CN118084878A - Fluorescent probe for high-specificity tumor imaging, preparation method and application thereof - Google Patents
Fluorescent probe for high-specificity tumor imaging, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent probe for high-specificity tumor imaging, a preparation method and application thereof, belonging to the technical field of fluorescent probes, wherein the structural formula of the fluorescent probe is shown as formula III and formula IV: Compared with the existing specific fluorescent probes, the fluorescent probes III and IV for high-specificity tumor imaging combine an attack system for tumor growth with a defense system for tumor, do not need to introduce a large volume of targeting groups in molecules, do not need to obtain specific sites through a large number of screening, and have better cost performance. Compared with unmodified fluorescent probes I and II, the fluorescent probe for high-specificity tumor imaging can effectively realize the distinction between normal cells and tumor cells at the cellular level.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, relates to a fluorescent probe for high-specificity tumor imaging, a preparation method and application thereof, and in particular relates to application of high-precision visual imaging of liver tumor boundaries by utilizing tumor growth, invasion and migration related peptidase and maintaining complementary connection between biomarkers for refractory growth of liver cancer cells in an environment with hypoxia and redox imbalance.
Background
Hepatocellular carcinoma is the most predominant subtype of liver cancer, and has great significance to its research. The specific fluorescent probe designed aiming at the focus over-expressed biomarker has the potential of visualizing the imaging tumor boundary and assisting in clinical high-quality tumor excision. Alanine aminopeptidase (APN/CD 13) assists in hydrolyzing basal membrane during tumor growth process so as to promote release of Vascular Endothelial Growth Factor (VEGF), so that blood vessels near tumor are dense to absorb nutrition from organism more easily, and extremely convenient condition is provided for invasion and metastasis of tumor as attack of tumor. In addition, in order to ensure the refractory growth of the tumor under the stimulation of hypoxia, an immune system and external drugs, the tumor-specific aerobic glycolysis metabolic mode can effectively reduce the generation of active oxygen with killing capacity in cells, and overexpress the biomarkers with reducibility such as quinone oxidoreductase (hNQO 1) and the like to provide a layer of powerful defense for the tumor. By utilizing the attack characteristic of tumor growth and the defense characteristic of tumor survival development, a series of small molecular fluorescent probes aiming at the tumor multidimensional characteristic are designed, and the fluorescent probes can improve the false positive signal problem existing in most of the current fluorescent probes, and have the capability of visually imaging tumor boundaries and assisting clinical high-quality tumor excision.
In recent years, fluorescence analysis techniques have been used in various fields of labeling of biomolecules, environmental monitoring, cell staining, clinical diagnosis, etc., wherein most of the fluorescent probes for tumor diagnosis have been reported to use only unilateral markers of tumors, which may result in insufficient probe specificity, causing false positive signals. This is a number of markers selected to design probes that play an important role in physiological environments in addition to playing an important role in pathological environments. Therefore, the relation of tumor multi-aspect markers is clarified, and the fluorescent probe without false positive signals is designed with high specificity, so that the method can provide the greatest help for distinguishing tumor boundaries and realizing high-precision visual imaging.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fluorescent probe for high-specificity tumor imaging, a preparation method and application thereof, wherein the fluorescent probe has high-precision visual boundary properties of hepatocellular carcinoma tissues and paracancerous tissues, and the fluorescent probe is designed and applied by utilizing biomarkers in two processes of tumor attack and defense, so that the fluorescent probe with high specificity is used for the visual near infrared fluorescence imaging of the boundary of the liver tumor, and a new choice is provided for realizing clinical high-precision operation.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention relates to a small molecule near infrared fluorescent probe which uses peptidase as a recognition substrate and is used for detecting the growth, invasion and migration of cancer cells, wherein the structural formula of the probe is shown as formula I and formula II:
The invention discloses a fluorescent probe for high-specificity tumor imaging, which has structural formulas shown in formulas III and IV:
Wherein, the fluorescent probes are respectively named III (ANQ), IV (FANQ) and have the characteristics of high sensitivity, high specificity, high space-time resolution and rapid imaging. The fluorescent probes III and IV are constructed by taking biomarker quinone oxidoreductase (hNQO 1) which can maintain refractory growth of liver cancer cells in the environment of hypoxia and redox imbalance as a recognition site.
