A class of pH-sensitive carboline pyridine fluorescent probes and preparation methods and applications thereof

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a class of pH-sensitive carboline pyridine fluorescent probes and a preparation method and application thereof. The carboline pyridine fluorescent probe has a structure shown in general formula I: The present invention utilizes the ICT principle to be activated in the acidic microenvironment of tumor tissue, and selectively and rapidly generates pH-sensitive near-infrared fluorescence at the tumor site. The specific implementation method is to spray the carboline pyridine solution of the present invention on the tumor lesion site, and use a fluorescent laparoscope or a living body imager to perform rapid and selective fluorescent imaging and tracing of the tumor lesion tissue, which has high selectivity for tumor tissue fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a class of pH-sensitive carboline pyridine fluorescent probes and a preparation method and application thereof. Background Art

[0002] Cancer is currently one of the greatest threats to human health. By 2035, the World Health Organization estimates that there could be 24 million new cases and 14.5 million tumor-related deaths each year. Early diagnosis and treatment could save approximately 30% of cancer-related deaths.

[0003] Tumor fluorescent probes are special molecular tools used to detect and image tumor tissues in vivo or in vitro. These probes are usually composed of molecules that can specifically bind to tumor-related biomarkers and emit fluorescent signals, thereby helping doctors and researchers locate and diagnose tumors. The design of fluorescent probes can be based on a variety of factors, including surface markers of tumor cells, metabolic activity within cells, or physiological environment in tissues. These probes have important application potential in tumor research and clinical diagnosis, and can improve the accuracy of early diagnosis of tumors and the precision of surgical treatment. Traditional fluorescent probes are based on structures such as hydrazone bonds, imine bonds, or acetals. Although they can respond to pH changes, their fluorescence reversibility is insufficient and cannot meet the needs of real-time, accurate, and rapid imaging diagnosis of tumors. In addition, the expression levels of enzymes in different types of cancer cells vary greatly, which limits the application of existing enzyme-activated fluorescent probes in a wide range of malignant tumors.

[0004] Among them, β-carboline alkaloids are a large class of naturally occurring indole alkaloids with a planar tricyclic ring of pyridine [3,4-b] indole, which gives them good fluorescence imaging potential. The nitrogen-containing heterocyclic ring gives them stronger protonation ability and enhances their sensitivity to pH.

[0005] The present invention designs a novel pH-sensitive fluorescent probe by modifying β-carboline. This probe utilizes the special structure of the β-carboline molecule to achieve reversible conversion of fluorescence signals under different pH conditions, thereby realizing real-time monitoring of the tumor microenvironment. Compared with traditional probes, this improved probe has higher fluorescence signal response sensitivity and better fluorescence reversibility, and can more accurately reflect the changes in acid-base balance inside tumor cells. In addition, the improved probe described in the present invention is not only dependent on the expression level of the enzyme, but is based on the unique metabolic characteristics of tumor cells, such as pH imbalance caused by aerobic glycolysis. Therefore, regardless of the type of tumor, this novel probe can be widely used in imaging diagnosis of various types of cancer, providing clinicians with a powerful tool to accurately monitor the biological activity and treatment response of tumors. In short, the present invention breaks through the limitations of traditional pH-sensitive fluorescent probes by structural modification of β-carboline, has broad application prospects, and is expected to become an important innovation in the field of tumor imaging diagnosis in the future.

[0006] The present invention aims to solve several key problems existing in current pH-sensitive fluorescent probes in tumor imaging diagnosis by modifying β-carboline. Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and propose a class of pH-sensitive carboline pyridine fluorescent probes and their preparation method and application, which have medical uses for selective fluorescence imaging of tumors in vivo and in vitro through pH sensitivity to guide surgical resection and / or drug treatment, and thus have important application significance for the rapid diagnosis and treatment of cancer.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A class of pH-sensitive carboline pyridine fluorescent probes having a structure shown in general formula Ⅰ:

[0010]

[0011] Wherein, R is selected from one of H and NH2.

[0012] Preferably, R═H, R═NH 2 .

