate and 3 mmol of compound 1 were placed in a round-bottom flask containing acetonitrile, activated at 35℃ for 30 min, then 1 mmol of compound 2 dissolved in acetonitrile was added dropwise, then the temperature was raised to 45℃ and reacted for 3 hours. After the reaction was completed, the solvent was removed by reduced pressure distillation, then the inorganic salt in the system was removed by dichloromethane extraction, the organic phase was collected, anhydrous sodium sulfate was used to remove a small amount of water, then the solvent was removed by reduced pressure distillation, and the reaction mixture was separated by column chromatography to obtain compound 3, with a yield of 50.8%.

[0096] (2) 6 mmol of EEDQ and 6 mmol of compound 5 were dissolved in 10 mL of dichloromethane, and the reaction system was activated for ten minutes, then 6 mmol of compound 4 was added, and a large amount of white solid was precipitated after 1 h of reaction. After filtration, the filter residue was washed with a small amount of dichloromethane to obtain compound 6, with a yield of 88%; 2.77 mmol of compound 6 obtained was placed in dichloromethane, a large amount of solid was not dissolved, then 5.54 mmol of phosphorus tribromide was added dropwise, the solid was dissolved, the reaction was carried out for 2 h, then the reaction system was poured into ice water, then dichloromethane was used for extraction, the organic phase was collected, dried with anhydrous sodium sulfate, and then the solvent was removed by reduced pressure distillation to obtain compound 7, with a yield of 75%.

[0097] (3) 0.587 mmol of compound 3, 0.8805 mmol of potassium carbonate and 0.8805 mmol of compound 7 were placed in a round-bottom flask and dissolved in acetonitrile, refluxed for 6 hours, then the solvent was removed by reduced pressure distillation, and then the reaction mixture was separated by column chromatography to obtain compound 8, with a yield of 76.3%.

[0098] (4) Put 0.22 mmol of compound 8 in a round bottom flask and dissolve with dichloromethane, drop 2.2 mmol of piperidine into the system under the condition of ice bath, after 1 hour of reaction, put the reaction system into saturated ammonium chloride solution, then extract with dichloromethane, repeatedly wash the obtained organic phase with saturated aqueous ammonium chloride solution, finally recover the organic phase, separate the mixture by column chromatography to obtain fluorescent probe I for detecting cancer cell growth, invasion and migration with peptidase as recognition substrate, the yield is 24.6%.

[0099] (5) Put 0.325 mmol of compound 9 and 0.325 mmol of triphosgene in a two-neck flask filled with nitrogen, inject super dry dichloromethane and 0.4 mmol of DIPEA into the reaction system through a syringe, and react for 12 hours under ice bath. Then dry the solvent in the system by membrane pump, inject 0.162 mmol of dichloromethane solution of fluorescent probe I and 0.2 mmol of dichloromethane solution of DIPEA into the reaction system through a syringe, dry the solvent in the system by membrane pump after 8 hours of reaction, separate the reaction mixture by column chromatography to obtain fluorescent probe III.

[0100] 1 H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 15.0 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.71 (s, 1H), 7.69 (s, 2H), 7.67 (s, 1H), 7.55 (s, 1H), 7.53 (s, 1H), 7.48 (s, 1H), 7.47 (s, 2H), 7.45 (s, 1H), 7.28 (s, 2H), 7.17 (s, 1H), 7.06 (d, J = 8.8 Hz, 1H), 6.56 (d, J = 15.1 Hz, 1H), 5.24 (s, 2H), 5.06 (s, 1H), 4.26 - 4.19 (m, 1H), 3.89 (s, 3H), 3.05 (s, 2H), 2.72 (d, J = 5.8 Hz, 2H), 2.67 (d, J = 6.3 Hz, 2H), 2.02 (s, 2H), 1.90 (s, 3H), 1.76 (s, 6H), 1.32 (s, 2H), 1.30 (d, J = 2.7 Hz, 6H), 1.27 (s, 6H), 1.24 (s, 3H). HRMS (ESI): m / z calc. for C59H63N4O8[M + 1] 955.4640; found 955.4634. As Figure 1 and Figure 3 shown.