The invention provides a preparation method of a small molecule near infrared fluorescent probe, which comprises the following steps:
(1) Adding potassium carbonate, resorcinol (compound 1) and a solvent into a first reactor for activation, dropwise adding compound 2, then raising the temperature to a preset temperature, and reacting to obtain a near infrared hemicyanine dye (compound 3);
(2) Placing a compound 3, potassium carbonate and 9H-fluoren-9-yl) methyl (1- ((4- (bromomethyl) phenyl) amino) -1-oxo-propan-2-yl) carbamate (compound 7) into a second reactor, adding a solvent, and dissolving and refluxing at a set temperature to obtain a compound 8;
(3) Placing the compound 8 in a third reactor, dissolving the compound by using dichloromethane, dropwise adding piperidine at a preset temperature, and reacting to obtain a fluorescent probe I;
The chemical reaction formula involved is:
wherein, in the preparation process of the small molecule near infrared fluorescent probe, acetonitrile is adopted as the solvent;
The molar ratio of the potassium carbonate to the resorcinol to the compound 2 is (2-5): 1;
the molar volume ratio of the compound 2 to the acetonitrile is 1: (5-10) mmol/mL;
the molar ratio of the compound 3 to the potassium carbonate to the compound 7 is 1 (1-3): (1-3);
The molar volume ratio of the compound 3 to the acetonitrile is 1: (5-10) mmol/mL;
The molar ratio of compound 8 to piperidine is 1: (10-20);
the molar volume ratio of compound 8 to dichloromethane is 1: (5-10) mmol/mL.
The invention provides a preparation method of a fluorescent probe for high-specificity tumor imaging, which comprises the following steps:
(1) Placing N- (4- (hydroxymethyl) phenyl) -N, 2-trimethyl-3- (2, 4, 5-trimethyl-3, 6-dioxocyclohexyl-1, 4-diene-1-yl) propionamide (compound 9) and triphosgene in a reaction vessel filled with inert atmosphere, injecting ultra-dry dichloromethane and DIPEA into the reaction system, and reacting for a set time under ice bath;
(2) Pumping the system, injecting a dichloromethane solution of the fluorescent probe I obtained in claim 3 and a dichloromethane solution of DIPEA, and reacting to obtain a fluorescent probe III (ANQ);
The chemical reaction formula involved is:
Wherein, in the preparation process of the fluorescent probe for high specificity tumor imaging, the molar ratio of the compound 9 to triphosgene to DIPEA is 1: (1-2): (1-2);
the molar ratio of the fluorescent probe I to the DIPEA is 1: (1-2);
the molar volume ratio of the fluorescent probe I to the dichloromethane is 1: (2-5) mmol/mL.
The inventor respectively carries out characterization by means of nuclear magnetic resonance hydrogen spectrum, mass spectrum, ultraviolet spectrum and the like, and shows that the fluorescent probe ANQ for imaging tumor boundaries with high specificity is successfully synthesized.
As a general inventive concept, the invention also provides a preparation method of the small molecule near infrared fluorescent probe, which comprises the following steps:
(1) Adding potassium carbonate, 4-fluororesorcinol (compound 10) and a solvent into a first reactor for activation, dropwise adding compound 2, then raising the temperature to a preset temperature, and reacting to obtain a near infrared hemicyanine dye (compound 11);
(2) Placing a compound 11, potassium carbonate and 9H-fluoren-9-yl) methyl (1- ((4- (bromomethyl) phenyl) amino) -1-oxo-propan-2-yl) carbamate (compound 7) into a second reactor, adding a solvent, and dissolving and refluxing at a set temperature to obtain a compound 12;
(3) Placing the compound 12 in a third reactor, dissolving the compound in dichloromethane, dropwise adding piperidine at a preset temperature, and reacting to obtain a fluorescent probe II;
The chemical reaction formula involved is:
wherein, in the preparation process of the small molecule near infrared fluorescent probe, acetonitrile is adopted as the solvent;
The molar ratio of the potassium carbonate to the 4-fluororesorcinol to the compound 2 is (2-5): 1;
the molar volume ratio of the compound 2 to the acetonitrile is 1: (5-10) mmol/mL;
the molar ratio of the compound 11 to the potassium carbonate to the compound 7 is 1 (1-3): (1-3);
The molar volume ratio of the compound 11 to the acetonitrile is 1: (5-10) mmol/mL;
the molar ratio of compound 12 to piperidine is 1: (10-20);
The molar volume ratio of compound 12 to dichloromethane is 1: (5-10) mmol/mL.
