Fluorescent probe for high-specificity diagnosis of tumor tissue as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for high-specificity diagnosis of tumor tissues, a preparation method and application thereof, compared with the existing fluorescent probe, the fluorescent probe HD-CTSC with high-specificity tumor imaging advantage combines cathepsin C with obvious content difference in benign / malignant tumors with HD dye, and the fluorescence probe HD-CTSC has the advantages of high specificity and high tumor imaging sensitivity. Other auxiliary analytes do not need to be introduced, large-volume targeting groups do not need to be introduced into molecules, synthesis is easy, the yield is stable, and better cost performance is achieved. Compared with the fluorescent probes RhD-CTSC and DCM-CTSC which are not improved, the near-infrared fluorescent probe HD-CTSC for high-specificity tumor imaging can effectively distinguish normal cells from tumor cells at the cellular level. The HD-CTSC can accurately distinguish benign tumor from malignant tumor in a complex clinical sample, so that a reliable basis is provided for rapid non-invasive detection of clinical tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and relates to a fluorescent probe for highly specific diagnosis of tumor tissues, a preparation method and an application thereof, and specifically relates to a class of visible light to near-infrared fluorescent probes activatable by cathepsin C, which are used to achieve benign / malignant differentiation of mouse and human breast tissues and real-time detection of primary breast cancer and lung metastases in vivo. Background Art

[0002] As an analytical tool with the advantages of non-invasive, real-time and convenient, fluorescent probes have been widely used in many fields such as chemistry, molecular medicine, and clinical medicine. They play an important role in detecting target content in life systems or the process of diseases (especially tumors). In recent years, in order to achieve the goal of rapid detection medicine, researchers have constructed fluorescent probes for detecting different biomarkers (mainly active small molecules or biological proteases) based on fluorescent dyes of different properties, and have achieved certain results in a variety of preclinical tumor models. However, many probes often show poor detection capabilities in benign / malignant tumors with high apparent similarity. This is mainly because the reported active small molecule activatable probes lack specificity, and the reported protease activatable probes have a long response time, which greatly reduces the sensitivity and accuracy of imaging or detection. This problem is particularly significant in near-infrared fluorescent probes. The lack of representativeness of biomarkers is the most fundamental reason why fluorescent probes cannot accurately identify benign / malignant tumors. Designing and constructing probes based on more representative biomarkers may be an effective way to improve the detection of benign / malignant tumors by probes.

[0003] Cathepsins, as a class of enzymes that degrade proteins, exist in all animals and other organisms and play an important role in maintaining cellular homeostasis. Classified by their structure and catalytic mechanism, there are more than a dozen members in this enzyme family. Among these members, cathepsin C (CTSC) has been shown to be closely associated with breast cancer metastasis to the lungs. At the same time, it has no obvious significance for primary tumor growth. Clinical studies have shown that high CTSC activity is associated with an increased incidence of brain and lung metastasis and a low survival rate in breast cancer patients. Further studies have shown that CTSC is more active in metastatic tumors than in primary tumors, and CTSC promotes the proliferation of lung cancer cells in the early stages of breast cancer metastasis. Considering that metastasis is an external manifestation of malignancy, we speculate that CTSC may be a potential candidate for identifying tumor carcinogenic characteristics.

[0004] Therefore, it is of great significance to develop a class of highly specific fluorescent probes from visible light to near-infrared that can be activated by CTSC to enhance the ability of the probes to identify benign / malignant tumors. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a highly specific fluorescent probe for diagnosing tumor tissue, a preparation method and its application. By using cathepsin C, a biomarker highly correlated with breast cancer lung metastasis, a highly specific fluorescent probe is synthesized for rapid diagnosis of breast tumors by visualized near-infrared fluorescence imaging. The fluorescent probe can achieve high-precision visual identification of the properties of complex breast tumor tissue (benign / malignant), providing a new option for achieving clinical tumor auxiliary grading and rapid diagnosis.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A type of highly specific fluorescent probe for diagnosing tumor tissues, which consists of a highly specific dipeptide substrate and a covalently linked near-infrared fluorophore. The structural formula of the highly specific dipeptide substrate is Formula A1 or Formula A2:

[0008]

[0009] The structural formula of the near-infrared fluorophore is any one of Formula B;

