pH-responsive iminocarboline quinolinium compound and preparation method and application thereof

Abstract

The present invention belongs to the field of medicine, and discloses a pH-responsive iminocarboline quinolinium compound and a preparation method and application thereof. The iminocarboline quinolinium compound disclosed in the present invention has the following structure: #imgabs0# The iminocarboline quinolinium compound can induce the cleavage of the imine bond in the acidic microenvironment of the tumor, release the β-carboline quinolinium fluorescent compound, realize the switching mode fluorescence, and can be applied to the preparation of tumor microenvironment selective fluorescent imaging reagents or tumor diagnostic reagents.

Description

Technical Field

[0001] The invention belongs to the field of medicines and relates to a pH-responsive iminocarboline quinolinium compound and a preparation method and application thereof. Background Art

[0002] In recent years, more and more studies have shown that the occurrence, growth and metastasis of tumors are related to the tumor microenvironment. The tumor microenvironment refers to the special environment that is conducive to tumor growth due to the interaction between tumor cells and extracellular matrix and various cells and molecules during the growth of the tumor.

[0003] Compared with normal tissues, the tumor microenvironment has obvious acidic characteristics. Glycolysis disorders in cancer cells lead to high lactic acid levels in tumor tissues, resulting in acidic pH values. There are many methods for detecting pH, such as the traditional glass electrode method, weak base weak acid step method, nuclear magnetic resonance method and atomic absorption spectrometry, but most of them have poor sensitivity, low selectivity and complex operation, and cannot be used in real-time diagnosis of tumors. In recent years, due to the high sensitivity and low cost of fluorescence spectroscopy, it has attracted more and more attention. Fluorescence molecular imaging relies on specific fluorescent molecular probes to label specific molecules or cells, thereby generating corresponding fluorescence under the excitation of external light sources, and its spatial resolution can be as high as millimeter level. In addition, fluorescence imaging technology also has the advantages of high efficiency, sensitivity, affordability and no ionizing radiation. It can be used to study a series of physiological and pathological changes occurring in tumors at the molecular, cellular and tissue levels, providing an effective means for early diagnosis of tumors in clinical practice.

[0004] β-Carboline compounds are a large class of naturally occurring indole alkaloids. Their structure is similar to the planar tricyclic framework of pyridoindole of carbazole. They have certain fluorescence properties and can be used to study and develop fluorescent probes. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a pH-responsive iminocarbolinequinolinium compound and a preparation method and application thereof. The iminocarbolinequinolinium compound can induce the cleavage of the imine bond in the acidic microenvironment of the tumor, release a β-carbolinequinolinium fluorescent compound, emit fluorescence, and realize the positioning and imaging of the tumor. It can be applied to pH-responsive fluorescent probes and can be applied to the preparation of tumor microenvironment selective fluorescent imaging reagents or tumor diagnostic reagents.

[0006] The present invention provides a pH-responsive iminocarboline quinolinium compound or a pharmaceutically acceptable salt thereof, wherein the structure of the iminocarboline quinolinium compound is as shown in the following formula:

[0007]

[0008] Wherein, R is one of H, OH and OMe.

[0009] Furthermore, the iminocarboline quinolinium compound has a structure shown in the general formula I1 or I2:

[0010] I1:

[0011] I2:

[0012] The present invention also provides a method for preparing an iminocarboline quinolinium compound, which specifically comprises: dissolving ((E)-4-(2-(6-amino-9-methyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodine salt (1) and 2-R-benzaldehyde (2) in anhydrous methanol containing an appropriate amount of HAc, refluxing for 2 to 5 hours, concentrating under reduced pressure, and separating by column chromatography to obtain an iminocarboline quinolinium compound I;

[0013] The synthetic route of the preparation method is as follows:

[0014]

[0015] Wherein, R is one of H, OH and OMe.

[0016] The present invention also provides a pH-responsive fluorescent probe, which is the above-mentioned iminocarbolinequinolinium compound or a pharmaceutically acceptable salt thereof, and is a fluorescent probe whose fluorescence intensity increases as the acidic pH value decreases.

[0017] The present invention also provides the use of the above-mentioned iminocarboline quinolinium compound or a pharmaceutically acceptable salt thereof in the preparation of a selective fluorescent imaging agent for tumor microenvironment or tumor metastasis site.

[0018] Furthermore, in the above application, the tumor microenvironment selective fluorescence imaging agent is an agent that achieves selective fluorescence imaging of the tumor microenvironment through single-photon and / or two-photon excitation.

[0019] The present invention also provides the use of the above-mentioned iminocarboline quinolinium compound or a pharmaceutically acceptable salt thereof in a tumor diagnostic reagent.

