(E)-3-(6-(Dimethylamino)naphthalen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivatives, their preparation methods and applications

Abstract

The present invention discloses an (E)-3-(6-(dimethylamino)naphthalen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivative, a preparation method thereof and an application thereof, belonging to the technical field of biochemical analysis. The chemical structure of the derivative is represented by formula (I). (I) The preparation method is as follows: 2'-hydroxyacetophenone, 6-(dimethylamino)-2-naphthaldehyde and an organic solvent are mixed, and then an alkali solution is added. After reacting at room temperature, the organic phase is concentrated, neutralized with an acid, and an intermediate is obtained. The intermediate reacts with 2,4-dinitrobenzenesulfonyl chloride in an organic solvent, and after separation and purification, the derivative (I) is obtained. The preparation method of the present invention is simple and feasible, and the obtained (E)-3-(6-(dimethylamino)naphthalen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivative (I) can achieve a selective fluorescence response to biological thiols, and can detect biological thiols in cells and in vivo in living mice. It has many advantages such as high sensitivity, rapid response, high specificity, etc., and has broad application prospects in the fields of biochemical analysis and detection, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical analysis, and particularly relates to an (E)-3-(6-(dimethylamino)naphthalen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivative, a preparation method thereof, and an application thereof. Background Art

[0002] The thiol group is one of the groups with the highest chemical activity in organisms, and its importance cannot be ignored. It plays a key role in biological redox systems and post-translational modification processes of proteins, affects the metabolism of living organisms, and plays an important regulatory role in the health of living organisms. The biological thiol compounds in living organisms include cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). These compounds have a wide range of cell biological functions and play an important role in the biochemical defense system of the human body. For example, Cys is an important thiol compound in cells, and abnormal concentrations thereof are related to diseases such as albinism, edema, and liver damage. The abnormal concentration of Hcy is also closely related to the occurrence of diseases such as cardiovascular diseases and Alzheimer's disease. GSH is an important antioxidant and detoxifying agent in the body, and changes in its concentration will affect the intracellular redox dynamic balance and are closely related to the occurrence and development of various diseases. Therefore, it is very important to maintain the balance of thiol compounds in the body. Excessive or too low levels of thiol compounds will have a negative impact on human health. Therefore, detecting thiol compounds in biological systems, especially tracking their processes in life activities, is of great significance. By monitoring the levels of biological thiols in the body, potential health problems can be detected in a timely manner, helping people better prevent and treat various diseases.

[0003] Currently, the commonly used methods for detecting biological thiols are mainly enzyme-linked immunosorbent assay, radioimmunoassay, electrochemiluminescence method, etc. These methods require expensive instruments and the cooperation of professional technical personnel, and may cause damage to living cells or tissues during the operation process. Therefore, real-time monitoring of the thiol concentration in biological systems cannot be achieved. In contrast, the near-infrared fluorescence probe technology has significant advantages. It can realize real-time in-situ detection of biological thiols, is simple to operate, has high sensitivity, and does not require damage to living cells or tissues, which is very suitable for clinical and scientific research. However, since most of the probes reported in the literature are in the short-wave region, they will cause damage to cells during excitation, and the autofluorescence in organisms also seriously interferes with the detection. Therefore, its practical application is limited to a certain extent. To solve these problems, it is of great significance to develop a near-infrared fluorescence probe for in-vivo detection of biological thiols, which can not only meet the needs of biological thiol detection in fields such as chemistry, environment, biology, and medicine, but also provide more research means for fields such as in-vivo imaging. Summary of the Invention

[0004] The object of the present invention is to provide an (E)-3-(6-(dimethylamino)naphthalen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivative, a preparation method thereof and an application thereof. When the derivative is used as a near-infrared probe for detecting biological thiols, it has the characteristics of high sensitivity, high selectivity and fast response speed. Another object of the present invention is to provide a preparation method of the above-mentioned derivative. The third object of the present invention is to provide an application of the above-mentioned derivative in the detection of biological thiols.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] An (E)-3-(6-(dimethylamino)naphthalen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivative for detecting biological thiols, the chemical structure of which is shown in (I):