The invention also provides a preparation method of the fluorescent probe for high-specificity tumor imaging, which comprises the following steps:
(1) Placing N- (4- (hydroxymethyl) phenyl) -N, 2-trimethyl-3- (2, 4, 5-trimethyl-3, 6-dioxocyclohexyl-1, 4-diene-1-yl) propionamide (compound 9) and triphosgene in a reaction vessel filled with inert atmosphere, injecting ultra-dry dichloromethane and DIPEA into the reaction system, and reacting for a set time under ice bath;
(2) Pumping the system, injecting a dichloromethane solution of the fluorescent probe II and a dichloromethane solution of DIPEA, and reacting to obtain a fluorescent probe IV (FANQ);
The chemical reaction formula involved is:
Wherein, in the preparation process of the fluorescent probe for high specificity tumor imaging, the molar ratio of the compound 9 to triphosgene to DIPEA is 1: (1-2): (1-2);
The molar ratio of the fluorescent probe II to the DIPEA is 1: (1-2);
The molar volume ratio of the fluorescent probe II to the dichloromethane is 1: (2-5) mmol/mL.
The inventor respectively performs characterization by means of nuclear magnetic resonance hydrogen spectrum, mass spectrum, ultraviolet spectrum and the like, and shows that the fluorescent probe FANQ for imaging tumor boundaries with high specificity is successfully synthesized.
The invention provides application of the small molecule near infrared fluorescent probe in reference probes.
The invention provides application of the fluorescent probe for high-specificity tumor imaging in detecting tumor specific markers.
The invention provides a fluorescent probe for high-specificity tumor imaging, which is obtained by comparing and screening a series of probes based on a high-efficiency tumor distinguishing strategy in vitro and in cells.
The fluorescent probe for high-specificity tumor imaging is applied to detection of tumor specific markers, and the probe is used for testing tumor specific substrates.
Compared with the prior art, the invention has the beneficial effects that:
Compared with the existing specific fluorescent probes, the fluorescent probes II and IV with high-specificity tumor imaging advantages combine an attack system for tumor growth with a defense system for tumor, do not need to introduce large-volume targeting groups in molecules, do not need to obtain specific sites through a large number of screening, and have better cost performance.
Compared with unmodified fluorescent probes I and III, the fluorescent probe for high-specificity tumor imaging can effectively realize the distinction between normal cells and tumor cells at the cellular level.
Fluorescent probes II and IV which have sufficient activity and healthy liver and kidney function can not activate high-specificity tumor imaging, and a reliable basis is provided for realizing the visualization of liver tumor boundaries;
the human tissue has more complex components and structures, the fluorescent probe designed based on the single aspect of tumor is difficult to show high selectivity tested in vitro, and the fluorescent probes II and IV for high-specificity tumor imaging can still show excellent selectivity in complex organisms and have excellent characteristics of visualizing tumor boundaries.
Drawings
FIG. 1 is a 1 H NMR spectrum of fluorescent probe III (ANQ);
FIG. 2 is a 1 H NMR spectrum of fluorescent probe IV (FANQ);
FIG. 3 is a high resolution mass spectrum of fluorescent probe III (ANQ);
FIG. 4 is a high resolution mass spectrum of fluorescent probe IV (FANQ);
FIG. 5 is a confocal imaging of fluorescent probes I (AN) and III after incubation in different cell types for the same period of time;
FIG. 6 is a fluorescence imaging of compound fluorescent probe II (FAN) and fluorescent probe IV (FANQ) by tail vein injection into healthy mice, respectively, in real time;
FIG. 7 is a fluorescence imaging of compound fluorescent probe II (FAN) and fluorescent probe IV (FANQ) respectively observed in clinical in situ hepatocellular carcinoma samples incubated by spray.
Detailed Description
The following examples further illustrate embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples.
The invention relates to a small molecule near infrared fluorescent probe which uses peptidase as a recognition substrate and is used for detecting the growth, invasion and migration of cancer cells, wherein the structural formula of the probe is shown as formula I and formula II:
The invention discloses a fluorescent probe for high-specificity tumor imaging, which has structural formulas shown in formulas III and IV:
Wherein, the fluorescent probes are respectively named III (ANQ), IV (FANQ) and have the characteristics of high sensitivity, high specificity, high space-time resolution and rapid imaging. The fluorescent probes III and IV are constructed by taking biomarker quinone oxidoreductase (hNQO 1) which can maintain refractory growth of liver cancer cells in the environment of hypoxia and redox imbalance as a recognition site.