[0010]

[0011] The present invention provides a highly specific fluorescent probe for diagnosing tumor tissues. The fluorescent probe uses cathepsin C as a recognition substrate and is used to quickly distinguish normal / cancer cells (tissues). The structural formula of the fluorescent probe is (RhD-CTSC):

[0012]

[0013] The present invention provides another highly specific fluorescent probe for diagnosing tumor tissues. The fluorescent probe uses cathepsin C as a recognition substrate and is used to quickly distinguish normal / cancer cells (tissues). The structural formula of the fluorescent probe is (DCM-CTSC):

[0014]

[0015] The present invention provides another highly specific fluorescent probe for diagnosing tumor tissues. The fluorescent probe uses cathepsin C as a recognition substrate and is used to quickly distinguish normal / cancer cells (tissues). The structural formula of the fluorescent probe is (HD-CTSC):

[0016]

[0017] The present invention provides a method for preparing a highly specific fluorescent probe (RhD-CTSC) for diagnosing tumor tissue, comprising the following steps:

[0018] RhD dye, sodium iodide, cesium carbonate, and (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate are placed in a first reactor, and a solvent is added, and the mixture is dissolved and stirred at a set temperature to obtain a first intermediate;

[0019] The first intermediate is placed in a second reactor and dissolved in dichloromethane, piperidine is added dropwise at a predetermined temperature, and the fluorescent probe RhD-CTSC is obtained after the reaction;

[0020] The chemical reaction involved is:

[0021]

[0022] In the preparation process of the first intermediate, acetonitrile is used as the solvent;

[0023] The molar ratio of the first intermediate to piperidine is 1:(10-20);

[0024] The molar volume ratio of the first intermediate to dichloromethane is 1: (5-10) mmol / mL.

[0025] The present invention also provides a method for preparing a fluorescent probe (DCM-CTSC) for diagnosing tumor tissue with high specificity, comprising the following steps:

[0026] DCM dye, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), (((9H-fluoren-9-yl)methoxy)carbonyl)glycylphenylalanine, and N,N-diisopropylethylamine (DIPEA) are placed in a first reactor, and a solvent is added, and the mixture is dissolved and stirred at a set temperature to obtain a second intermediate;

[0027] The second intermediate is placed in a second reactor and dissolved in dichloromethane, and piperidine is added dropwise at a predetermined temperature to obtain a fluorescent probe DCM-CTSC after the reaction;

[0028]

[0029] The chemical reaction involved is:

[0030] In the preparation process of the second intermediate, dichloromethane is used as the solvent;

[0031] The molar ratio of HATU, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylphenylalanine and DIPEA is (2-5):1:(2-5);

[0032] The molar ratio of the second intermediate to piperidine is 1:(10-20);

[0033] The molar volume ratio of the second intermediate to dichloromethane is 1: (5-10) mmol / mL.

[0034] The present invention also provides a method for preparing a fluorescent probe for highly specific diagnosis of tumor tissue (HD-CTSC), comprising the following steps:

[0035] Putting HD dye (compound 3), sodium iodide, cesium carbonate, and (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate into a first reactor, adding a solvent, and dissolving and stirring at a set temperature to obtain a third intermediate;

[0036] The third intermediate is placed in a second reactor and dissolved in dichloromethane, piperidine is added dropwise at a predetermined temperature, and the fluorescent probe HD-CTSC is obtained after the reaction;

[0037] The chemical reaction involved is:

[0038]

[0039] During the preparation of the third intermediate, acetonitrile is used as the solvent;

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

[0041] The molar ratio of the third intermediate to piperidine is 1:(10-20);

[0042] The molar volume ratio of the third intermediate and dichloromethane is 1: (5-10) mmol / mL;

[0043] The molar ratio of HD dye (compound 3), cesium carbonate (CsCO3), sodium iodide (NaI) and (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate is 1:(1~3):(1~3):(1~3).