[0020] Furthermore, in the above application, the tumor is one of colon cancer, breast cancer, lung cancer, cervical cancer, bladder cancer and gastric cancer.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The invention discloses a type of beta-carboline quinolinium in the form of quaternary ammonium salts. Different from conventional quaternary ammonium salt-type quinolinium salt compounds (which are always on under excitation light, have no "on-off" mode fluorescence, and have no tumor selective fluorescence imaging), the invention introduces a pH-sensitive aromatic imine fragment at the 6-position of beta-carboline quinolinium to form an imine carboline quinolinium compound; the compound of the invention has a fluorescence intensity that continuously increases with decreasing pH value, can generate pH-sensitive fluorescence, and realizes "on-off" mode fluorescence; can be activated in an acidic microenvironment of tumor tissue, and can selectively generate acidic pH-activated fluorescence at a tumor site by single-photon and / or two-photon excitation using the ICT principle, has high tumor tissue fluorescence imaging selectivity and low background fluorescence interference, can accurately diagnose tumors, and guide tumor surgery and / or drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the ultraviolet absorption spectrum of compound I1 prepared in Example 1 of the present invention at different pH values;

[0024] Figure 2 is the fluorescence spectrum of compound I1 prepared in Example 1 of the present invention at different pH values;

[0025] Figure 3 is the fluorescence spectrum of compound I2 prepared in Example 2 of the present invention at different pH values;

[0026] Figure 4 is a two-photon absorption cross-sectional diagram of compound I1 in Example 1 of the present invention at pH 4.0;

[0027] Figure 5 This is a fluorescence imaging image of the compound I1 in Example 1 of the present invention on tumor cells HT29;

[0028] Figure 6 This is a fluorescence imaging diagram of the compound I1 in Example 1 of the present invention on normal intestinal epithelial cells CCD841;

[0029] Figure 7 This is a fluorescence imaging diagram of the tumor cell Mcf-7 by compound I2 in Example 2 of the present invention;

[0030] Figure 8 This is a fluorescence imaging diagram of the compound I2 in Example 2 of the present invention on tumor cells A549;

[0031] Fig. 9 This is the fluorescence imaging diagram of the compound I2 in Example 2 of the present invention on normal cells HFL-1. DETAILED DESCRIPTION

[0032] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention, and should not be understood as limiting the present invention.

[0033] Example 1: Preparation of 4-((E)-2-(6-(((E)-2-hydroxybenzylidene)amino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodine salt (I1)

[0034] Dissolve (E)-4-(2-(6-amino-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodide (445.59 mg, 1.0 mmol) and 2-hydroxybenzaldehyde (122.12 mg, 1.0 mmol) in anhydrous methanol (10 ml), add 1-2 drops of acetic acid, reflux for 2 hours, and after the reaction is completed as monitored by TLC, concentrate under reduced pressure and perform column chromatography. The yield is 72%.

[0035] The spectrum data is: 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H,OH),8.93(s,1H,HC=N),8.71(d,J=15.7Hz,1H,C=CH),8.47(d,J= 8.5Hz,1H,ArH),8.32-8.26(m,2H,2ArH),8.23(d,J=8.2Hz,1H,ArH),8.15(d,J=8.9Hz,1H,ArH),7.99 (d,J=15.7Hz,1H,C=CH),7.84-7.80(m,1H,ArH),7.74-7.70(m,2H,2ArH),7.65-7.62(m,2H,2ArH),7. 41-7.38(m,2H,2ArH),7.29-7.27(m,1H,ArH),7.19-7.16(m,1H,ArH),4.26(s,3H,CH3),3.06(s,3H,CH 3) ,2.94(s,3H,CH3),2.07(s,6H,2CH3).

[0036] Example 2: Preparation of 4-((E)-2-(6-(((E)-benzyl)amino)-9-methyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodine salt (I2)

[0037] Dissolve (E)-4-(2-(6-amino-9-methyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodide (445.59 mg, 1.0 mmol) and benzaldehyde (122.12 mg, 1.0 mmol) in anhydrous methanol (10 ml), add 1-2 drops of acetic acid, reflux for 2 hours, and after the reaction is completed as monitored by TLC, concentrate under reduced pressure and perform column chromatography. The yield is 78%.

[0038] The spectrum data is: 1 H NMR (400MHz, DMSO-d6) δ11.21(s,1H,NH),8.81(s,1H,HC=N),8.70(d,J=15.6Hz,1H,C=CH) ,8.36-8.22(m,3H,3ArH),8.13(d,J=8.3Hz,1H,ArH),8.02(d,J=8.9Hz,1H,ArH),7.86-7.6 2(m,2H,2ArH),7.54-7.48(m,1H,ArH),7.32(d,J=15.6Hz,1H,C=CH),7.22-7.14(m,2H,2A rH),7.10-7.06(m,2H,2ArH),7.00-6.88(m,2H,2ArH),4.26(s,3H,CH3),3.15(s,3H,CH3).