Fluorescent probes III (ANQ) and IV (FANQ) are used as specific substrate probes of tumor invasive alanine Aminopeptidase (APN) and tumor reduction resistant biomolecules, do not have fluorescence under excitation of 660nm, release fluorescence at 705nm after induction of recognition site leaving after sequential reaction with the tumor reduction resistant biomolecules and tumor invasive alanine aminopeptidase, and the activity or content of various biomarkers is detected by quantitatively detecting the fluorescence intensity of reaction products in unit time.
The invention provides the fluorescent probe for high-specificity tumor imaging, and the fluorescent probe is most suitable for being applied to living bodies and clinic high-efficiency tumor specificity probes through in-vitro and in-cell layer-by-layer tests.
The synthesis of the ANQ probe is as follows:
The maximum absorption of the fluorescent probe III is 658nm when no recognition substrate is added, the fluorescent probe III has extremely low background fluorescence at 705nm, and the fluorescent probe III still has the characteristic of weak fluorescence when only alanine aminopeptidase or quinone oxidoreductase is added. If and only if alanine aminopeptidase and quinone oxidoreductase and coenzyme NADH are present simultaneously the absorbance at 658 is reduced and the maximum absorbance and maximum emission red shift to 680nm and 705nm, respectively.
As a fluorescent probe with high specificity, the fluorescent probe III can not only distinguish normal cells from tumor cells by the difference of the expression quantity and the activity of APN and hNQO 1.
The synthesis of FANQ probe is as follows:
The maximum absorption of the fluorescent probe IV (FANQ) is 658nm when no recognition substrate is added, the fluorescent probe IV has extremely low background fluorescence at 705nm, and the fluorescent probe IV still shows the characteristic of weak fluorescence when only alanine aminopeptidase or spread oxidoreductase is added. The absorbance at 658 is reduced if and only if alanine Aminopeptidase (APN) and quinone oxidoreductase (hNQO 1) and coenzyme NADH are present simultaneously, the maximum absorbance and maximum emission red shifted to 680nm and 705nm, respectively.
As a fluorescent probe with high specificity, the fluorescent probe IV can distinguish normal cells from tumor cells by the difference of the expression quantity and the activity of APN and hNQO.
The fluorescent probe III and the fluorescent probe IV are adopted as specific substrate probes of alanine aminopeptidase and quinone oxidoreductase in tumor environment, the fluorescent probes do not have fluorescence under excitation of 660nm, fluorescence is released at 705nm after the fluorescent probes react with the sequence of the quinone aminopeptidase and the alanine aminopeptidase to induce a recognition site to leave, and the activity or content detection of various biomarkers is realized by quantitatively detecting the fluorescence intensity of a reaction product in unit time.
The specific application method of the fluorescent probe for high-specificity tumor imaging is as follows:
in the system, a compound III and a compound IV are used as probes, the reaction temperature is between 30 and 38 ℃ in a PBS buffer solution of 20 percent EtOH, the incubation pH environment is between 3 and 9, the reaction time is between 0 and 240 minutes, the corresponding recognition site leaving of the probes is ensured to reach quantitative online, and the high-specificity detection of the probes and fluorescent dye can be realized simultaneously by an ultraviolet spectrophotometer and a fluorescent detector; fluorescent detection conditions: the excitation wavelength was 660nm and the maximum emission wavelength was 705nm.
Further, the reaction temperature is preferably 37 ℃; the incubation pH environment is preferably 7.
The invention is different from the prior fluorescent probe based on unilateral tumor design, and takes factors of tumor root taking into consideration at multiple angles to realize the leap.
The biological molecule over-expressed by the tumor cells in the growth and proliferation process shows important pathological properties, but plays an important role in the normal physiological environment, and the alanine aminopeptidase can promote angiogenesis to accelerate the growth and proliferation of tumors, and simultaneously promote the reabsorption of amino acids of metabolic organs such as liver and kidney, so that the fluorescent probe of the alanine aminopeptidase only aims at the problem of false positive in treating liver cancer or renal cancer, and the differentiation of tumor boundaries cannot be realized. Therefore, the signal accuracy is effectively improved by combining the tumor attack system and the defense system for reinforcing and defending the reductive environment, and the clinical accurate excision is realized.
