CN117466876B - Near-infrared small molecule biosensor material for detecting apyrimidine and apurinic sites in tumor cell DNA, and synthesis and application thereof - Google Patents

Abstract

A near-infrared small molecule biosensor material for detecting apyrimidinic and apurinic sites in tumor cell DNA, wherein the near-infrared small molecule biosensor material is 2-((4-(2-(7-(4-ethylpiperazine-1-yl)-4-methyl-2-oxo-2H-chromene-3-yl)-2-oxoethyl)-1-methyl-1,4-dihydroquinolin-3-yl)methylene)malononitrile, and the synthesis method is as follows: Compound 3-acetyl-7-(4-ethylpiperazine-1-yl)-4-methyl-2H-chromene-2-one and compound B 3-(2,2-dicyanovinyl)-1-methylquinolin-1-ium are added to a reactor containing ethanol and water in a volume ratio of 1:1 and completely dissolved with sodium carbonate for reaction. The present invention does not require a metal catalyst and an oxidant.

Description

Near-infrared small molecules for detecting apyrimidine and apurine sites in tumor cell DNA Biosensor materials, synthesis and applications

Technical Field

The invention relates to a near-infrared biosensor small molecule material for detecting apyrimidine and apurine sites in tumor cells, and belongs to the synthesis and application of compounds.

Background Art

Methyl coumarin and its derivatives are commonly used biomass fluorescent luminescent materials in the past two decades. Compared with traditional inorganic luminescent materials, this luminescent material shows some outstanding advantages. At present, most of the research and development of abasic detection reagents are limited to the detection of the total amount of abasic by the introduction of fluorescent signals. There is no simple detection reagent that can achieve quantitative detection of apurinic and apyrimidinic sites, and how to efficiently and low-costly detect tumors caused by DNA base deletion is a hot topic today. The biosensor small molecule materials used to detect apyrimidinic and apurinic sites in tumor cell DNA are mainly reported as follows:

1. Kim et al. reported the use of biotinylated aldehyde-reactive probes and streptavidin conjugated with fluorescent dyes to label abasic sites. Scanning near-field optical microscopy imaging can directly observe two abasic sites within 120 nm on a single DNA molecule. FEBS Lett. 2003, 555, 611-615;

2. Talpaert et al. reported the reaction of ¹⁴ C-methoxyamine with the aldehyde group present in the deoxyribose moiety after base loss, and the detection of base-deficient sites was achieved by complex radioactivity detection Biophysica Acta (BBA)-GeneStructure and Expression 1983, 740, 410-416;

There are no reports in the literature on the use of 2-((4-(2-(7-(4-ethylpiperazin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-1-methyl-1,4-dihydroquinolin-3-yl)methylene)malononitrile (FN) small molecule near-infrared fluorescent material for detecting the content of apyrimidinic and apurinic sites in tumor cells.

Summary of the invention

The purpose of the present invention is to provide a near-infrared small molecule biosensor material, synthesis and application for detecting apyrimidine and apurine sites in tumor cell DNA.

In order to achieve the purpose of the present invention, the following technical scheme is adopted: a near-infrared small molecule biosensor material for detecting apyrimidinic and apurinic sites in tumor cell DNA, wherein the near-infrared small molecule biosensor material is 2-((4-(2-(7-(4-ethylpiperazine-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-1-methyl-1,4-dihydroquinolin-3-yl)methylene)malononitrile, and the structural formula is shown in the following formula A:

.

A near-infrared small molecule biosensor material for detecting apyrimidinic and apurinic sites in tumor cell DNA is synthesized. The near-infrared small molecule biosensor material adopts the above-mentioned 2-((4-(2-(7-(4-ethylpiperazine-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-1-methyl-1,4-dihydroquinolin-3-yl)methylene)malononitrile, the structural formula of which is shown in formula A, and the synthesis route is as follows:

Compound C, 3-acetyl-7-(4-ethylpiperazin-1-yl)-4-methyl-2H-chromen-2-one and compound B, 3-(2,2-dicyanovinyl)-1-methylquinolin-1-ium, were added to a reactor filled with ethanol and water in a volume ratio of 1:1 and completely dissolved with sodium carbonate, stirred at 45°C for 6 hours, extracted the mixture twice with DCM, combined the organic layers and dried with sodium sulfate, and purified by a silica gel column with methanol/DCM=1:10 to obtain;

The synthetic route of the compound of formula B, 3-(2,2-dicyanovinyl)-1-methylquinolin-1-ium, is as follows:

Add acetonitrile, 3-quinoline formaldehyde and methyl iodide into a reactor, then heat the mixture under reflux at 60 degrees with stirring, continue stirring for 12 hours under nitrogen, cool the mixture to room temperature, precipitate to obtain a solid crude product, add malononitrile, piperazine and ethanol to the crude product, heat to 60 degrees and stir for 6 hours to obtain a compound shown in formula B;

The synthetic route of the compound of formula C, 3-acetyl-7-(4-ethylpiperazine-1-yl)-4-methyl-2H-chromene-2-one, is as follows:

1-Ethylpiperazine is added to a solution of 4-fluoro-2-hydroxybenzaldehyde dissolved in DMSO, and the mixture is stirred at 110°C under nitrogen for 12-25 h. Then, the reaction mixture is added to an aqueous sodium carbonate solution and extracted with DCM. The combined organic phase is washed with water and brine, dried over sodium sulfate, and concentrated under vacuum to obtain a compound of formula D, 4-(4-ethylpiperazin-1-yl)-2-hydroxybenzaldehyde;

4-(4-ethylpiperazin-1-yl)-2-hydroxybenzaldehyde, ethyl acetoacetate, and piperidine were dissolved in anhydrous ethanol, and the mixture was then refluxed at 78°C for 6 hours under nitrogen. After cooling to room temperature, the bright yellow precipitate was purified by silica gel column chromatography, as shown in the formula C: 3-acetyl-7-(4-ethylpiperazin-1-yl)-4-methyl-2H-chromen-2-one.

Application of near-infrared small molecule biosensor material for detecting apyrimidine and apurine sites in tumor cell DNA. The near-infrared small molecule biosensor material is used to detect apyrimidine and apurine sites in tumor cell DNA. The tumor cells include breast cancer cells MCF-7, esophageal cancer cells EC10, colorectal cancer cells SW480, and cervical cells Hela.

The positive and beneficial technical effects of the present invention are: compared with the prior art, the method of the present invention does not require metal catalysts and oxidants, and there are no harmful substances in the product, thereby reducing environmental pollution; the in vitro cell test involved in the detection requires simple preliminary processing of the sample and is easy to operate; using traditional fluorescence detection methods, it has high sensitivity to the target, strong response signal and rapid reaction; such small molecule materials have good membrane permeability, strong cell penetration, and are easy to contact and react with targets released from the cell body; under 350nm light, they will react differently with the apyrimidine and apurine sites in DNA, and are easy to construct into various types of in vitro diagnostic reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the absorption spectrum of near-infrared biosensor polymer materials.

Figure 2 shows the fluorescence spectrum characteristics of near-infrared biosensor polymer materials.

FIG. 3 is a quantitative standard curve for determining the content of apurinic sites in DNA.

FIG. 4 is a quantitative standard curve for determining the content of apyrimidinic sites in DNA.

FIG. 5 is a diagram showing the mechanism of the present invention.

DETAILED DESCRIPTION

In order to more fully explain the implementation of the present invention, implementation examples of the present invention are provided. These implementation examples are merely elaborations of the present invention and do not limit the scope of the present invention.

The equipment and models used in the embodiments of the present invention are as follows: the absorption spectrum is detected by Varian Cary 50 Bio full-power detector; the fluorescence spectrum is detected by Hitachi F4600; the NMR spectrum is detected by nuclear magnetic resonance spectrometer (Bruker ARX 400MHz). High-resolution mass spectrometry is performed by Agilent Technologies 6410.

1. Synthesis of near-infrared small molecule biosensor material with structural formula A: 2-((4-(2-(7-(4-ethylpiperazine-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-1-methyl-1,4-dihydroquinolin-3-yl)methylene)malononitrile

1.1: Synthesis of 3-(2,2-dicyanovinyl)-1-methylquinolin-1-ium of formula B:

In a 100 mL round-bottom flask, 10 mL of acetonitrile, 2.5 g (16 mmol) of 3-formaldehyde quinoline, and 11.4 g (80 mmol) of methyl iodide were added. The mixture was then heated to reflux under stirring and stirred for 12 hours under nitrogen. The mixture was cooled to room temperature and precipitated to obtain an orange solid. The final product can be obtained by filtering and washing with cold ethanol. Subsequently, 3.5 g of malononitrile, 0.5 mL of piperazine and 40 mL of ethanol were added to the crude product, heated to 60 °C and stirred for 6 hours to obtain a light yellow product 1. (4.5 g, 96% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.31 (s, 1H),10.02 (d, J = 1.8 Hz, 1H), 9.84 (s, 1H), 8.70 (dd, J = 8.3, 1.5 Hz, 1H), 8.62(d, J = 8.9 Hz, 1H), 8.45 (ddd, J = 8.8, 7.1, 1.5 Hz, 1H), 8.25 – 8.13 (m,1H), 4.74 (s, 3H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 189.45, 150.44, 149.12, 140.05, 138.42, 132.65, 131.38, 129.39, 128.95, 120.18, 46.27.