[0044] The preparation process of HD dye is as follows:

[0045] Potassium carbonate, compound 1, and a solvent are added to a third reactor for activation, compound 2 is added dropwise, and then the temperature is raised to a predetermined temperature, and a near-infrared HD dye (compound 3) is obtained after reaction;

[0046] The chemical reaction involved is:

[0047]

[0048] In the preparation of HD dye, acetonitrile was used as solvent;

[0049] The molar ratio of potassium carbonate, compound 1 and compound 2 is (2-5):(2-5):1;

[0050] The molar volume ratio of compound 2 and acetonitrile is 1: (5-10) mmol / mL;

[0051] The present invention also provides application of the fluorescent probe for diagnosing tumor tissue with high specificity in detecting analytes in body fluids.

[0052] Compared with the existing technology, the present invention has the following beneficial effects:

[0053] Compared with the existing fluorescent probes, the fluorescent probe HD-CTSC of the present invention has the advantage of high specificity for tumor imaging. It combines cathepsin C, which has obvious differences in content in benign and malignant tumors, with HD dye. It does not require the introduction of other auxiliary analytes or large-volume targeting groups in the molecule. It is easy to synthesize, has a stable yield, and has a better cost-effectiveness.

[0054] Compared with the unimproved fluorescent probes RhD-CTSC and DCM-CTSC, the near-infrared fluorescent probe HD-CTSC for highly specific tumor imaging can effectively distinguish between normal cells and tumor cells at the cellular level.

[0055] HD-CTSC can accurately distinguish benign / malignant tumors in complex clinical samples, which will provide a reliable basis for rapid and non-invasive detection of clinical tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the hydrogen NMR spectrum of the dye intermediate 2 prepared in Example 1.

[0057] Figure 2 This is the NMR spectrum and carbon spectrum of the dye intermediate 2 prepared in Example 1.

[0058] Figure 3 This is the hydrogen NMR spectrum of the HD-CTSC prepared in Example 4.

[0059] Figure 4 This is the NMR spectrum and carbon spectrum of the HD-CTSC prepared in Example 4.

[0060] Figure 5 This is the hydrogen NMR spectrum of the RhD-CTSC prepared in Example 2.

[0061] Figure 6 This is the NMR spectrum and carbon spectrum of the RhD-CTSC prepared in Example 2.

[0062] Figure 7 This is the hydrogen NMR spectrum of DCM-CTSC prepared in Example 3.

[0063] Figure 8 This is the NMR spectrum and carbon spectrum of DCM-CTSC prepared in Example 3.

[0064] Fig. 9 This is the high resolution mass spectrum of the dye intermediate 2 prepared in Example 1.

[0065] Fig.10 This is the high-resolution mass spectrum of the HD-CTSC prepared in Example 4.

[0066] Fig.11 This is the high-resolution mass spectrum of RhD-CTSC prepared in Example 2.

[0067] Fig.12 This is the high-resolution mass spectrum of DCM-CTSC prepared in Example 3.

[0068] Fig.13 Confocal fluorescence image of the HD-CTSC prepared in Example 4 used to distinguish normal cells from cancer cells.

[0069] Fig.14 This is an in vivo fluorescence imaging image of the HD-CTSC prepared in Example 4 used to distinguish between mammary hyperplasia and in situ breast cancer in mice.

[0070] Fig.15Confocal fluorescence images of the RhD-CTSCs prepared in Example 2, the DCM-CTSCs prepared in Example 3, and the HD-CTSCs prepared in Example 4 used to distinguish between benign and malignant human breast tissues. DETAILED DESCRIPTION

[0071] The specific embodiments of the present invention are further described below by way of examples, but the specific embodiments of the present invention are not limited to the following examples.

[0072] Example 1

[0073] Synthesis of CTSC-recognizable dipeptide linkers:

[0074] Step 1: Glycyl-phenylalanine (12 mmol), N,N-diisopropylethylamine (12 mmol) and fluorenylmethoxycarbonyl chloride (12 mmol) were dissolved in analytical grade methanol (10 mL), heated to 40°C for 6 h, and then the reaction system was concentrated under reduced pressure, a small amount of dichloromethane (2 mL) and a large amount of petroleum ether (100 mL) were added thereto, and a large amount of solid precipitates were filtered, washed, dried, and separated by column chromatography to obtain dye intermediate 1;

[0075] Step 2: The dipeptide intermediate 1 (1 mmoL), 4-aminobenzyl alcohol (1.1 mmoL) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.2 mmoL) were dissolved in anhydrous tetrahydrofuran (10 mL), reacted overnight at room temperature, the solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain the dye intermediate 2 for the subsequent synthesis of the fluorescent probe.