[0101] Example 2

[0102] Synthesis of fluorescent probe IV:

[0103] (1) 1.5 mmol of potassium carbonate and 1.5 mmol of compound 10 were placed in a round bottom flask with acetonitrile and activated for 30 min at 35°C, then 1 mmol of compound 2 dissolved in acetonitrile was added dropwise, and then the temperature was raised to 45°C for 3 h. After the reaction was completed, the solvent was removed by reduced pressure distillation, and the inorganic salts in the system were removed by extraction with dichloromethane. After the organic phase was collected, anhydrous sodium sulfate was used to remove a small amount of water, and then the solvent was removed by reduced pressure distillation. The reaction mixture was separated by column chromatography to obtain compound 11 with a yield of 65.6%.

[0104] (2) 6 mmol of EEDQ and 6 mmol of compound 5 were dissolved in 10 mL of dichloromethane, and after the reaction system was activated for 10 min, 6 mmol of compound 4 was added. After 1 h of reaction, a large amount of white solid was precipitated, which was filtered and washed with a small amount of dichloromethane to obtain compound 6 with a yield of 88%. 2.77 mmol of compound 6 obtained was placed in dichloromethane, and a large amount of solid was not dissolved. Then 5.54 mmol of phosphorus tribromide was added dropwise, and the solid was dissolved. After 2 h of reaction, the reaction system was poured into ice water, and then extracted with dichloromethane. After the organic phase was collected and dried with anhydrous sodium sulfate, the solvent was removed by reduced pressure distillation to obtain compound 7 with a yield of 75%.

[0105] (3) 0.567 mmol of compound 11, 0.85 mmol of potassium carbonate, and 0.85 mmol of compound 7 were placed in a round bottom flask and dissolved in acetonitrile. After refluxing for 6 h, the solvent was removed by reduced pressure distillation, and then the reaction mixture was separated by column chromatography to obtain compound 12 with a yield of 85.6%.

[0106] (4) 0.215 mmol of compound 12 was placed in a round bottom flask and dissolved in 2 mL of dichloromethane. 0.2 mL of piperidine was added dropwise under ice bath conditions. After 1 h of reaction, the reaction system was placed in a saturated ammonium chloride solution, and then extracted with dichloromethane. The obtained organic phase was repeatedly washed with saturated aqueous ammonium chloride solution, and finally the organic phase was recovered. The mixture was separated by column chromatography to obtain fluorescent probe II for detecting cancer cell growth, invasion, and migration with a yield of 34%.

[0107] (5) 0.108 mmol of compound 9 and 0.108 mmol of triphosgene were placed in a nitrogen-filled two-necked flask, 2 mL of super-dry dichloromethane and 0.135 mmol of DIPEA were injected into the reaction system by a syringe, and the reaction was carried out under ice-bath for 12 hours. Then the solvent in the system was pumped dry by a membrane pump, 0.054 mmol of the dichloromethane solution of fluorescent probe II and 0.0675 mmol of the dichloromethane solution of DIPEA were injected into the reaction system by a syringe, and the reaction was carried out for 8 hours. After the solvent in the system was pumped dry by a membrane pump, the reaction mixture was separated by column chromatography to obtain fluorescent probe IV with a yield of 12%.

[0108] 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 15.1 Hz, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.72 - 7.70 (m, 1H), 7.69 (s, 1H), 7.68 (d, J = 3.5 Hz, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.53 - 7.51 (m, 1H), 7.49 (d, J = 2.5 Hz, 2H), 7.47 (d, J = 1.5 Hz, 1H), 7.43 (d, J = 6.9 Hz, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.28 - 7.24 (m, 1H), 6.57 (d, J = 15.1 Hz, 1H), 5.32 (s, 2H), 5.03 (s, 1H), 4.21 (t, J = 6.5 Hz, 1H), 3.88 (s, 3H), 3.03 (s, 2H), 2.71 (d, J = 6.1 Hz, 2H), 2.65 (d, J = 6.7 Hz, 2H), 2.00 (s, 2H), 1.88 (s, 3H), 1.76 (d, J = 1.8 Hz, 6H), 1.32 (s, 2H), 1.28 (t, J = 3.6 Hz, 6H), 1.24 (d, J = 3.7 Hz, 6H), 1.22 (s, 3H). HRMS (ESI): m / z calc. for C59H62FN4O8[M + 1] 973.4546; found 973.4553. Figure 2 and Figure 4 as shown.