[0007]

[0008] The preparation method of the above-mentioned derivative for detecting biological thiols has the following synthetic route:

[0009]

[0010] The specific preparation steps are as follows:

[0011] (1) Mix compound 1, compound 2 and an organic solvent, then add a base, stir and react at room temperature, concentrate the organic phase, neutralize with an acid, and purify to obtain intermediate 3;

[0012] (2) Mix intermediate 3, compound 4 and an organic solvent, add a base, stir and react at room temperature. After the reaction is completed, concentrate the organic phase and purify to obtain the derivative (I) for detecting biological thiols.

[0013] Furthermore, the organic solvent in step (1) and step (2) is methanol, ethanol, acetonitrile, dichloromethane, chloroform, tetrahydrofuran or N,N-dimethylformamide.

[0014] Furthermore, the molar ratio of compound 1, compound 2 and the base in step (1) is 1:(0.8 - 1.2):(0.05 - 1); the base is pyrrolidine, piperidine, sodium hydroxide or potassium hydroxide.

[0015] Furthermore, the acid in step (1) is an inorganic acid or an organic acid. The inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid, and the organic acid is selected from citric acid, malic acid or ascorbic acid.

[0016] Further, in the step (2), the molar ratio of compound 3, compound 4 and the base is 1:(1-2):(1-5); the base is sodium carbonate, potassium carbonate, triethylamine, pyrrolidine, piperidine or N,N-diisopropylethylamine (DIPEA).

[0017] Further, the purification includes washing, recrystallization or column chromatography, and the solvent used is selected from any single-component solvent or a mixed solvent of two or more of ethanol, methanol, ethyl acetate, ether, chloroform, dichloromethane, petroleum ether and n-hexane.

[0018] Use of the above derivatives in the preparation of a fluorescent probe for detecting thiol content.

[0019] Further, the fluorescent probe for detecting thiol content refers to a fluorescent detection, visual qualitative detection, cell imaging and in vivo imaging of mice for detecting biological thiols.

[0020] The preparation method and product of the present invention have the following advantages:

[0021] 1. The derivative of the present invention has a short reaction step, simple post-treatment and high yield.

[0022] 2. The derivative of the present invention is used for the response of biological thiols, with high sensitivity and rapid response. The fluorescence change can be directly observed under a 365 nm portable ultraviolet lamp.

[0023] 3. The derivative of the present invention can be used for imaging detection of biological thiols in living cells or in vivo in living mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the synthetic route of the derivative (I) belonging to the present invention;

[0025] Figure 2 is the figure of intermediate 3 under white light and 365 nm ultraviolet light;

[0026] Figure 3 is the single crystal analysis result figure of intermediate 3;

[0027] Figure 4 is the solid-state fluorescence spectrum figure of intermediate 3;

[0028] Figure 5 is the nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) figure of the derivative (I) of the present invention;

[0029] Figure 6 is the high-resolution mass spectrum (HRMS) figure of the derivative (I) of the present invention;

[0030] Figure 7It is a graph of the solution of the derivative (I) obtained in Example 1 added to different amino acid solutions under ultraviolet light at 365 nm;

[0031] Figure 8 It is a graph of the fluorescence intensity of the derivative (I) obtained in Example 1 at 670 nm after adding different amino acids;

[0032] Figure 9 It is a graph showing the change of the fluorescence spectrum of the derivative (I) obtained in Example 1 with the concentration of Cys;

[0033] Figure 10 It is a linear relationship graph between the intensity at 670 nm fluorescence of the derivative (I) obtained in Example 1 and the concentration of Cys;

[0034] Figure 11 It is a linear relationship graph between the intensity at 670 nm fluorescence of the derivative (I) obtained in Example 1 and the concentration of Hcy;

[0035] Figure 12 It is a linear relationship graph between the intensity at 670 nm fluorescence of the derivative (I) obtained in Example 1 and the concentration of GSH;

[0036] Figure 13 It is an image of live cell imaging of the derivative (I) obtained in Example 1;

[0037] Figure 14 It is an imaging graph after co-incubating intermediate 3 with live cells;

[0038] Figure 15 It is an imaging graph of the hind limb of a live mouse of the derivative (I) obtained in Example 1;

[0039] Figure 16 It is an imaging graph of the abdomen of a live mouse of the derivative (I) obtained in Example 1. Detailed implementation mode

[0040] The present invention will be further described below in conjunction with examples and drawings, but the implementation modes of the present invention are not limited thereto.

