Tumor acidic pH response type carboline naphthalene indole fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a tumor acidic pH response type carboline naphthalene indole fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The carboline naphthalene indole fluorescent probe has a structure as shown in a general formula I: # imgabs0 #, wherein R is selected from one of H, NO2 and NH2. The carboline naphthyl indole fluorescent probe can be specifically activated in a tumor acidic environment, and can generate a pH response type near-infrared fluorescence signal at a tumor part.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a class of tumor acidic pH-responsive porphyrin naphthalene indole fluorescent probes, their preparation methods and applications. Background Art

[0002] Surgical resection remains the preferred option for the treatment of solid tumors. However, traditional surgeries face difficulties in identifying tumor boundaries and are prone to missing small lesions. Therefore, developing new imaging agents to assist intraoperative navigation is of great significance: precise localization of tumor boundaries and identification of small metastatic foci can be achieved through real-time fluorescence imaging, thereby improving the surgical accuracy and reducing the risk of postoperative recurrence, providing strong guarantees for improving the prognosis of patients.

[0003] Tumor fluorescent probes are a class of molecular probes with specific recognition functions, capable of achieving precise detection and visualization imaging of tumor tissues both in vitro and in vivo. These probes generate detectable fluorescence signals by specifically recognizing tumor-related biomarkers (such as surface receptors, metabolites, or microenvironmental characteristics), providing important bases for the localization diagnosis of tumors.

[0004] However, traditional fluorescent probes designed based on hydrazone bonds, imine bonds, or acetal structures, although having pH-responsive properties, have limitations such as poor fluorescence reversibility and slow response speed, and are difficult to meet the requirements of real-time dynamic imaging of tumors. At the same time, due to significant differences in enzyme expression levels among different types of cancer cells, the application scope of existing enzyme-activated fluorescent probes in the diagnosis and treatment of malignant tumors is greatly restricted. These limitations have prompted researchers to continuously explore new design strategies for fluorescent probes to overcome the deficiencies of existing technologies. Summary of the Invention

[0005] In view of the deficiencies of existing fluorescent probes, the present invention has developed a class of tumor acidic pH-responsive porphyrin naphthalene indole fluorescent probes, their preparation methods and applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided a class of tumor acidic pH-responsive porphyrin naphthalene indole fluorescent probes, and the porphyrin naphthalene indole fluorescent probes have the structure shown in General Formula Ⅰ:

[0008]

[0009] Wherein, R is selected from one of H, NO2, and NH2, and the corresponding compound codes and structures are shown in Table 1.

[0010] Table 1 Compound codes and corresponding structures of partial compounds in General Formula Ⅰ

[0011]

[0012] The codes of some compounds of the above general formula I and their corresponding compound names are as follows:

[0013] I 1： (E)-2-(2-(1,9-Dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylbenzo[cd]indol-1-ium.

[0014] I 2： (E)-2-(2-(1,9-Dimethyl-6-nitro-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylbenzo[cd]indol-1-ium.

[0015] I 3： (E)-2-(2-(1,9-Dimethyl-6-amino-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylbenzo[cd]indol-1-ium.

[0016] In some embodiments of the present invention, a preparation method of the above-mentioned carboline naphthalene indole fluorescent probe is provided. The preparation method is as follows: Compound 1 (1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carbaldehyde or 1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carbaldehyde) and Compound 2 (1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carbaldehyde) are heated under reflux in the presence of a catalytic amount of piperidine to undergo a Knoevenagel condensation reaction to obtain Compound I, where R is selected from H or NO2. (When R = H, it is Compound I1; when R = NO2, it is Compound I2)

[0017] In some embodiments of the present invention, a preparation method of the above-mentioned carboline naphthalene indole fluorescent probe is provided, where R represents NH2. The preparation method includes the following steps:

[0018] S1. 1,9-Dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carbaldehyde and 1,2-dimethylbenzo[cd]indol-1-ium are heated under reflux in the presence of a catalytic amount of piperidine to undergo a Knoevenagel condensation reaction to obtain Compound I2;

[0019] S2. Compound I2 is reduced by heating under reflux in the presence of iron powder and ammonium chloride to obtain Compound I3.