[0109] Example 3

[0110] The fluorescent probes I (AN) and II (ANQ) were applied to the fluorescence imaging analysis in different normal cells and cancer cells:

[0111] The above probe is dissolved in DMSO to prepare a 1 mM stock solution for use. When staining, the medium is used to dilute the staining solution to a final concentration of 5 μM. Different kinds of normal cells (L02, HK-2) and cancer cells (MCF-7, HepG-2) are inoculated in a confocal dish and incubated in the staining solution for 1 hour. After that, confocal imaging is directly performed without washing, and the obtained confocal images are shown in Figure 5 The excitation wavelength is 640 nm, and the collected emission channel is 663-738 nm. The probe ANQ based on our strategy shows better performance than the probe AN, and can improve the distinction between normal cells and cancer cells.

[0112] The obtained compound fluorescent probes II (FAN) and IV (FANQ) are injected into healthy mice through the tail vein to observe the real-time fluorescence imaging images;

[0113] The above two probes are dissolved in DMSO to prepare a 10 mM stock solution for use. Before the probes are injected into healthy mice through the tail vein, the probes are diluted to a final concentration of 100 μM working solution using a PBS buffer solution containing 20% ethanol at pH = 7.4. Then, 100 μL of the above two probes are injected into multiple healthy mice through the tail vein, and then the in vivo fluorescence of the mice is observed through real-time detection. After 4 hours of real-time imaging, the mice are sacrificed according to the requirements of animal ethics, and different organs are taken out as the basis for in vitro imaging. The probe FANQ based on our strategy shows better performance than the probe FAN, which is different from other fluorescent probes that need to be metabolized for a long time. The probe designed based on our strategy can remain silent in the body for a long time, greatly reducing the false positive signal, as shown in Figure 6 The excitation wavelength is 675 nm, and the collected emission channel is Cy5.5.

[0114] Example 4

[0115] The fluorescent probes II (FAN) and IV (FANQ) are sprayed and incubated in clinical in situ hepatocellular carcinoma samples to observe the fluorescence imaging images:

[0116] The clinical hepatocellular carcinoma sample in situ obtained from the First People's Hospital of Hunan Province is placed in a clean glass dish, then the probe is diluted into a working solution with a final concentration of 50 μM by spraying a PBS buffer solution containing 20% ethanol with pH = 7.4 uniformly on the entire hepatocellular carcinoma sample, and after incubation for ten minutes, the fluorescence imaging image is obtained by imaging, and the clinical sample is sent for hematoxylin staining, then we compare the fluorescence imaging image obtained and the hematoxylin staining image, and find that our fluorescent probe can realize specific imaging of the tumor, and the obvious tumor boundary can be observed, which will greatly assist the success of intraoperative resection. In addition, we immerse the small volume hepatocellular carcinoma clinical sample obtained in the probe working solution of 50 μM, and after the same time of incubation, we obtain the same result in the fluorescence imaging image as shown in Figure 7 , which indicates that our fluorescent probe avoids the problem of false positive signal existing in the current fluorescent probe.

[0117] A series of small molecule fluorescent probes are disclosed, which are composed of a near-infrared fluorophore covalently connected to the substrate of a hepatocellular carcinoma cell growth, invasion, migration related peptidase and a biomarker for maintaining the stubborn growth of hepatocellular carcinoma cells in anoxic and redox imbalance environment. The present application first innovatively utilizes the complementary relationship of multiple markers in the tumor microenvironment, designs, synthesizes and applies this type of small molecule fluorescent probe with high specificity for the visualization of near-infrared fluorescence imaging of liver tumor boundary, and provides an effective method and practical tool for realizing high-precision surgery in clinic.

[0118] The above embodiment is a preferred embodiment of the present application, but the embodiment of the present application is not limited by the above embodiment, and any change, modification, substitution, combination, simplification made without departing from the spirit and principles of the present application should be an equivalent replacement mode, and all are included in the protection scope of the present application.