[0039] Example 3: UV absorption spectrum test

[0040] The compound I1 prepared in Example 1 of the present invention was dissolved in a 5% DMSO aqueous solution to prepare a test solution with a pH of 3.6-7.4. The ultraviolet absorption spectrum data of the compound I1 under different pH conditions was tested using an ultraviolet-visible spectrophotometer. The results are as follows: Figure 1 As shown. The results show that the maximum ultraviolet absorption wavelength of the fluorescent compound of the present invention is in the range of 450-550nm. The ultraviolet absorption peak of the compound decreases as the pH decreases. The results show that the compound of the present invention has a pH-sensitive ultraviolet absorption spectrum.

[0041] Example 4: Fluorescence emission spectrum test

[0042] The compounds I1 and I2 prepared in Example 1 of the present invention were dissolved in a 5% DMSO aqueous solution to prepare a test solution with a pH of 3.6-7.6. The fluorescence emission spectrum data under different pH conditions were tested using a fluorescence spectrometer. The results are as follows: Figure 2 and Figure 3 The results show that the maximum emission wavelength of the fluorescent compound of the present invention is in the range of 550-680 nm. The intensity of the fluorescence peak in the range of 550-680 nm increases with the decrease of pH value ( Figure 2 and Figure 3 ). The results show that the compounds of the present invention have significant pH-sensitive fluorescence properties.

[0043] Example 5: Using femtosecond fluorescence measurement technology to detect two-photon absorption cross sections at different wavelengths

[0044] The compound I1 of the present invention was dissolved in PBS buffer at pH = 4.0 (5 μM), and the compound I1 of the present invention and the reference compound Ru(bpy)3 were tested. 2+ The fluorescence intensity under two-photon excitation from 900nm to 1100nm. The two-photon absorption cross section of the reduction product at different wavelengths is calculated using the formula: δ = δr × (Fs × фr × nr) / (Fr × фs × ns). The results are as follows Figure 3 As shown, where δ, F, ф and n are two-photon absorption cross section, spectral integrated area, quantum yield and concentration respectively; s and r represent the compound of the present invention and the control compound respectively. The results show that the compound I1 of the present invention has the largest two-photon absorption cross section (δmax=115GM) at 1000nm ( Figure 4 ).

[0045] Example 6: Cell imaging using confocal microscopy

[0046] Fluorescence imaging of human lung cancer cells (A549), colon cancer cells (HT29), breast cancer cells (Mcf-7), and normal human colon epithelial cells (CCD841) or human embryonic lung fibroblasts (HFL-1) was performed using a confocal microscope. Figure 5-9 Specifically, after the cells were cultured in a laser confocal dish for 24 hours, 1-10 μM of the compound of the present invention was added to the cells, and the cells were placed in a cell culture incubator at 37°C and 5% CO2 for half an hour. Then, after washing three times with a phosphate buffer solution of pH=7.4, 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=450-600nm, λex=550-680nm.

[0047] from Figure 5-9 The cell imaging results in the experiment show that the compounds of the present invention can selectively illuminate multiple tumor cells and produce significant fluorescence imaging of tumor cells, while the fluorescence imaging of normal human colon epithelial cells and human embryonic lung fibroblasts is very weak, with almost no fluorescence visible. This indicates that the compounds of the present invention can selectively perform imaging diagnosis on a variety of human tumor cells, providing a feasible means for in vivo and in vitro tumor tissue or cell imaging studies.

[0048] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A pH-responsive iminocarboline quinolinium compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the iminocarboline quinolinium compound is shown below: Wherein, R is H or OH.

2. The iminocarbolinequinolinium compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The iminocarboline quinolinium compound has a structure shown in the general formula I1 or I2: I1： I2： 3. A method for preparing an iminocarboline quinolinium compound, characterized in that: The preparation method is specifically as follows: dissolving ((E)-4-(2-(6-amino-9-methyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodine salt (1) and 2-R-benzaldehyde (2) in anhydrous methanol containing an appropriate amount of HAc, refluxing for 2 to 5 hours, concentrating under reduced pressure, and separating by column chromatography to obtain iminocarboline quinolinium compound I; The synthetic route of the preparation method is shown in the figure below: Wherein, R is H or OH.

4. A pH-responsive fluorescent probe, characterized in that: The pH-responsive fluorescent probe is the iminocarbolinequinolinium compound or a pharmaceutically acceptable salt thereof according to claim 1, and the pH-responsive fluorescent probe is a fluorescent probe whose fluorescence intensity increases as the acidic pH value decreases.

5. Use of the iminocarboline quinolinium compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a selective fluorescent imaging agent for tumor microenvironment or tumor metastasis site.

6. The use according to claim 5, characterized in that: The selective fluorescent imaging agent is an agent that realizes selective fluorescent imaging of tumor microenvironment or tumor metastasis site through single-photon and / or two-photon excitation.

7. Use of the iminocarbolinequinolinium compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a reagent for tumor diagnosis.

8. The use according to any one of claims 5 to 7, characterized in that: The tumor is one of colon cancer, breast cancer, lung cancer, cervical cancer, bladder cancer and gastric cancer.

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