[0020] In the present invention, the synthesis route of the preparation process of the carboline naphthalene indole fluorescent probe is shown by the following formula:

[0021]

[0022] In a third aspect of the present invention, there is provided an application of the above-mentioned carboline naphthalene indole fluorescent probe in the preparation of a pH-responsive fluorescent imaging reagent.

[0023] In some embodiments of the present invention, the fluorescent imaging reagent is used for selective fluorescent imaging in tumor tissues or tumor cells.

[0024] In some embodiments of the present invention, the fluorescent imaging reagent is used in the following manner: the above-mentioned fluorescent imaging reagent is configured into a solution and then sprayed or locally injected. Specifically, the solution containing the fluorescent probe is used to achieve rapid and real-time detection and imaging of tumors by spraying or local injection.

[0025] In some embodiments of the present invention, the solution containing the above-mentioned fluorescent probe is applied to the tissues at and around the tumor lesion site before or during surgery, and with the aid of a fluorescence endoscope or in vivo imaging system, precise localization and real-time dynamic tracing of tumor tissues are achieved to guide surgery and / or drug treatment.

[0026] In some embodiments of the present invention, the solution containing the above-mentioned fluorescent probe is prepared from the above-mentioned fluorescent imaging reagent, a solvent, a co-solvent, and a surfactant.

[0027] In some embodiments of the present invention, in the solution containing the above-mentioned fluorescent probe, the solvent is H2O, the co-solvent is selected from 1,2-propanediol, DMSO or ethanol, and the surfactant is selected from Tween 20, Tween 40 or Tween 80.

[0028] Preferably, the tumor is liver cancer, colon cancer, breast cancer or lung cancer.

[0029] The fluorescent probe provided by the present invention is based on the ICT (intramolecular charge transfer) principle and is specifically activated in the acidic microenvironment unique to tumor tissues, and can generate a pH-responsive near-infrared fluorescent signal at the tumor site. By locally spraying the carboline naphthalene indole compound solution on the lesion area and with the aid of a fluorescence endoscope or in vivo imaging system, precise localization and real-time dynamic tracing of tumor tissues are achieved. This novel fluorescent probe has the characteristic of specific response to the tumor microenvironment, can remain stable under physiological pH conditions, and is rapidly activated in the acidic tumor microenvironment to generate a significant near-infrared fluorescent signal, thus providing a highly sensitive imaging method for the visual detection of tumor lesions.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. By modifying the structure of the β-carboline skeleton, the present invention develops a novel pH-responsive fluorescent probe. The probe realizes the reversible switching of the fluorescent signal in the acidic microenvironment of tumors, and its pH-responsive fluorescence is improved compared with traditional probes, and can selectively light up tumor cells.

[0032] 2. The present invention breaks through the limitations of traditional enzyme-activated probes. Its mechanism of action is based on the pH imbalance characteristics caused by aerobic glycolysis (Warburg effect) commonly present in tumor cells, rather than relying on the expression level of specific enzymes. Therefore, this acidic pH-activated probe has broad applicability to multiple tumors and can be widely used for selective fluorescence imaging of various malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the ultraviolet absorption spectrum diagram of partial compound I2 of the fluorescence probe of the present invention at different pH values;

[0034] Figure 2 It is the fluorescence emission spectrum diagram of partial compound I1 of the fluorescence probe of the present invention at different pH values;

[0035] Figure 3 It is the fluorescence emission spectrum diagram of partial compound I2 of the fluorescence probe of the present invention at different pH values;

[0036] Figure 4 It is the fluorescence emission spectrum diagram of partial compound I3 of the fluorescence probe of the present invention at different pH values;

[0037] Figure 5 It is the application schematic diagram of selective fluorescence imaging of in vitro and in vivo tumor cells by compound I1 of the present invention;

[0038] Figure 6 It is the application schematic diagram of selective fluorescence imaging of in vitro and in vivo tumor cells by compound I2 of the present invention;

[0039] Figure 7 It is the application schematic diagram of selective fluorescence imaging of in vitro and in vivo tumor cells by compound I3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus make 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, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention.

