A near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, its preparation method and application

Abstract

The present invention relates to the field of fluorescent probe technology, and in particular to a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, as well as its preparation method and application. The present invention provides a fluorescent probe TM-N2 with good water solubility and near-infrared red light for detecting nitroreductase. The probe has good water solubility, a wavelength that can reach the near-infrared band, and a simple preparation method. The probe has high sensitivity and good selectivity. The probe has achieved detection of nitroreductase under hypoxic conditions in zebrafish and in tumor cells, and has broad application prospects in the fields of in vivo imaging analysis and tumor detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, and a preparation method and application thereof. Background Art

[0002] With current medical technology, cancer has become a leading cause of death. Cancer detection relies on late-stage tissue biopsies, which can lead to delayed detection. Therefore, developing methods for early diagnosis and identification of cancer is crucial. When substantial tumorigenesis develops in human tissue, the expression level of nitroreductase in tumor cells is directly correlated with the cells' hypoxic state. Therefore, monitoring tumor cells is essential, as they produce nitroreductase under hypoxic conditions. Detecting nitroreductase can confirm hypoxia and, in turn, help detect substantial tumorigenesis in vivo.

[0003] In recent years, the detection of the tumor marker nitroreductase has received widespread attention. Fluorescent probes have important applications in the detection of nitroreductase due to their high specificity, high sensitivity, and excellent biocompatibility. Currently, most probes for detecting nitroreductase have a wavelength of around 600 nm. Short-wavelength probes have poor biological penetration, while red light detection has good biological penetration and less damage. Secondly, other types of probes have poor water solubility and require more co-solvents to dissolve them. More co-solvents are more harmful to organisms, while probes with good water solubility can enter the body through water transport, causing less damage to organisms and not requiring large amounts of chemical reagents. Therefore, the present invention designs a near-infrared fluorescent probe for detecting nitroreductase with good water solubility, which is very important for achieving sensitive detection of nitroreductase. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, as well as its preparation method and application. The fluorescent probe has the characteristics of high sensitivity, good stability, and little damage to organisms. It has a high yield and is simple to synthesize. It can be used to prepare reagents for detecting hypoxic lesions in organisms and can also be used to prepare tumor cell detection reagents.

[0005] The present invention provides a near-infrared fluorescent probe and TM-N2 with good water solubility for detecting nitroreductase. The chemical structure of the fluorescent probe TM-N2 is:

[0006] .

[0007] The present invention provides a method for preparing the above-mentioned near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, comprising the following steps:

[0008] S1, dissolving 4-diethylamino keto acid and 4-aminoacetophenone in concentrated sulfuric acid, heating under reflux for reaction, and after cooling to room temperature, adding dropwise to an ice-water mixture, then adding perchloric acid and standing for precipitation, and filtering to obtain an intermediate product;

[0009] S2. The intermediate product prepared in S1 is mixed with K2CO3, dissolved in acetonitrile, and then 4-nitrobenzyl bromide is added. The mixture is reacted at room temperature and separated by column chromatography to obtain the desired product TM-N2.

[0010] According to the preparation method provided by the present invention, the molar volume ratio of 4-diethylaminoketo acid, 4-aminoacetophenone, concentrated sulfuric acid, ice-water mixture and perchloric acid in S1 is (1-2) mmol: (1-2) mmol: (5-10) mL: (20-40) g: (1-2) mL, the concentration of the concentrated sulfuric acid is 98%, and the mass fraction of the perchloric acid is 70%.

[0011] According to the preparation method provided by the present invention, the temperature of the heating reflux reaction in S1 is 90° C., and the time of the heating reflux reaction is 0.5 to 5 h.

[0012] According to the preparation method provided by the present invention, the molar volume ratio of the intermediate product, K2CO3, acetonitrile and 4-nitrobenzyl bromide in S2 is (0.334~0.835) mmol: (0.334~0.835) mmol: (1.3~2) mL: (0.334~0.835) mmol.