Further description will be made by way of specific examples and accompanying drawings.
Example 1
Synthesis of fluorescent probe iii (ANQ):
(1) 3.5mmol of potassium carbonate and 3mmol of Compound 1 were placed in a round-bottomed flask containing acetonitrile and activated at 35℃for 30 minutes, then 1mmol of Compound 2 dissolved in acetonitrile was dropwise added, followed by reaction at 45℃for 3 hours, after completion of the reaction, the solvent was removed by distillation under reduced pressure, the inorganic salts in the system were removed by extraction with methylene chloride, the organic phase was collected, a small amount of water was removed by use of anhydrous sodium sulfate, followed by distillation under reduced pressure, and the reaction mixture was separated by column chromatography to give Compound 3 in a yield of 50.8%.
(2) Dissolving 6mmol of EEDQ and 6mmol of compound 5 in 10mL of dichloromethane, adding 6mmol of compound 4 after ten minutes of activation in a reaction system, reacting for 1h, precipitating a large amount of white solid, filtering, and washing filter residues with a small amount of dichloromethane to obtain compound 6 with the yield of 88%; the resulting 2.77mmol of Compound 6 was placed in methylene chloride, a large amount of solids was not dissolved, then 5.54mmol of phosphorus tribromide was added dropwise, the solids were dissolved, the reaction system was poured into ice water after 2 hours of reaction, followed by extraction with methylene chloride, and the organic phase was collected, and after drying over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure to give Compound 7 in 75% yield.
(3) 0.587Mmol of Compound 3 and 0.8805mmol of potassium carbonate and 0.8805mmol of Compound 7 were placed in a round-bottomed flask and dissolved by adding acetonitrile, after refluxing for 6 hours, the solvent was removed by distillation under reduced pressure, and then the reaction mixture was separated by column chromatography to give Compound 8 in a yield of 76.3%.
(4) 0.22Mmol of compound 8 was placed in a round bottom flask and dissolved with methylene chloride, 2.2mmol of piperidine was dropwise added under ice bath conditions, after 1 hour of reaction, the reaction system was placed in a saturated ammonium chloride solution, followed by extraction with methylene chloride, the resulting organic phase was then washed with an aqueous solution of the organic phase instead of the saturated ammonium chloride, and finally the organic phase was recovered, and the mixture was separated by column chromatography to give a fluorescent probe I for detecting growth, invasion and migration of cancer cells using peptidase as a recognition substrate in a yield of 24.6%.
(5) 0.325Mmol of Compound 9 and 0.325mmol of triphosgene were placed in a two-necked flask filled with nitrogen gas, and ultra-dry methylene chloride and 0.4mmol of DIPEA were injected into the reaction system via a syringe and reacted under an ice bath for 12 hours. And then pumping the solvent in the system by a diaphragm pump, injecting a dichloromethane solution of 0.162mmol of fluorescent probe I and a dichloromethane solution of 0.2mmol of DIPEA into the reaction system by a syringe, reacting for 8 hours, pumping the solvent in the system by the diaphragm pump, and separating the reaction mixture by column chromatography to obtain the fluorescent probe III.
1H NMR(400MHz,DMSO-d6)δ8.57(d,J＝15.0Hz,1H),7.77(d,J＝7.4Hz,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＝8.8Hz,1H),6.56(d,J＝15.1Hz,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＝5.8Hz,2H),2.67(d,J＝6.3Hz,2H),2.02(s,2H),1.90(s,3H),1.76(s,6H),1.32(s,2H),1.30(d,J＝2.7Hz,6H),1.27(s,6H),1.24(s,3H).HRMS(ESI):m/z calc.for C59H63N4O8[M+1]955.4640;found 955.4634. As shown in fig. 1 and 3.
Example 2
Synthesis of fluorescent probe IV:
(1) After 1.5mmol of potassium carbonate and 1.5mmol of compound 10 were placed in a round bottom flask charged with acetonitrile and activated at 35℃for 30 minutes, 1mmol of compound 2 dissolved in acetonitrile was added dropwise, followed by a reaction at 45℃for 3 hours, after the completion of the reaction, the solvent was removed by distillation under reduced pressure, the inorganic salts in the system were removed by extraction with methylene chloride, the organic phase was collected, a small amount of water was removed by use of anhydrous sodium sulfate, followed by distillation under reduced pressure, and the reaction mixture was separated by column chromatography to give compound 11 in a yield of 65.6%.