1.2: Synthesis of 4-(4-ethylpiperazine-1-yl)-2-hydroxybenzaldehyde of D-structure:

To a solution of 4-fluoro-2-hydroxybenzaldehyde (1.40 g, 10.0 mmol) in 30 mL of DMSO was added 1-ethylpiperazine (1.19 g, 10.5 mmol) and the mixture was stirred at 110 °C under nitrogen for 20 h. The reaction mixture was then added to aqueous Na2CO3 solution (500 mL) and extracted with DCM (3 x 100 mL). The combined organic phases were washed with water and brine, dried over Na2SO4, and concentrated under vacuum to give the product as a brown solid, (0.98 g, 3.92 mmol, 39.2%). ¹ H NMR (400 MHz, Chloroform-d) δ 11.49 (s, 1H), 9.56 (s, 1H), 3.61 – 3.32 (m,4H), 2.59 – 2.53 (m, 4H), 2.47 (q, J = 7.2 Hz, 2H), 1.13 (t, J = 7.2 Hz, 3H). ¹³ C NMR (101 MHz, Chloroform-d) δ 192.77, 164.16, 156.60, 135.14, 112.78,106.13, 99.45, 52.33, 52.27, 46.71, 11.94. HRMS (ESI): calcd for C13H18N2O2 ⁺ [M+H] ⁺ : 234.1368; found: 234.1362.

1.3: Synthesis of 3-acetyl-7-(4-ethylpiperazin-1-yl)-4-methyl-2H-chromen-2-one of formula C:

4-(4-Ethylpiperazin-1-yl)-2-hydroxybenzaldehyde (0.43 g, 1.8 mmol), ethyl acetoacetate (0.28 g, 2.16 mmol) and 0.1 ml of piperidine were dissolved in 25 ml of absolute ethanol. The mixture was then refluxed under nitrogen for 6 hours. After cooling to room temperature, the bright yellow precipitate was purified by silica gel column chromatography to give the title compound (0.32 g, 1.07 mmol). Yield: 59.4%. ¹ H NMR (400 MHz, Chloroform-d) δ 8.45 (s, 1H), 7.45(d, J = 8.9 Hz, 1H), 6.83 (dd, J = 8.9, 2.5 Hz, 1H), 6.67 (d, J = 2.4 Hz,1H), 3.48 (t, J = 5.2 Hz, 4H), 2.70 (s, 3H), 2.61 (t, J = 5.2 Hz, 4H), 2.50(q, J = 7.2 Hz, 2H), 1.17 – 1.10 (m, 3H). ¹³ C NMR (101 MHz, Chloroform-d) δ195.70, 160.50, 158.31, 155.29, 147.71, 131.55, 118.05, 111.56, 109.60,99.17, 52.24, 46.96, 30.61, 11.97. (ESI): calcd for C17H20N2O3 ⁺ [M+H] ⁺ :300.1474; found: 300.1476.

1.4: Synthesis of near-infrared small molecule biosensor material 2-((4-(2-(7-(4-ethylpiperazin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-1-methyl-1,4-dihydroquinolin-3-yl)methylene)malononitrile with structure A:

Compound C (0.3 mmol, 90 mg) and compound B (0.45 mmol, 135 mg) were slowly added to a round bottle containing 6 mL EtOH/H2O (1:1) and Na2CO3 (65 mg) was completely dissolved in advance. Then, the reaction was stirred at 45 °C for 6 hours. The mixture was extracted twice with DCM (20 mL). The organic layers were combined and dried over Na2SO4. Purification by silica gel column (methanol/DCM＝1∶10) gave a yellow solid (76 mg, 55% yield). ¹ H NMR (400 MHz, Chloroform-d) δ9.13 (s, 1H), 8.38 (s, 1H), 7.50 – 7.45 (m, 1H), 7.40 (d, J = 8.9 Hz, 1H),7.17 (td, J = 7.8, 1.6 Hz, 1H), 7.06 (td, J = 7.5, 1.1 Hz, 1H), 6.99 (s, 1H),6.87 (d, J = 8.1 Hz, 1H), 6.79 (dd, J = 8.9, 2.4 Hz, 1H), 6.60 (d, J = 2.4Hz, 1H), 4.73 (t, J = 6.0 Hz, 1H), 3.61 (dd, J = 15.5, 5.9 Hz, 1H), 3.45 (t,J = 5.4 Hz, 4H), 3.40 (s, 3H), 3.06 (dd, J = 15.5, 6.2 Hz, 1H), 2.60 (t, J =5.2 Hz, 4H), 2.49 (q, J = 7.2 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H). ¹³ C NMR (101MHz, Chloroform-d) δ 195.38, 187.41, 160.24, 158.07, 155.07, 151.75, 147.89,138.22, 131.44, 130.03, 127.39, 127.24, 124.48, 118.13, 114.96, 112.72,111.51, 109.81, 99.12, 52.20, 52.16, 51.88, 46.86, 39.36, 30.79, 11.87. HRMS(ESI): calcd for C28H30N3O4 ⁺ [M+H] ⁺ : 472.2231; found: 472.2235.

Test Example:

1. First, dissolve the near-infrared small molecule biosensor material probe in a stock solution (10.0 mM) using DMSO (HPLC grade) for later use.

2. For each test, the solution was mixed thoroughly and data were collected at room temperature after 10 minutes. 25 mM PBS solution was used as the test solution. Fluorescence spectra were obtained at its excitation wavelength at a scanning speed of 1200 nm/min (slit 5/5 nm), and absorption spectra were recorded in the wavelength range of 300 nm to 700 nm.

Preparation of tumor cell DNA samples

1. On a clean laboratory bench, culture esophageal cancer (EC109) tumor cell samples in the laboratory. Take 200 μL of tumor cells and add them to a 1.5 mL centrifuge tube. Add 1 mL of 0.1 M PBS PH=7.4 buffer and pre-incubate at 37°C for 3 minutes.

2. Add 30 ml of cell lysis buffer to a 50 ml sterilized centrifuge tube, gently shake the cell sample tube until the sample is completely mixed, transfer the cell sample (10 ml) to the tube with cell lysis buffer, invert the tube several times to mix, incubate at room temperature for 10 min (invert 2-3 times during the period) to lyse the cells, centrifuge at 2000g for 10 min at room temperature, remove the supernatant, and obtain the resuspended cells below;

3. Add 10 ml of nuclear lysis solution to the test tube containing the resuspended cells. If there are clumping cells, incubate the solution at 37°C until the cell clumps are completely destroyed. Add 3 ml of protein precipitation solution to the lysate. After vigorous shaking for 10-20 seconds, small protein clumps may be seen. Centrifuge at 2000g for 10 minutes and retain the supernatant;

4. Transfer the above supernatant to a 50ml clean test tube containing 10ml isopropanol, gently invert the mixed solution until the white linear DNA is deformed into visible blocky DNA, centrifuge at 2000g for 1min, pour out the supernatant, add 10ml 70% ethanol, gently invert the test tube several times to wash the DNA precipitate and the test tube wall, centrifuge, aspirate the ethanol, and obtain the DNA sample.

Preparation of DNA apyrimidinic and apurinic samples:

1. Synthesis of depurine samples: Add 10 μg of phosphoramidite to the DNA sample obtained above, shake well for 30 min, and treat the uracil-containing DNA with 5 μg/mL uracil-N-glycosylase (UNG enzyme) to generate DNA containing depurine sites;

2. Synthesis of apyrimidinic samples: Add 10 μg of uracil phosphoramidite to the DNA sample obtained above, shake well for 30 min, and finally treat the adenine-containing DNA with 5 μg/mL adenine-N-glycosylase (TNG enzyme) to generate DNA containing apyrimidinic sites.

3. DNA was isolated and purified using 10 mg/L polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC). The preparation of the above DNA samples, DNA apyrimidinic and apurinic samples is prior art, and this application does not involve specific DNA sequences.

Test Method

1. At room temperature, prepare the small molecule FN (infrared small molecule biosensor material) into a 10 mM solution using DMSO and store it in a refrigerator at 4°C. Dilute the prepared apyrimidine and apurine samples with double distilled water in different ratios (1:1, 1:2, 1:5, 1:20, 1:50, 1:100, 1:200). After dilution, divide each sample into 4 portions, 2 ml each; add them to the cuvette and let stand.