[0013] According to the preparation method provided by the present invention, the column chromatography conditions are: in a silica gel column, methanol / dichloromethane is used as the eluent, and the elution ratio is 1 / (2-15).

[0014] The present invention also provides an application of the above-mentioned near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase. The fluorescent probe TM-N2 can be used to prepare a reagent for detecting hypoxic lesions in an organism, and can also be used to prepare a tumor cell detection reagent.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The present invention provides a water-soluble near-infrared red fluorescent probe TM-N2 for detecting nitroreductase. The probe has good water solubility, a wavelength that reaches the near-infrared range, and a simple preparation method. The probe exhibits high sensitivity and good selectivity. It has been demonstrated to detect nitroreductase in zebrafish under hypoxic conditions and in tumor cells, and has broad application prospects in in vivo imaging analysis and tumor detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 is the H NMR spectrum of probe TM-N2;

[0019] Figure 2 is the carbon NMR spectrum of probe TM-N2;

[0020] Figure 3 is the mass spectrum of probe TM-N2;

[0021] Figure 4 This is the fluorescence spectrum of probe TM-N2 to nitroreductase at different times;

[0022] Figure 5 is the fluorescence spectrum of probe TM-N2 to different concentrations of nitroreductase;

[0023] Figure 6 is a schematic diagram of the selectivity of probe TM-N2, and the ordinate is the fluorescence intensity of probe TM-N2 at 650 nm;

[0024] Figure 7 This is a diagram showing the changes of probe TM-N2 on nitroreductase in zebrafish;

[0025] Figure 8 These are confocal images of HeLa cells co-cultured with TM-N2 for 30 minutes under different oxygen content conditions. DETAILED DESCRIPTION

[0026] Example 1

[0027] This example provides a method for preparing a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, and the specific steps are as follows:

[0028] S1. Place 1 mmol of 4-diethylaminoketo acid and 1 mmol of 4-aminoacetophenone in a 25 mL round-bottom flask, add 5 mL of 98% concentrated sulfuric acid (the concentration of concentrated sulfuric acid is 98%), and react at 90°C for 2 h. After cooling to room temperature, add dropwise to a 20 g ice-water mixture, then add 1 mL of 70% perchloric acid and let stand for 12 h to precipitate. Filter to obtain the desired compound TM as a purple solid.

[0029]

[0030] S2. Dissolve 0.334 mmol of TM prepared in S1 and 0.334 mmol of K2CO3 in 1.3 mL of acetonitrile. Add 0.334 mmol of 4-nitrobenzyl bromide and react at room temperature for 12 h. After the reaction is completed, separate the mixture by column chromatography to obtain the desired product TM-N2 as a purple solid.

[0031] The column chromatography conditions are as follows: using a silica gel column with methanol / dichloromethane as the eluent at an elution ratio of 1 / (2-15).

[0032]

[0033] Example 2

[0034] This example provides a method for preparing a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, and the specific steps are as follows:

[0035] S1. Place 2 mmol of 4-diethylaminoketo acid and 2 mmol of 4-aminoacetophenone in a 25 mL round-bottom flask, add 10 mL of concentrated sulfuric acid, and react at 90°C for 2 h. After cooling to room temperature, add dropwise to 40 g of ice-water mixture, then add 2 mL of 70% perchloric acid and let stand for 12 h to precipitate. Filter to obtain the desired compound TM as a purple solid.

[0036] S2. Dissolve 0.334 mmol of TM prepared in S1 and 0.835 mmol of K2CO3 in 2 mL of acetonitrile. Add 0.835 mmol of 4-nitrobenzyl bromide and react at room temperature for 12 h. After the reaction is completed, separate the mixture by column chromatography to obtain the desired product TM-N2 as a purple solid.

[0037] The column chromatography conditions are as follows: using a silica gel column with methanol / dichloromethane as the eluent at an elution ratio of 1 / (2-15).