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

[0042] Dissolve the 1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carbaldehyde compound 1a (220 mg, 1.0 mmol) and 1,2-dimethylbenzo[cd]indol-1-ium in absolute ethanol (574.07 mg, 1.0 mmol), add 0.1 mL of piperidine, reflux at 85 °C for 12 hours. After monitoring the reaction by TLC until completion, filter by suction and purify by recrystallization to obtain compound I1 with a yield of 77%.

[0043] (I1) The spectral data are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 7.6 Hz, 1H, ArH), 8.13 (m, 2H, ArH), 7.76 (m, 2H, ArH), 7.63 (m, 2H, ArH), 7.51 (s, 1H, ArH), 7.46 (d, J = 15.2 Hz, 2H, CH), 7.33 - 7.39 (m, 2H, ArH), 3.98 (s, 3H, CH3), 3.39 (s, 3H, CH3), 2.83 (s, 3H, CH3).

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

[0045] Dissolve 1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carbaldehyde 1b (270 mg, 1.0 mmol) and 1,2-dimethylbenzo[cd]indol-1-ium in absolute ethanol (574.07 mg, 1.0 mmol), add 0.1 mL of piperidine, reflux at 85 °C for 12 hours. After monitoring the reaction by TLC until completion, filter by suction and purify by recrystallization to obtain I2 with a yield of 81%.

[0046] (I2) The spectral data are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 7.4 Hz, 1H, ArH), 8.07 (d, J = 8.0 Hz, 2H, ArH), 7.69 (d, J = 8.1 Hz, 2H, ArH), 7.60 (d, J = 7.4 Hz, 2H, ArH), 7.53 (s, 1H, ArH), 7.44 (d, J = 15.2 Hz, 2H, CH), 7.04 (s, 1H, CH), 3.95 (s, 3H, CH3), 3.36 (s, 3H, CH3), 2.75 (s, 3H, CH3).

[0047] Example 3: Preparation of (E)-2-(2-(1,9-dimethyl-6-amino-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylbenzo[cd]indol-1-ium (I3)

[0048] Compound I2 (200 mg, 0.36 mmol) prepared in Example 2 and iron powder (67.2 mg, 1.2 mmol) were added to a single-necked flask, dissolved in ethanol (5 mL) containing ammonium chloride, refluxed at 85 °C for 1 - 2 h. After the reaction was monitored by TLC and completed, it was filtered by suction and purified by column chromatography to obtain compound I3 with a yield of 67%.

[0049] (I3) Spectral data are as follows: 1 H NMR (400 MHz, DMSO) δ 8.72 - 7.53 (m, 4H, 4ArH), 7.52 (d, J = 15.9 Hz, 1H, CH=), 7.50 (d, J = 15.6 Hz, 1H, CH=), 7.33 (s, 1H, ArH), 7.23 - 6.87 (m, 3H, 3ArH), 4.32 (s, 2H, NH2), 3.89 (s, 3H, CH3), 3.38 (s, 3H, CH3), 2.81 (s, 3H, CH3).

[0050] Example 4: UV absorption spectrum test of the fluorescent probe of the fluorescent compound I2 under different pH conditions

[0051] The fluorescent compound I2 of the present invention was dissolved in an ethanol aqueous solution containing 50% to prepare a detection solution with a pH of 3.5 - 7.4 and a concentration of 10 μM. The UV absorption spectrum data were measured using a UV-visible spectrophotometer, and the results are as Figure 1 shown. The results show that as the pH of the compound I2 of the present invention decreases, the absorption peak at the maximum UV absorption wavelength of 500 nm is significantly enhanced, and the peak value of the maximum absorption peak is enhanced by nearly a hundred times under decreasing pH.