(2) Dissolving 6mmol of EEDQ and 6mmol of compound 5 in 10mL of dichloromethane, adding 6mmol of compound 4 after ten minutes of activation in a reaction system, reacting for 1h, precipitating a large amount of white solid, filtering, and washing filter residues with a small amount of dichloromethane to obtain compound 6 with the yield of 88%; the resulting 2.77mmol of Compound 6 was placed in methylene chloride, a large amount of solids was not dissolved, then 5.54mmol of phosphorus tribromide was added dropwise, the solids were dissolved, the reaction system was poured into ice water after 2 hours of reaction, followed by extraction with methylene chloride, and the organic phase was collected, and after drying over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure to give Compound 7 in 75% yield.
(3) 0.567Mmol of compound 11 and 0.85mmol of potassium carbonate and 0.85mmol of compound 7 were put into a round-bottomed flask and dissolved by adding acetonitrile, after refluxing for 6 hours, the solvent was removed by distillation under reduced pressure, and then the reaction mixture was separated by column chromatography to obtain compound 12 in a yield of 85.6%.
(4) 0.215Mmol of compound 12 is placed in a round-bottom flask and dissolved with 2mL of dichloromethane, 0.2mL of piperidine is dropwise added under ice bath conditions, after 1 hour of reaction, the reaction system is placed in a saturated ammonium chloride solution, then the saturated ammonium chloride aqueous solution is extracted with dichloromethane, the obtained organic phase is reversely reused and washed, finally the organic phase is recovered, the mixture is separated by column chromatography, and a fluorescent probe II which takes peptidase as a recognition substrate and is used for detecting the growth, invasion and migration of cancer cells is obtained, and the yield is 34%.
(5) 0.108Mmol of Compound 9 and 0.108mmol of triphosgene were placed in a two-necked flask filled with nitrogen, and 2mL of ultra-dry dichloromethane and 0.135mmol of DIPEA were injected into the reaction system via syringe and reacted under ice bath for 12 hours. Then the solvent in the system is pumped out by a diaphragm pump, 0.054mmol of the dichloromethane solution of the fluorescent probe II and 0.0675mmol of the dichloromethane solution of the DIPEA are injected into the reaction system by a syringe, the solvent in the system is pumped out by the diaphragm pump after the reaction is carried out for 8 hours, and the fluorescent probe IV is obtained by separating the reaction mixture by column chromatography, and the yield is 12%.
1H NMR(400MHz,DMSO-d6)δ8.52(d,J＝15.1Hz,1H),7.79(d,J＝7.4Hz,1H),7.72–7.70(m,1H),7.69(s,1H),7.68(d,J＝3.5Hz,1H),7.64(d,J＝7.5Hz,1H),7.54(d,J＝1.2Hz,1H),7.53–7.51(m,1H),7.49(d,J＝2.5Hz,2H),7.47(d,J＝1.5Hz,1H),7.43(d,J＝6.9Hz,1H),7.34(d,J＝8.5Hz,2H),7.28–7.24(m,1H),6.57(d,J＝15.1Hz,1H),5.32(s,2H),5.03(s,1H),4.21(t,J＝6.5Hz,1H),3.88(s,3H),3.03(s,2H),2.71(d,J＝6.1Hz,2H),2.65(d,J＝6.7Hz,2H),2.00(s,2H),1.88(s,3H),1.76(d,J＝1.8Hz,6H),1.32(s,2H),1.28(t,J＝3.6Hz,6H),1.24(d,J＝3.7Hz,6H),1.22(s,3H).HRMS(ESI):m/z calc.for C59H62FN4O8[M+1]973.4546;found 973.4553. As shown in fig. 2 and 4.
Example 3
Fluorescent imaging analysis of fluorescent probes i (AN), ii (ANQ) in different normal and cancer cells:
The above probe was dissolved in DMSO to prepare a 1mM stock solution, and the stock solution was diluted with a medium to a final concentration of 5. Mu.M for use in staining. Different normal cells (L02, HK-2) and cancer cells (MCF-7, hepG-2) are inoculated by copolymerization Jiao Min and incubated for 1 hour in a staining solution, confocal imaging is directly carried out without washing, the obtained confocal image is shown in figure 5, the excitation wavelength is 640nm, and the collected emission channel is 663-738nm. Wherein the probe ANQ based on our strategy shows better phenomenon than the probe AN, and can realize the improvement of the differentiation of normal cells and cancer cells.