[0038] Example 3

[0039] This example provides a method for preparing a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, and the specific steps are as follows:

[0040] S1. Place 1.5 mmol of 4-diethylaminoketo acid and 1.5 mmol of 4-aminoacetophenone in a 25 mL round-bottom flask, add 7.5 mL of concentrated sulfuric acid, and react at 90°C for 2 h. After cooling to room temperature, add dropwise to a 30 g ice-water mixture, then add 1.5 mL of 70% perchloric acid and let stand for 12 h to precipitate. Filter to obtain the desired compound TM as a purple solid.

[0041] S2. Dissolve 0.334 mmol of TM prepared in S1 and 0.58 mmol of K2CO3 in 1.7 mL of acetonitrile. Add 0.58 mmol of 4-nitrobenzyl bromide and react at room temperature for 12 h. After the reaction is completed, column chromatography is used to obtain the desired product TM-N2 as a purple solid.

[0042] The column chromatography conditions are as follows: using a silica gel column with methanol / dichloromethane as the eluent at an elution ratio of 1 / (2-15).

[0043] Example 4

[0044] This example provides the responsiveness of the probe TM-N2 prepared in Example 1 to nitroreductase;

[0045] The probe TM-N2 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. Nitroreductase was prepared with a pH 7.4 PBS stock solution to a concentration of 500 μg / mL, and reduced coenzyme I (NADH) was prepared to a 1 mM NADH stock solution.

[0046] In a PBS solution of probe TM-N2 with a concentration of 10 μM, the fluorescence signal of the probe itself was low. After adding 250 μL NADH stock solution and 2 μg / mL nitroreductase, a strong fluorescence signal was emitted at 650 nm.

[0047] As shown in the figure, Figure 4 The fluorescence spectra before and after adding nitroreductase are shown in Figure 2. Figure 5 The fluorescence images are those with different concentrations of nitroreductase added;

[0048] The comparative experimental results showed that the probe TM-N2 can achieve high-sensitivity response to nitroreductase, and it was found that the fluorescence signal was significantly enhanced after the compound TM-N2 was added to nitroreductase.

[0049] Example 5

[0050] This example provides the selectivity of the probe TM-N2 prepared in Example 1;

[0051] Common interfering substances, such as inorganic ions (K + Mg 2+ , Ca 2+ 、HSO3 – 、SO3 2– , HS – ), glucose, Vc, VB6, HSA, Glu, Arg, Ser, Asp, Pro, active oxygen (HClO, 1O2, H2O2, ·OH) and biological thiols (GSH, Cys, Hcy and DTT) were used to investigate the selectivity of the probe for nitroreductase.

[0052] like Figure 6 As shown, add the above interfering substances such as inorganic ions (K + Mg 2+ , Ca 2+ 、HSO3 – 、SO3 2– , HS – ), glucose, Vc, VB6, HSA, Glu, Arg, Ser, Asp, Pro, active oxygen (HClO, 1 After the addition of thiols (O2, H2O2, ·OH) and biothiols (GSH, Cys, Hcy and DTT), the fluorescence of the probe at 650 nm was basically not enhanced, indicating that the probe has a high selectivity for nitroreductase.

[0053] Example 6

[0054] This embodiment provides an application of the probe TM-N2 prepared in Example 1 for zebrafish imaging.

[0055] like Figure 7 As shown, the first group is the control group; the second group is added with the probe TM-N2 (10 μM); the third group is added with the probe TM-N2 and then placed for 30 minutes, and the zebrafish are changed from normoxia to hypoxia; the fourth group is added with the probe TM-N2 (10 μM) and the nitroreductase inhibitor dicoumarol (50 μM), and the zebrafish are incubated for 30 minutes.

[0056] As can be seen from the figure, the third group of zebrafish were incubated in a 10 μM concentration of probe TM-N2 and PBS solution for 20 minutes. The probe TM-N2 had strong fluorescence in specific parts of the zebrafish body. During the 30 minutes of observation out of water, the fish changed from a normoxic state to a hypoxic state, the content of nitroreductase in the fish body changed, and the fluorescence intensity gradually increased.