[0041] The nuclear magnetic resonance spectrum was measured using a Bruker AV-300 nuclear magnetic resonance spectrometer in the United States, with deuterated chloroform as the solvent. The fluorescence spectrum was measured using a fluorescence spectrophotometer from Agilent Technologies in the United States, and the ultraviolet spectrum was measured using a UV9100A ultraviolet spectrophotometer from Labtech Instruments Co., Ltd. in Beijing. Other parameters can refer to conventional instruments.

[0042] Example 1

[0043] For the derivative (I) of this example, the synthesis route is as Figure 1 shown, and its synthesis steps are as follows:

[0044] 272 mg of 2'-hydroxyacetophenone (2 mmol) was added to 20 mL of an anhydrous ethanol solution containing 398 mg of 6-(dimethylamino)-2-naphthaldehyde (2 mmol), and 0.2 mmol of pyrrolidine was added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was evaporated, and the residue was adjusted to neutral with a 10% (v / v) dilute hydrochloric acid solution. The mixture was filtered, and the filter cake was washed three times with 1 mL of ethanol each time and dried to obtain intermediate 3. Intermediate 3 (533 mg, 2 mmol) and 2,4-dinitrobenzenesulfonyl chloride (2 mmol) were dissolved in 40 mL of dichloromethane, and 202 mg of triethylamine (2 mmol) was slowly added dropwise with stirring. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the mixture was concentrated, and the residue was recrystallized from anhydrous ethanol to obtain 880 mg of derivative (I) with a yield of 80%. The identification data of intermediate 3 and derivative (I) are as follows:

[0045] Intermediate 3, red needle crystals ( Figure 2 a and 2b), showing bright red fluorescence under ultraviolet light at 365 nm ( Figure 2 c and 2d), showing the property of crystallization-induced luminescence. The results of single crystal analysis are shown in Figure 3 ; the maximum emission wavelength of the solid-state fluorescence of its crystal is greater than 675 nm. The solid-state fluorescence spectrum is shown in Figure 4 ; melting point: 146.7 - 148.7 °C; 1 H NMR (300 MHz, CDCl3) δ 13.05 (s, 1H), 8.07 (d, J = 15.3 Hz, 1H), 8.01–7.90 (m, 2H), 7.78–7.64 (m, 4H), 7.50 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.94 (t, J = 8.4 Hz, 1H), 6.90 (s, 1H), 3.11 (s, 6H); 13 C NMR (75 MHz, CDCl3) δ 193.6, 163.5, 149.7, 146.4, 136.6, 136.0, 131.6, 129.9, 129.5, 128.1, 126.9, 126.0, 124.2, 120.3, 118.7, 118.5, 117.4, 116.3, 105.8, 40.5. HRMS (ESI): Calcd. for C 21 H 20 NO2 + : 318.1489, found: 318.1514 [M + H] + .

[0046] Derivative (I), black-purple crystals, melting point: 189.2 - 190.6 °C; 11H NMR (300 MHz, CDCl3) δ 8.24 (s, 2H), 8.13 (d, J = 9.0 Hz, 1H), 7.71–7.58 (m, 4H), 7.50 (d, J = 8.7 Hz, 3H), 7.43–7.33 (m, 2H), 7.16 (d, J = 9.6 Hz, 1H), 6.96 (d, J = 15.6 Hz, 1H), 6.85 (s, 1H), 3.12 (s, 6H). 13 13C NMR (75 MHz, CDCl3) δ 190.2, 146.6, 133.3, 132.6, 131.3, 130.6, 129.8, 128.1, 127.0, 126.3, 124.0, 123.7, 122.9, 120.0, 116.5, 105.6, 40.5; HRMS (ESI) m / z: Calcd. for C 27 H 21 N3NaO8S + : 570.0942, found: 570.0978 [M+Na] + 。 1 1H NMR and HRMS spectra are shown in Figure 5 and Figure 6 。