Performing real-time observed fluorescence imaging images of the compound fluorescence probes II (FAN) and IV (FANQ) obtained through comparison by tail vein injection into healthy mice;
The above two probes were dissolved in DMSO to prepare a 10mM stock solution to be used, and the probes were diluted to a final concentration of 100 μm using PBS buffer solution containing 20% ethanol at ph=7.4 before being injected into healthy mice via tail vein. Subsequently, 100. Mu.L of each of the above two probes was injected into a plurality of healthy mice via tail vein, and then, in vivo fluorescence of the mice was observed by real-time detection. After 4 hours of real-time imaging, after mice were sacrificed by animal ethical requirements, different organs were removed as imaging basis in vitro. Wherein the probe FANQ based on our strategy shows better phenomenon than the probe FAN, unlike other fluorescent probes requiring long-term metabolism, the probe designed based on our strategy can keep silent in the organism for a long time, greatly reducing false positive signals, as shown in FIG. 6. The excitation wavelength was 675nm and the emission channel collected was Cy5.5.
Example 4
Fluorescence imaging patterns observed in clinical in situ hepatocellular carcinoma samples were incubated with fluorescent probe II (FAN), fluorescent probe IV (FANQ) probes by spraying:
Clinical in-situ hepatocellular carcinoma samples obtained from the first people's hospitals in Hunan province were placed in clean glass dishes, then a PBS buffer solution with pH=7.4 containing 20% ethanol was used to dilute the probe to a final concentration of 50 μm and evenly sprayed on the whole hepatoma cell samples, after ten minutes of incubation, a fluorescence imaging image was obtained by imaging with an imager, and the clinical samples were sent to be stained with hematoxylin, then we compared the obtained fluorescence imaging image with the hematoxylin stained image to find that our fluorescence probe can realize specific imaging of tumors and can observe obvious tumor boundaries, which would greatly assist in the success of excision in clinical surgery. In addition, the obtained small-volume liver cancer clinical sample is soaked in 50 mu M probe working solution, and the same result is obtained in a fluorescence imaging chart after incubation for the same time, as shown in fig. 7, which shows that the fluorescent probe avoids the problem of false positive signals existing in the current fluorescent probe.
The invention discloses a series of small molecular fluorescent probes, which are formed by covalent connection of substrates of biological markers for the growth, invasion and migration of liver cancer cells and for maintaining the refractory growth of the liver cancer cells in the environment of hypoxia and redox imbalance, wherein the small molecular fluorescent probes with high specificity are designed, synthesized and applied for the first time by creatively utilizing the complementary relationship of various markers in the tumor microenvironment, and are used for the visualization near infrared fluorescence imaging of liver tumor boundaries, thereby providing an effective method and a practical tool for realizing clinical high-precision operation.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The small molecule near infrared fluorescent probe is characterized in that the structural formula of the probe is shown as formula I and formula II:
2. The fluorescent probe for high-specificity tumor imaging is characterized in that the structural formula of the fluorescent probe is shown as formula III and formula IV:
3. the preparation method of the small molecule near infrared fluorescent probe is characterized by comprising the following steps of:
(1) Adding potassium carbonate, resorcinol (compound 1) and a solvent into a first reactor for activation, dropwise adding compound 2, then raising the temperature to a preset temperature, and reacting to obtain a near infrared hemicyanine dye (compound 3);
(2) Placing a compound 3, potassium carbonate and 9H-fluoren-9-yl) methyl (1- ((4- (bromomethyl) phenyl) amino) -1-oxo-propan-2-yl) carbamate (compound 7) into a second reactor, adding a solvent, and dissolving and refluxing at a set temperature to obtain a compound 8;
(3) Placing the compound 8 in a third reactor, dissolving the compound by using dichloromethane, dropwise adding piperidine at a preset temperature, and reacting to obtain a fluorescent probe I;
The chemical reaction formula involved is:
4. the method for preparing a small molecule near infrared fluorescent probe according to claim 3, wherein acetonitrile is used as the solvent;
The molar ratio of the potassium carbonate to the resorcinol to the compound 2 is (2-5): 1;
the molar volume ratio of the compound 2 to the acetonitrile is 1: (5-10) mmol/mL;
the molar ratio of the compound 3 to the potassium carbonate to the compound 7 is 1 (1-3): (1-3);
The molar volume ratio of the compound 3 to the acetonitrile is 1: (5-10) mmol/mL;
The molar ratio of compound 8 to piperidine is 1: (10-20);
the molar volume ratio of compound 8 to dichloromethane is 1: (5-10) mmol/mL.