[0013] Table 1 Codes of some compounds of general formula I and their corresponding structures

[0014]

[0015] The compound codes of the above general formula I and their corresponding compound names are as follows:

[0016] I1: (E)-1,9-dimethyl-3-(2-(pyridin-4-yl)vinyl)-9H-pyrido[3,4-b]indole;

[0017] I2: (E)-1,9-dimethyl-3-(2-(pyridin-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-amine.

[0018] Another object of the present invention is to provide a method for preparing a pH-sensitive carboline pyridine fluorescent probe, comprising the following steps:

[0019] Step 1, 1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carbaldehyde (1) and 4-methylpyridine (2) are heated to reflux in the presence of a catalytic amount of piperidine to undergo Knoevenagel condensation reaction to obtain compound I1;

[0020] Step 2, 1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carboxaldehyde (3) and 4-methylpyridine (2) are heated under reflux in the presence of a catalytic amount of piperidine to undergo a Knoevenagel condensation reaction to obtain compound 4; compound 4 is heated under reflux under the catalysis of iron powder and ammonium chloride to undergo reduction to obtain compound I2;

[0021] Its synthetic route is as follows:

[0022]

[0023] The present invention also provides a use of the pH-sensitive carboline pyridine fluorescent probe in preparing a pH-responsive reagent for achieving selective fluorescent imaging of tumor tissues or tumor cells.

[0024] Preferably, the fluorescent probe is a fluorescent probe that is formulated into a solution and can be sprayed or locally injected to achieve rapid, real-time detection and imaging of tumors.

[0025] Preferably, the preparation method of the solution is as follows:

[0026] The method uses H2O as a solvent, 1,2-propylene glycol, DMSO or ethanol as a cosolvent, and Tween 20, Tween 40 or Tween 80 as a surfactant; wherein the volume percentage of 1,2-propylene glycol is 1-30%, the volume percentage of DMSO is 1-30%, the volume percentage of ethanol is 1-30%, the volume percentage of Tween 20 is 1-30%, the volume percentage of Tween 40 is 1-30%, and the volume percentage of Tween 80 is 1-30%.

[0027] Preferably, pH-sensitive fluorescence imaging of tumor cells and tissues can be achieved.

[0028] Preferably, the fluorescent probe solution is sprayed or locally injected on the tumor lesion site and surrounding tissues before or during surgery, and the tumor lesion tissue is rapidly and selectively imaged and tracked using a fluorescent laparoscope or in vivo imager to guide surgery and / or drug treatment.

[0029] Preferably, the tumor is liver cancer, colon cancer, breast cancer or lung cancer.

[0030] By adopting the above technical solution: utilizing the ICT principle, activating in the acidic microenvironment of tumor tissue, selectively and rapidly generating pH-sensitive near-infrared fluorescence at the tumor site, the specific implementation method is to spray or locally inject the solution of the compound of the present invention on the tumor lesion site and surrounding tissues before or during surgery, and use a fluorescent laparoscope or in vivo imager to perform rapid and selective fluorescence imaging and tracing of the tumor lesion tissue, which has high tumor tissue fluorescence imaging selectivity and low background fluorescence interference, and can accurately diagnose tumors to guide surgery and / or drug treatment.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention designs a pH-sensitive fluorescent probe by modifying β-carboline. This probe utilizes the special structure of the β-carboline molecule to achieve reversible conversion of fluorescence signals under different pH conditions, thereby achieving real-time monitoring of the tumor microenvironment. Compared with traditional probes, this improved probe has higher fluorescence signal response sensitivity and better fluorescence reversibility, and can more accurately reflect the changes in acid-base balance inside tumor cells.

[0033] 2. The improved probe prepared by the present invention does not rely solely on the expression level of the enzyme, but is based on the unique metabolic characteristics of tumor cells, such as pH imbalance caused by aerobic glycolysis. Therefore, regardless of the tumor type, this probe can be widely used in imaging diagnosis of various types of cancer, providing clinicians with a powerful tool to accurately monitor the biological activity and treatment response of tumors.