[0057] The fourth group of zebrafish was treated with 50 μM dicoumarol, a nitroreductase inhibitor. After the nitroreductase in the fish was eliminated, there was no strong fluorescence, and the probe TM-N2 only had a strong fluorescence signal in specific areas, indicating that the probe TM-N2 has high selectivity and sensitivity in vivo and has the potential to detect hypoxia in organisms.

[0058] Example 7

[0059] This example provides an application of the probe TM-N2 prepared in Example 1 for Hela cell imaging.

[0060] Hela cells were seeded into 15 mm culture dishes and cultured in an incubator at 37 °C and 5% CO2 for 12 h. After the cells adhered to the wall, they were divided into three groups for culture;

[0061] The three conditions were: 21% oxygen content, 1% oxygen content, and 1% oxygen content with the inhibitor dicoumarol added;

[0062] After culturing for 6 h, 10 μM probe TM-N2 was added to the cells under the three culture conditions, and the cells were cultured for another 30 min. The cell culture medium was discarded, and the cells were washed three times with PBS buffer. The cells under the three culture conditions were photographed using a confocal microscope. The pictures are as follows: Figure 8 shown.

[0063] from Figure 8 It was found that the fluorescence intensity of the cells cultured in the second group with an oxygen content of 1% was significantly stronger after being exposed to the probe TM-N2;

[0064] The fluorescence intensity of the cells in the third group cultured at an oxygen content of 1% and with the addition of the inhibitor dicoumarol was significantly reduced.

[0065] It was demonstrated that under hypoxic conditions, the content of nitroreductase in cells changed, and the probe TM-N2 could effectively detect changes in nitroreductase in Hela cells.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, characterized in that: The chemical structural formula of the fluorescent probe TM-N2 is: 。 2. A method for preparing the water-soluble near-infrared fluorescent probe TM-N2 for detecting nitroreductase as claimed in claim 1, characterized in that: The following steps are involved: S1, dissolving 4-diethylamino keto acid and 4-aminoacetophenone in concentrated sulfuric acid, heating under reflux for reaction, and after cooling to room temperature, adding dropwise to an ice-water mixture, then adding perchloric acid and standing for precipitation, and filtering to obtain an intermediate product; S2. The intermediate product prepared in S1 is mixed with K2CO3, dissolved in acetonitrile, and then 4-nitrobenzyl bromide is added. The mixture is reacted at room temperature and separated by column chromatography to obtain the desired product TM-N2.

3. The preparation method according to claim 2, characterized in that The molar volume ratio of 4-diethylaminoketo acid, 4-aminoacetophenone, concentrated sulfuric acid, ice-water mixture and perchloric acid in S1 is (1-2) mmol: (1-2) mmol: (5-10) mL: (20-40) g: (1-2) mL, the concentration of the concentrated sulfuric acid is 98%, and the mass fraction of the perchloric acid is 70%.

4. The preparation method according to claim 2, characterized in that The temperature of the heating reflux reaction in S1 is 90° C., and the time of the heating reflux reaction is 0.5 to 5 h.

5. The preparation method according to claim 2, characterized in that: The molar volume ratio of the intermediate product, K2CO3, acetonitrile and 4-nitrobenzyl bromide in S2 is 0.334 mmol: (0.334-0.835) mmol: (1.3-2) mL: (0.334-0.835) mmol.

6. The preparation method according to claim 2, characterized in that: The column chromatography conditions are as follows: using methanol / dichloromethane as eluent in a silica gel column at an elution ratio of 1 / (2-15).

7. A use of the near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase as claimed in claim 1, characterized in that: The fluorescent probe TM-N2 can be used to prepare a reagent for detecting hypoxic lesions in an organism, and can also be used to prepare a reagent for detecting tumor cells.

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