2. Let the four samples in each portion stand for 10 min, 30 min, 1 h, and 2 h, respectively, add 5 μL (10 mM) of FN small molecules, shake well, and filter using a 0.24 μm filter membrane. Keep the solution for later use.

3. At room temperature, perform fluorescence detection on all cuvettes (Ex = 530 nm, Em = 653/712nm), test the fluorescence intensity and yield in each cuvette, and compare the average value of each group with the control group of normal cells in the infrared receiving window; after irradiation with 350nm light, calculate the fluorescence intensity of the product and the abasic content (relative concentration of apyrimidine and apurine) to make a standard curve, and count and record at least three sets of data. Finally, it was concluded that the sensitivity of this method to the apyrimidine and apurine content was 0.15 μM and 0.26 μM, respectively.

Preferably, the tumor cells are breast cancer (MCF-7), esophageal cancer (EC109), colorectal cancer (SW480), cervical cancer (Hela)

After describing the implementation mode of the present invention in detail, people familiar with the technology can clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned patent application. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are within the scope of the technical solution of the present invention, and the present invention is not limited to the implementation mode of the examples given in the specification.

Claims (3)

1. A near-infrared small molecule biosensor material for detecting apyrimidinic and apurinic sites in tumor cell DNA, characterized in that the near-infrared small molecule biosensor material is 2-((4-(2-(7-(4-ethylpiperazine-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-1-methyl-1,4-dihydroquinolin-3-yl)methylene)malononitrile, with a structural formula as shown in Formula A below: . 2. The method for synthesizing a near-infrared small molecule biosensor material for detecting apyrimidine and apurine sites in tumor cell DNA according to claim 1, wherein the synthesis route is as follows: ; Compound C, 3-acetyl-7-(4-ethylpiperazin-1-yl)-4-methyl-2H-chromen-2-one and compound B, 3-(2,2-dicyanovinyl)-1-methylquinolin-1-ium, were added to a reactor filled with ethanol and water in a volume ratio of 1:1 and completely dissolved with sodium carbonate, and stirred at 45°C for 6 hours. The mixture was extracted twice with DCM, the organic layers were combined and dried with sodium sulfate, and purified by a silica gel column with methanol/DCM=1:10 to obtain; The synthetic route of the compound of formula B, 3-(2,2-dicyanovinyl)-1-methylquinolin-1-ium, is as follows: ; Add acetonitrile, 3-quinoline formaldehyde and methyl iodide into a reactor, then heat the mixture under reflux at 60 degrees with stirring, continue stirring for 12 hours under nitrogen, cool the mixture to room temperature, precipitate to obtain a solid crude product, add malononitrile, piperazine and ethanol to the crude product, heat to 60 degrees Celsius and stir for 6 hours to obtain a compound shown in formula B; The synthetic route of the compound of formula C, 3-acetyl-7-(4-ethylpiperazine-1-yl)-4-methyl-2H-chromene-2-one, is as follows: ; 1-Ethylpiperazine is added to a solution of 4-fluoro-2-hydroxybenzaldehyde dissolved in DMSO, and the mixture is stirred at 110° C. under nitrogen for 12-25 h. Then, the reaction mixture is added to an aqueous sodium carbonate solution and extracted with DCM. The combined organic phase is washed with water and brine, dried over sodium sulfate, and concentrated under vacuum to obtain a compound of formula D, 4-(4-ethylpiperazine-1-yl)-2-hydroxybenzaldehyde; 4-(4-ethylpiperazin-1-yl)-2-hydroxybenzaldehyde, ethyl acetoacetate and piperidine were dissolved in anhydrous ethanol, and the mixture was then refluxed at 78°C for 6 hours under nitrogen. After cooling to room temperature, the bright yellow precipitate was purified by silica gel column chromatography to obtain 3-acetyl-7-(4-ethylpiperazin-1-yl)-4-methyl-2H-chromen-2-one represented by formula C. 3. The use of the near-infrared small molecule biosensor material for detecting apyrimidine and apurine sites in tumor cell DNA as claimed in claim 1, wherein the application excludes use in disease diagnosis and treatment methods, and is characterized in that the near-infrared small molecule biosensor material is used to detect apyrimidine and apurine sites in tumor cell DNA, and the tumor cells are breast cancer cells MCF-7, esophageal cancer cells EC10, colorectal cancer cells SW480, and cervical cells Hela.