[0076] The chemical reaction involved is:

[0077]

[0078] Using nuclear magnetic resonance ( Figure 1 and Figure 2 ) and high resolution mass spectrometry analysis of intermediate 2 ( Fig. 9 )structure. 1 H NMR (400MHz, DMSO-d6) δ10.16(s,2H),8.29(s,2H),7.53(d,J=8.0Hz,5H),7.28(s,2H),7.26(s,5H),7.2 4(s,2H),7.20(d,J=9.4Hz,2H),4.73(s,1H),4.45(s,3H),3.18(s,2H),3.12(s,2H),3.06–2.95(m,2H). 13C NMR (100MHz, DMSO-d6) δ172.78,170.23,169.48,156.96,144.29,143.87,141. 17,140.25,139.12,138.11,137.93,137.75,129.66,128.57,128.10,127.55, 127.36,126.87,125.72,122.41,121.75,120.58,119.60,117.97,66.21,65.1 7,63.04,55.21,49.07,47.05,43.68,39.75,37.52,35.38.ESI-MS(m / z):calcd C 33 H 31 N3O5[M+Na+]:572.2264,found:572.2155.

[0079] Example 2

[0080] Synthesis of CTSC-activatable, highly specific fluorescent probes designed based on RhD dyes:

[0081] RhD (1 mmoL), cesium carbonate (3 mmoL), and sodium iodide (3 mmoL) were dissolved in acetonitrile (10 mL), and the mixture was stirred at room temperature for 15 min. (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate (1.5 mmoL) was added to the mixture, and stirring continued overnight at room temperature. The mixture was poured into ice water and extracted three times with dichloromethane. The organic phase was dried and concentrated, and the residue was redissolved in dichloromethane (2 mL). Piperidine (0.5 mL) was added thereto, and the mixture was stirred at room temperature for 60 min. The organic phase was removed under reduced pressure, and the residue was separated by column chromatography to obtain the probe RhD-CTSC.

[0082] The chemical reaction involved is:

[0083]

[0084] Using nuclear magnetic resonance ( Figure 5 and Figure 6 ) and high resolution mass spectrometry ( Fig.11 )Analyze the structure of the compound. 1H NMR(400MHz,Chloroform-d)δ8.00(d,J=7.4Hz,1H),7.62(dt,J=13.3,7.3Hz,2H),7.50( d,J=8.4Hz,2H),7.30(s,4H),7.24(s,3H),7.17(d,J=7.5Hz,2H),6.78(s,1H),6.65(d,J= 8.8Hz,1H),6.62–6.54(m,2H),6.44(s,1H),6.36(d,J=8.9Hz,1H),4.97(s,2H),4.83(s, 1H), 3.46 (s, 1H), 3.35 (q, J = 7.3Hz, 4H), 3.19 (s, 1H), 3.05 (s, 2H), 1.17 (t, J = 7.1Hz, 6H). 13 C NMR(100MHz,Chloroform-d)δ169.76,168.52,160.39,160.28,158.99,154.47,149.92,144.01 ,143.79,137.24,136.39,134.58,132.80,129.95,129.40,129.16,128.94,128.66,128.25,127 .02,126.78,124.97,124.86,124.23,123.71,120.27,112.20,112.10,111.66,111.25,107.66, 105.22,101.85,97.55,69.83,55.37,44.50,31.52,29.71,22.07,14.14,12.53.ESI-MS(m / z):C 42 H 41 N4O6+calcd:697.3021,found:697.3029.

[0085] Example 3

[0086] Synthesis of CTSC-activatable, highly specific fluorescent probe based on DCM dye:

[0087] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylphenylalanine (1mmoL), HATU (2mmoL), and DIPEA (2mmoL) were dissolved in dichloromethane (10mL), and the mixture was stirred at room temperature for 30min. DCM (1.5mmoL) was added to the mixture, and stirring was continued at room temperature overnight. After stopping the reaction, the organic phase was concentrated, and the residue was redissolved in dichloromethane (2mL). Piperidine (0.5mL) was added thereto, and the mixture was stirred at room temperature for 60min. The organic phase was removed under reduced pressure, and the residue was separated by column chromatography to obtain the probe DCM-CTSC.