[0034] 3. The present invention breaks through the limitations of traditional pH-sensitive fluorescent probes by modifying the structure of β-carboline, has broad application prospects, and is expected to become an important innovation in the field of tumor imaging diagnosis in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The ultraviolet absorption spectra of the fluorescent probe compound I1 of the present invention at different pH values ​​are shown in Figure 1, where the abscissa is the wavelength and the ordinate is the absorbance value;

[0036] Figure 2 The fluorescence emission spectra of the fluorescent probe compound I1 of the present invention at different pH values ​​are shown in the figure, where the abscissa is the wavelength and the ordinate is the fluorescence intensity;

[0037] Figure 3 The fluorescence emission spectra of the fluorescent probe compound I2 at different pH values ​​of the present invention are shown in the figure, where the abscissa is the wavelength and the ordinate is the fluorescence intensity;

[0038] Figure 4 Schematic diagram of the application of compound I1 of the present invention to achieve selective fluorescence imaging of tumor cells in vivo and in vitro. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0040]

[0041] Example 1: Preparation of (E)-1,9-dimethyl-3-(2-(pyridin-4-yl)vinyl)-9H-pyrido[3,4-b]indole (I1)

[0042] 1,9-Dimethyl-9H-pyrido[3,4-b]indole-3-carbaldehyde (500 mg, 1.0 mmol) and 4-methylpyridine (1.0 mmol) were added into a single-necked bottle and dissolved with anhydrous ethanol (10 ml). Then, 1-2 drops of piperidine were added and refluxed at 80°C for 12 h. After the reaction was completed as monitored by TLC, the mixture was filtered and purified by recrystallization to obtain compound I1 with a yield of 83%.

[0043] (I1) Spectral data is: 1 H NMR (400MHz, DMSO) δ8.72-7.53(m,4H,4ArH),7.52(d,J=15.9Hz,1H,CH=),7.58(d,J=15.6H z,1H,CH=),7.53(s,1H,ArH),7.36-6.98(m,4H,4ArH),3.79(s,3H,CH3),2.79(s,3H,CH3).

[0044] Example 2: Preparation of (E)-1,9-dimethyl-3-(2-(pyridin-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-amine (I2)

[0045] Referring to the synthesis method of (I1) in Example 1, 1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carboxaldehyde was used instead of 1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carboxaldehyde to obtain compound 4. Compound 4 (496 mg, 1.0 mmol), iron powder (4.0 mmol) and ammonium chloride (523 mg, 8.0 mmol) were added to a single-necked bottle, dissolved with anhydrous ethanol (20 ml), refluxed at 80° C. for 5 h, and after the reaction was completed as monitored by TLC, the reaction was filtered, the filtrate was spin-dried, and purified by column chromatography to obtain compound I2 with a yield of 75%.

[0046] (I2) Spectral data is: 1 H NMR (400MHz, DMSO) δ8.72-7.53(m,4H,4ArH),7.52(d,J=15.9Hz,1H,CH=),7.50(d,J=15.6Hz,1H,CH =),7.33(s,1H,ArH),7.23-6.87(m,3H,3ArH),4.32(s,2H,NH2),3.82(s,3H,CH3),2.89(s,3H,CH3).

[0047] Example 3: Ultraviolet absorption spectrum test of the fluorescent probe under different pH conditions of the present invention

[0048] The fluorescent compound of the present invention was dissolved in a 50% ethanol aqueous solution to prepare a test solution with a pH of 4.5-7.4 and a concentration of 10 μM. The ultraviolet absorption spectrum data was tested using an ultraviolet-visible spectrophotometer. The results showed that the ultraviolet maximum absorption wavelength of the fluorescent compound of the present invention was in the range of 450-600 nm. The ultraviolet absorption peak of compound I1 at around 532 nm increased as the pH decreased ( Figure 1 ).

[0049] Example 4: Fluorescence spectroscopy test of pH response of some compounds of the present invention

[0050] The fluorescent compound of the present invention was dissolved in a 50% ethanol aqueous solution to prepare a detection solution with a pH of 4.5-7.4. The fluorescence emission spectrum data was tested using a fluorescence spectrometer. The results showed that the maximum emission wavelength of the fluorescent compound of the present invention was in the range of 650-750nm. The fluorescence peak of compound I1 at around 632nm increased as the pH decreased, and the fluorescence signal at pH = 4.56 was significantly higher than the fluorescence value at pH = 7.42 ( Figure 2 ), a similar phenomenon was also observed for compound I2 at 650nm where the fluorescence peak increased with decreasing pH ( Figure 3 ).