5. The preparation method of the fluorescent probe for high-specificity tumor imaging is characterized by comprising the following steps of:
(1) Placing N- (4- (hydroxymethyl) phenyl) -N, 2-trimethyl-3- (2, 4, 5-trimethyl-3, 6-dioxocyclohexyl-1, 4-diene-1-yl) propionamide (compound 9) and triphosgene in a reaction vessel filled with inert atmosphere, injecting ultra-dry dichloromethane and DIPEA into the reaction system, and reacting for a set time under ice bath;
(2) Pumping the system, injecting a dichloromethane solution of the fluorescent probe I obtained in claim 3 and a dichloromethane solution of DIPEA, and reacting to obtain a fluorescent probe III (ANQ);
The chemical reaction formula involved is:
6. The method for preparing a fluorescent probe for high specificity tumor imaging according to claim 5, wherein the molar ratio of the compound 9 to triphosgene to DIPEA is 1: (1-2): (1-2);
the molar ratio of the fluorescent probe I to the DIPEA is 1: (1-2);
the molar volume ratio of the fluorescent probe I to the dichloromethane is 1: (2-5) mmol/mL.
7. The preparation method of the small molecule near infrared fluorescent probe is characterized by comprising the following steps of:
(1) Adding potassium carbonate, 4-fluororesorcinol (compound 10) and a solvent into a first reactor for activation, dropwise adding compound 2, then raising the temperature to a preset temperature, and reacting to obtain a near infrared hemicyanine dye (compound 11);
(2) Placing a compound 11, potassium carbonate and 9H-fluoren-9-yl) methyl (1- ((4- (bromomethyl) phenyl) amino) -1-oxo-propan-2-yl) carbamate (compound 7) into a second reactor, adding a solvent, and dissolving and refluxing at a set temperature to obtain a compound 12;
(3) Placing the compound 12 in a third reactor, dissolving the compound in dichloromethane, dropwise adding piperidine at a preset temperature, and reacting to obtain a fluorescent probe II;
The chemical reaction formula involved is:
8. The method for preparing a small molecule near infrared fluorescent probe according to claim 7, wherein acetonitrile is used as the solvent;
The molar ratio of the potassium carbonate to the 4-fluororesorcinol to the compound 2 is (2-5): 1;
the molar volume ratio of the compound 2 to the acetonitrile is 1: (5-10) mmol/mL;
the molar ratio of the compound 11 to the potassium carbonate to the compound 7 is 1 (1-3): (1-3);
The molar volume ratio of the compound 11 to the acetonitrile is 1: (5-10) mmol/mL;
the molar ratio of compound 12 to piperidine is 1: (10-20);
The molar volume ratio of compound 12 to dichloromethane is 1: (5-10) mmol/mL.
9. The preparation method of the fluorescent probe for high-specificity tumor imaging is characterized by comprising the following steps of:
(1) Placing N- (4- (hydroxymethyl) phenyl) -N, 2-trimethyl-3- (2, 4, 5-trimethyl-3, 6-dioxocyclohexyl-1, 4-diene-1-yl) propionamide (compound 9) and triphosgene in a reaction vessel filled with inert atmosphere, injecting ultra-dry dichloromethane and DIPEA into the reaction system, and reacting for a set time under ice bath;
(2) Pumping the system, injecting a dichloromethane solution of the fluorescent probe II and a dichloromethane solution of DIPEA, and reacting to obtain a fluorescent probe IV (FANQ);
The chemical reaction formula involved is:
the molar ratio of compound 9, triphosgene and DIPEA was 1: (1-2): (1-2);
The molar ratio of the fluorescent probe II to the DIPEA is 1: (1-2);
The molar volume ratio of the fluorescent probe II to the dichloromethane is 1: (2-5) mmol/mL.
10. Use of a fluorescent probe for high specificity tumor imaging according to claim 2 for detecting tumor specific markers.
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