[0088] The chemical reaction involved is:

[0089]

[0090] Using nuclear magnetic resonance ( Figure 7 and Figure 8 ) and high resolution mass spectrometry ( Fig.12 )Analyze the structure of the compound. 1 H NMR(400MHz,Chloroform-d)δ7.50(d,J=8.6Hz,2H),7.45(d,J=11.4Hz,2H),7 .23(d,J=7.2Hz,2H),7.19(d,J=7.7Hz,3H),7.04(d,J=16.1Hz,1H),6.93(d,J= 16.1Hz,1H),6.79(d,J=8.2Hz,1H),4.71(t,J=7.4Hz,1H),3.30(dd,J=16.0,8 .4Hz, 4H), 2.58 (d, J = 8.3Hz, 2H), 2.48 (d, J = 6.0Hz, 2H), 1.07 (d, J = 8.4Hz, 6H). 13 C NMR(101MHz,Chloroform-d)δ177.06,174.05,173.87,165.67,158.78,143.14,141.54,140.86,140.16,135.69,133.33,133.10,132. 40,132.29,132.08,130.87,127.75,126.93,124.22,123.72,117.47,116.70,58.98,47.60,46.92,43.01,42.34,35.91,33.51,31.68.

[0091] ESI-MS (m / z): C 30 H 31N5O2 calcd:493.2478,found:494.2553.

[0092] Example 4

[0093] Synthesis of CTSC-activatable, highly specific fluorescent probes designed based on HD dyes:

[0094] HD (1mmoL), cesium carbonate (3mmoL), and sodium iodide (3mmoL) were dissolved in acetonitrile (10mL), and the mixture was stirred at room temperature for 15min. (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate (1.5mmoL) was added to the mixture, and stirring was continued at room temperature overnight. The mixture was poured into ice water, extracted three times with dichloromethane, and the organic phase was dried and concentrated. The residue was redissolved in dichloromethane (2mL), and piperidine (0.5mL) was added thereto. The mixture was stirred at room temperature for 60min, and the organic phase was removed under reduced pressure. The residue was separated by column chromatography to obtain the probe HD-CTSC.

[0095] The chemical reaction involved is:

[0096]

[0097] Using nuclear magnetic resonance ( Figure 3 and Figure 4 ) and high resolution mass spectrometry ( Fig.10 )Analyze the structure of the compound. 1 H NMR (400MHz, Methanol-d4): δ8.70(d,J=14.9Hz,1H),7.71(d,J=7.4Hz,1H),7.60–7.53(m,4H), 7.46(d,J=8.2Hz,3H),7.28(d,J=10.9Hz,2H),7.26–7.23(m,4H),7.20–7.16(m,1H),6.52(s,1H ),5.31(s,2H),4.75(t,J=7.4Hz,1H),4.39(q,J=6.9Hz,2H),3.37(d,J=7.9Hz,2H),3.16(m,1H) ,3.00(m,1H),2.72(m,4H),1.91(d,J=9.2Hz,2H),1.82(d,J=2.3Hz,6H),1.47(t,J=8.1Hz,4H). 13C NMR(100MHz,Methanol-d4)δ177.62,170.44,161.15,150.22,145.82,142.40,141.07,13 8.17,136.56,132.37,131.86,128.93,128.86,128.55,128.12,128.07,127.16,126.53,1 22.55,120.08,114.33,112.94,112.73,112.37,103.45,102.55,71.02,55.42,50.76,47. 59,41.87,40.10,37.95,29.35,28.71,26.84,26.57,23.61,20.11,11.52.ESI-MS(m / z):C 45 H 46 FN4O4+[M]calcd:725.3498,found:725.3498.

[0098] Example 5

[0099] Apply the fluorescent probe (HD-CTSC) to fluorescence imaging analysis in single cells and mixed cells (normal / cancer cells):

[0100] The above probes were dissolved in DMSO to prepare a 1mM stock solution for use. When staining, the medium was used to dilute the staining solution to a final concentration of 5μM. Normal cells (HEK293), cancer cells (4T1) and mixed culture cells (HEK293 / 4T1) were inoculated in the staining solution for 1 hour, and then directly confocal imaging was performed after washing with DPBS three times. The confocal images obtained are shown in Figure 2. Fig.13 As shown, the excitation wavelength is 640nm, and the collected emission channel is 663-738nm. Based on the higher expression of CTSC in cancer cells, the highly specific probe is activated, which can achieve efficient differentiation between normal cells and cancer cells.