Claims

1. A small molecule near-infrared fluorescent probe, characterized in that, The structural formula of the probe is shown as formula I, formula II: 。 2. A fluorescent probe for high specificity tumor imaging, characterized by, The structural formula of the fluorescent probe is shown as formula III, formula IV: 。 3. A method for preparing a small molecule near-infrared fluorescent probe, characterized in that, The method comprises the following steps: (1) adding potassium carbonate, compound 1 and a solvent into a first reactor to activate, dropwise adding compound 2, then increasing the temperature to a predetermined temperature, and obtaining compound 3 after reaction; (2) putting compound 3, potassium carbonate and compound 7 into a second reactor, and adding a solvent to dissolve and reflux at a set temperature to obtain compound 8; (3) putting compound 8 into a third reactor and dissolving with dichloromethane, dropwise adding piperidine at a predetermined temperature, and obtaining fluorescent probe I after reaction; The chemical reaction formula involved is as follows: 。 4. The method for preparing a small molecule near-infrared fluorescent probe according to claim 3, characterized in that, The solvent is acetonitrile; The molar ratio of the potassium carbonate, resorcinol and compound 2 is (2-5):(2-5): 1; The molar volume ratio of the compound 2 and acetonitrile is 1:(5-10) mmol / mL; The molar ratio of the compound 3, potassium carbonate and compound 7 is 1:(1-3):(1-3); The molar volume ratio of the compound 3 and acetonitrile is 1:(5-10) mmol / mL; The molar ratio of compound 8 and piperidine is 1:(10-20); The molar volume ratio of compound 8 and dichloromethane is 1:(5-10) mmol / mL.

5. A method for preparing a fluorescent probe for high specificity tumor imaging, characterized by, The method comprises the following steps: (1) putting compound 9 and triphosgene into a reaction container filled with inert gas, and injecting super-dry dichloromethane and DIPEA into the reaction system, and reacting under ice bath for a set time; (2) then, the system is pumped dry, and a dichloromethane solution of the fluorescent probe I obtained in claim 3 and a dichloromethane solution of DIPEA are injected, and the fluorescent probe ANQ is obtained after reaction; The chemical reaction formula involved is as follows: 。 6.The method of claim 5, wherein the method is characterized by, The molar ratio of the compound 9, triphosgene and DIPEA is 1:(1-2):(1-2); The molar ratio of the fluorescent probe I and DIPEA is 1:(1-2); The molar volume ratio of the fluorescent probe I and dichloromethane is 1:(2-5) mmol / mL.

7. A method for preparing a small molecule near-infrared fluorescent probe, characterized in that, The method comprises the following steps: (1) adding potassium carbonate, compound 10 and a solvent into a first reactor to activate, dropwise adding compound 2, then increasing the temperature to a predetermined temperature, and obtaining compound 11 after reaction; (2) putting compound 11, potassium carbonate and compound 7 into a second reactor, and adding a solvent to dissolve and reflux at a set temperature to obtain compound 12; (3) putting compound 12 into a third reactor and dissolving with dichloromethane, dropwise adding piperidine at a predetermined temperature, and obtaining fluorescent probe II after reaction; The chemical reaction formula involved is as follows: 。 8. The method for preparing a small molecule near-infrared fluorescent probe according to claim 7, characterized in that, The solvent is acetonitrile; The molar ratio of the potassium carbonate, 4-fluororesorcinol and compound 2 is (2-5):(2-5): 1; The molar volume ratio of the compound 2 and acetonitrile is 1:(5-10) mmol / mL; The molar ratio of the compound 11, potassium carbonate and compound 7 is 1:(1-3):(1-3); The molar volume ratio of the compound 11 and acetonitrile is 1:(5-10) mmol / mL; The molar ratio of compound 12 and piperidine is 1:(10-20); The molar volume ratio of compound 12 and dichloromethane is 1:(5-10) mmol / mL. The molar volume ratio of compound 12 and dichloromethane is 1: (5-10) mmol / mL.

9. A method for preparing a fluorescent probe for high specificity tumor imaging, characterized by, The method comprises the following steps: (1) Compound 9, triphosgene are placed in a reaction container filled with an inert atmosphere, ultra-dry dichloromethane and DIPEA are injected into the reaction system, and the reaction is set for a time under an ice bath; (2) Then the system is pumped dry, dichloromethane solution of fluorescent probe II and dichloromethane solution of DIPEA are injected, and fluorescent probe FANQ is obtained after reaction; The chemical reaction formula involved is: The molar ratio of compound 9, triphosgene and DIPEA is 1: (1-2): (1-2); The molar ratio of fluorescent probe II and DIPEA is 1: (1-2); The molar volume ratio of fluorescent probe II and dichloromethane is 1: (2-5) mmol / mL.

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