[0052] Example 5: Fluorescence spectrum test of the pH response of the fluorescent compound of the present invention

[0053] The fluorescent compounds I1, I2, and I3 of the present invention were dissolved in an ethanol aqueous solution containing 50% to prepare a detection solution with a pH of 3.5 - 7.4. The fluorescence emission spectrum data were measured using a fluorescence spectrometer, and the results are as Figures 2 - 4 shown. Figure 2 is the fluorescence emission spectrum diagram of compound I1 of the fluorescent probe part of the present invention at different pH values; Figure 3 is the fluorescence emission spectrum diagram of compound I2 of the fluorescent probe part of the present invention at different pH values; Figure 4This is the fluorescence emission spectrum of Compound I3, a part of the fluorescent probe of the present invention, at different pH values. The results show that as the pH decreases, the compounds of the present invention gradually produce significant fluorescence peaks in the range of 650 - 690 nm, and among them, the fluorescence peak intensity of I2 is the strongest.

[0054] Example 6: Cell imaging was performed using a confocal microscope

[0055] For cell imaging using a confocal microscope, one day before imaging, tumor cells: HCT116 cells, A549 cells, Mcf cells or normal cells CCD841 cells, HFL1 cells were cultured in DEME or 1640 culture medium, placed in a laser confocal dish, and then 1 - 25 μM of the test compound was added to the cells. The cells were placed in an incubator for half an hour. The next day, the incubated cells were placed on the stage of the confocal microscope for confocal fluorescence imaging. The excitation wavelength of the test compound was set as: λex = 550 - 600 nm, λem = 650 - 690 nm. The fluorescence imaging diagram is as Figures 5 - 7 shown.

[0056] Cell imaging showed that the compounds of the present invention can selectively perform fluorescence imaging on multiple tumor cells (HCT116 cells, A549 cells, Mcf cells), while the fluorescence imaging of normal intestinal cells CCD841 and normal lung epithelial cells HFL1 is very weak, providing a feasible means for the study of selective fluorescence imaging of tumor tissues or cells in vivo and in vitro. It shows that the fluorescent probe provided by the present invention has the characteristic of specific response to the tumor microenvironment, can remain stable under physiological pH conditions, and can be rapidly activated in the acidic tumor microenvironment to produce significant near-infrared fluorescence signals.

[0057] According to the above test results, it can be seen that the present invention designed a tumor acidic 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 selective fluorescence imaging of tumor cells.

Claims

1. A class of tumor acidic pH-responsive porphyrin naphthalene indole fluorescent probes, characterized in that, The carbazole naphthalene indole-based fluorescent probe has the structure shown in General Formula I: Wherein, R is selected from one of H, NO2 and NH2.

2. A preparation method of the carbazole naphthalene indole fluorescent probe as described in claim 1, characterized in that, The preparation method is as follows: Compound 1 and Compound 2 are heated under reflux in the presence of a catalytic amount of piperidine to undergo a Knoevenagel condensation reaction to obtain Compound |; The synthetic route of the preparation method is as follows: Wherein, R is selected from H or NO2.

3. A preparation method of the carbazole naphthalene indole fluorescent probe as described in claim 1, characterized in that, When R represents NH2, the preparation method includes the following steps: S1. 1,9-Dimethyl-6-nitro-9H-pyrido[3,4-b]indole-3-carbaldehyde and 1,2-dimethylbenzo[cd]indol-1-ium undergo a Knoevenagel condensation reaction under reflux in the presence of a catalytic amount of piperidine to obtain Compound I2; The structure of Compound l2 is shown in the following formula: S2. Compound I2 is heated under reflux in the presence of iron powder and ammonium chloride as catalysts to obtain Compound I by reduction 3； 4. Use of a carbazole naphthalene indole-based fluorescent probe as described in claim 1 in the preparation of a pH-responsive fluorescent imaging reagent.

5. The application according to claim 4, characterized in that The fluorescent imaging reagent is used for selective fluorescent imaging in tumor tissues or tumor cells.

6. The application according to claim 5, wherein The tumor is liver cancer, colon cancer, breast cancer or lung cancer.

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