[0101] The compound fluorescent probe (HD-CTSC) obtained by comparison was injected into breast hyperplasia mice and breast cancer mice by in situ injection, and the real-time fluorescence imaging was observed ( Fig.14 );

[0102] The above two probes were dissolved in DMSO to prepare a 10mM mother solution for use. Before the probes were injected into well-off mice through the tail vein, the probes were diluted to a working solution with a final concentration of 200μM using a PBS buffer solution of pH=7.4 containing 10% DMSO. Subsequently, 30μL of the probes were injected in situ into the mammary tissues of different mice, and then the in vivo fluorescence of the mice was observed through real-time detection. After the real-time imaging was completed, the accuracy of HD-CTSC was verified using potassium salt of fluorescein as a luminescent substrate for breast cancer (4T1-Luc). After the mice were killed using methods required by animal ethics, the corresponding mammary tissues were taken out as a basis for in vitro imaging. The probe based on the high-specific activation of CTSC showed excellent detection ability, with good fluorescence onset in malignant breast tissue, while remaining silent in benign breast tissue, greatly reducing false positive signals. The excitation wavelength was 710nm, and the emission channel collected was ICG.

[0103] Example 6

[0104] The confocal fluorescence images of the frozen sections of clinical human benign and malignant breast tissues were observed by spraying and incubating the fluorescent probes RhD-CTSC, DCM-CTSC, and HD-CTSC. Fig.15 ):

[0105] The clinical breast tissue samples obtained from the First People's Hospital of Hunan Province were placed in a clean glass dish, embedded with OTC glue and fixed in a -80 degree ultra-low temperature refrigerator. The tissue was processed into 20 μm tissue sections using a freezing microtome, and then the probe was diluted to a final concentration of 20 μM in a PBS buffer solution with pH = 7.4 containing 20% ​​DMSO and sprayed evenly on the entire section. After incubation for 20 minutes, confocal imaging images were obtained, and clinical samples were stained with hematoxylin. Then we analyzed the obtained confocal imaging images, hematoxylin staining and clinical pathological diagnosis one by one, and found that our fluorescent probes (RhD-CTSC, DCM-CTSC, HD-CTSC) can significantly distinguish different breast tissues, showing strong fluorescence in malignant breast tumors, and maintaining weak fluorescence in benign breast tumors. Among them, HD-CTSC showed the strongest differentiation ability, which may be due to its longer wavelength to avoid the interference of tissue autofluorescence to the greatest extent. Three CTSC-activatable high-specificity probes with different wavelengths all showed the ability to distinguish benign from malignant tumors in human breast tissue samples, indicating that our fluorescent probes significantly improve the accuracy of rapid detection of benign and malignant breast tumors and have the potential for further transformation.

[0106] The present invention discloses a series of small molecule fluorescent probes, which are composed of a highly specific dipeptide substrate (glycyl-phenylalanine) of cathepsin C that drives malignant breast cancer cells to invade and metastasize, covalently linked to a near-infrared fluorophore. This is the first innovative design, synthesis and application of this type of highly specific small molecule fluorescent probe based on the specific substrate of CTSC, and is used for the rapid diagnosis of clinical human benign and malignant breast tissues, providing an effective method and practical tool for achieving rapid, non-invasive and accurate detection of clinical tissues.

[0107] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A type of highly specific fluorescent probe for diagnosing tumor tissue, characterized in that: This type of fluorescent probe is composed of a high-specificity dipeptide substrate and a covalently linked near-infrared fluorophore. The structural formula of the high-specificity dipeptide substrate is Formula A1 or Formula A2: The structural formula of the near-infrared fluorophore is any one of Formula B; 2. A highly specific fluorescent probe for diagnosing tumor tissue according to claim 1, characterized in that: The structural formula of the fluorescent probe is RhD-CTSC:

3. A fluorescent probe for diagnosing tumor tissue with high specificity according to claim 1, characterized in that: The structural formula of the fluorescent probe is DCM-CTSC:

4. A fluorescent probe for diagnosing tumor tissue with high specificity according to claim 1, characterized in that: The structural formula of the fluorescent probe is HD-CTSC:

5. The method for preparing the highly specific fluorescent probe RhD-CTSC for diagnosing tumor tissue according to claim 2, characterized in that: The following steps are involved: RhD dye, sodium iodide, cesium carbonate, and (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate are placed in a first reactor, and a solvent is added, and the mixture is dissolved and stirred at a set temperature to obtain a first intermediate; The first intermediate is placed in a second reactor and dissolved in dichloromethane, piperidine is added dropwise at a predetermined temperature, and the fluorescent probe RhD-CTSC is obtained after the reaction; The chemical reaction involved is:

6. The method for preparing the highly specific fluorescent probe RhD-CTSC for diagnosing tumor tissue according to claim 5, characterized in that: In the preparation process of the first intermediate, acetonitrile is used as the solvent; The molar ratio of the first intermediate to piperidine is 1:(10-20); The molar volume ratio of the first intermediate to dichloromethane is 1: (5-10) mmol / mL.

7. The method for preparing the fluorescent probe DCM-CTSC for diagnosing tumor tissue with high specificity according to claim 3, characterized in that: The following steps are involved: DCM dye, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), (((9H-fluoren-9-yl)methoxy)carbonyl)glycylphenylalanine, and N,N-diisopropylethylamine (DIPEA) are placed in a first reactor, and a solvent is added, and the mixture is dissolved and stirred at a set temperature to obtain a second intermediate; The second intermediate is placed in a second reactor and dissolved in dichloromethane, and piperidine is added dropwise at a predetermined temperature to obtain a fluorescent probe DCM-CTSC after the reaction; The chemical reaction involved is:

8. The method for preparing the fluorescent probe DCM-CTSC for diagnosing tumor tissue with high specificity according to claim 7, characterized in that: In the preparation process of the second intermediate, dichloromethane is used as the solvent; The molar ratio of HATU, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylphenylalanine and DIPEA is (2-5):1:(2-5); The molar ratio of the second intermediate to piperidine is 1:(10-20); The molar volume ratio of the second intermediate to dichloromethane is 1: (5-10) mmol / mL.

9. The method for preparing the fluorescent probe HD-CTSC for diagnosing tumor tissue with high specificity according to claim 4, characterized in that: The following steps are involved: Putting HD dye (compound 3), sodium iodide, cesium carbonate, and (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate into a first reactor, adding a solvent, and dissolving and stirring at a set temperature to obtain a third intermediate; The third intermediate is placed in a second reactor and dissolved in dichloromethane, piperidine is added dropwise at a predetermined temperature, and the fluorescent probe HD-CTSC is obtained after the reaction; The chemical reaction involved is:

10. The method for preparing the fluorescent probe HD-CTSC for highly specific diagnosis of tumor tissue according to claim 9, characterized in that: During the preparation of the third intermediate, acetonitrile is used as the solvent; The molar volume ratio of HD dye and acetonitrile is 1:(5-10)mmol / mL; The molar ratio of the third intermediate to piperidine is 1:(10-20); The molar volume ratio of the third intermediate and dichloromethane is 1: (5-10) mmol / mL; The molar ratio of HD dye, cesium carbonate (CsCO3), sodium iodide (NaI) and (9H-fluoren-9-yl)methyl(2-((1-((4-(bromomethyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)carbamate is 1:(1~3):(1~3):(1~3).

11. The method for preparing the fluorescent probe HD-CTSC for diagnosing tumor tissue with high specificity according to claim 9, characterized in that: The preparation process of HD dye is: Potassium carbonate, compound 1, and a solvent are added to a third reactor for activation, compound 2 is added dropwise, and then the temperature is raised to a predetermined temperature, and a near-infrared HD dye (compound 3) is obtained after reaction; The chemical reaction involved is: In the preparation of HD dye, acetonitrile was used as solvent; The molar ratio of potassium carbonate, compound 1 and compound 2 is (2-5):(2-5):1; The molar volume ratio of compound 2 and acetonitrile is 1: (5-10) mmol / mL.

12. Use of the fluorescent probe for diagnosing tumor tissue with high specificity according to any one of claims 1 to 11 in the detection of analytes in body fluids.

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