[0051] Example 5: Cell imaging using confocal microscopy

[0052] Reference Figure 4 , cell imaging was performed using a confocal microscope. One day before imaging, A549 cells, 4T1 cells or LO2 cells were cultured in DEME or 1640 culture medium and placed in a laser confocal dish. Then 1-25 μM of the test compound was added to the cells and placed in a cell culture incubator for half an hour. The incubated cells were placed on the stage of a confocal microscope for confocal fluorescence imaging, and the excitation wavelength of the test compound was set to: λem=450-500nm, λex=600-640nm.

[0053] Cell imaging shows that the compound of the present invention can selectively perform fluorescence imaging on multiple tumor cells, while the fluorescence imaging on normal liver cells is very weak, thus providing a feasible means for in vivo and in vitro tumor tissue or cell imaging research.

[0054] In summary, the present invention designs a pH-sensitive fluorescent probe by modifying β-carboline. This probe utilizes the special structure of the β-carboline molecule to achieve reversible conversion of fluorescence signals under different pH conditions, thereby achieving real-time monitoring of the tumor microenvironment. Compared with traditional probes, this improved probe has higher fluorescence signal response sensitivity and better fluorescence reversibility, and can more accurately reflect the changes in acid-base balance inside tumor cells.

[0055] The description and practice disclosed in the present invention are easy to think and understand for ordinary technicians in the technical field, and several improvements and modifications can be made without departing from the principles of the present invention. Therefore, modifications or improvements made without departing from the spirit of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A pH-sensitive carboline pyridine fluorescent probe, characterized in that: It has the structure shown in general formula I: Wherein, R is selected from one of H and NH2.

2. The method for preparing a pH-sensitive carboline pyridine fluorescent probe according to claim 1, characterized in that: The following steps are involved: Step 1, 1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carbaldehyde (1) and 4-methylpyridine (2) are heated to reflux in the presence of a catalytic amount of piperidine to undergo Knoevenagel condensation reaction to obtain compound I1; Step 2, 1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carboxaldehyde (3) and 4-methylpyridine (2) are heated under reflux in the presence of a catalytic amount of piperidine to undergo a Knoevenagel condensation reaction to obtain compound 4; compound 4 is heated under reflux under the catalysis of iron powder and ammonium chloride to undergo reduction to obtain compound I2; Its synthetic route is as follows:

3. Use of the pH-sensitive carboline pyridine fluorescent probe according to claim 1 in preparing a reagent for pH response and achieving selective fluorescence imaging of tumor tissues or tumor cells.

4. The use according to claim 3, characterized in that: The fluorescent probe is prepared into a solution and can realize rapid and real-time detection and imaging of tumors by spraying or local injection.

5. The use according to claim 4, characterized in that: The preparation method of the solution is specifically as follows: The method uses H2O as a solvent, 1,2-propylene glycol, DMSO or ethanol as a cosolvent, and Tween 20, Tween 40 or Tween 80 as a surfactant; wherein the volume percentage of 1,2-propylene glycol is 1-30%, the volume percentage of DMSO is 1-30%, the volume percentage of ethanol is 1-30%, the volume percentage of Tween 20 is 1-30%, the volume percentage of Tween 40 is 1-30%, and the volume percentage of Tween 80 is 1-30%.

6. The use according to claim 3, characterized in that: It can realize pH-sensitive fluorescence imaging of tumor cells and tissues.

7. The use according to claim 4, characterized in that: The fluorescent probe solution is sprayed or locally injected on the tumor lesion site and surrounding tissues before or during surgery, and the tumor lesion tissue is quickly and selectively imaged and tracked using a fluorescent laparoscope or in vivo imager to guide surgery and / or drug treatment.

8. The use according to any one of claims 3 to 7, characterized in that: The tumor is liver cancer, colon cancer, breast cancer or lung cancer.

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