[0047] Example 2

[0048] The derivative (I) of this example was synthesized as follows:

[0049] The preparation method was the same as that of Example 1, except that in step (1), 10% aqueous sodium hydroxide solution (5 mL) was used instead of pyrrolidine, and in step (2), anhydrous potassium carbonate (5 mmol) was used instead of triethylamine. Finally, a black-purple crystal was obtained with a yield of 74%, and the identification data was the same as that of Example 1.

[0050] Example 3

[0051] The derivative (I) of this example was synthesized as follows:

[0052] The preparation method was the same as that of Example 1, except that in step (1), 10% aqueous potassium hydroxide solution (5 mL) was used instead of pyrrolidine, and in step (2), DIPEA (4 mmol) was used instead of triethylamine. Column chromatography was used for separation instead of recrystallization, and the eluent was a mixed solvent of dichloromethane and methanol with a volume ratio of 50:1. Finally, a black-purple solid was obtained with a yield of 71%, and the identification data was the same as that of Example 1.

[0053] Performance test:

[0054] (1) Visual detection and selectivity of derivative (I)

[0055] 1.0 mmol of the derivative (I) prepared in Example 1 was weighed and dissolved in anhydrous dimethyl sulfoxide. Each type of amino acid (1.0 mmol) was dissolved in pure water, and stock solutions with a concentration of 10 mmol / L were prepared. When used, they were diluted with a mixed solution of tetrahydrofuran and phosphate buffer solution (pH 7.4) (volume ratio 1:1). 2 mL of the derivative (I) with a concentration of 20 μmol / L and 2 mL of each type of amino acid with a concentration of 40 μmol / L were added to transparent glass bottles. The blank control was replaced with a mixed solution of tetrahydrofuran and phosphate buffer instead of the amino acid solution. After mixing and standing for 20 minutes, visual observation was carried out under a 365 nm hand-held ultraviolet lamp, as Figure 7 shown. When no amino acid was added to the derivative solution (blank), and when other amino acids without sulfhydryl groups (leucine Leu, tyrosine Tyr, proline Pro, threonine Thr, sarcosine Sar, valine Val, serine Ser, methionine Met, glutamic acid Glu, lysine Lys, aspartic acid Asp, histidine His, alanine Ala, phenylalanine Phe, glycine Gly, arginine Arg without sulfhydryl groups) were added, the solution was colorless; while when amino acids with sulfhydryl groups such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) were added, the color changed significantly, showing a bright red color. The fluorescence intensity of each solution at 670 nm was recorded using a fluorescence spectrometer. The fluorescence intensity increased by about 100 times after the addition of the three biothiols, as Figure 8 shown. The above results confirm that the derivative (I) has a good selective fluorescence response to biothiols and can achieve naked-eye detection.

[0056] (2) Variation of the fluorescence intensity of derivative (I) with the concentration of biothiols

[0057] Using the above stock solutions and mixed solutions for dilution, the derivative (I) was diluted to 10 μmol / L, and the Cys concentration range was 0 - 50 μmol / L. The fluorescence spectra at different Cys concentrations were recorded using a fluorescence spectrometer, as Figure 9 shown. As Figure 9 can be seen, the fluorescence intensity of the derivative (I) was very weak under 441 nm excitation. After adding Cys, a new emission peak appeared at 670 nm. In the range of ion concentration from 0 to 50 μmol / L, as the ion concentration increased, the fluorescence intensity gradually increased. After replacing Cys with Hcy or GSH, the spectra were Figure 9 basically the same.

[0058] (3) Detection limit of derivative (I) for biothiols

[0059] The detection limit was determined by fluorescence titration experiment with an excitation wavelength of 441 nm. Different concentrations of Cys (0 - 10 μmol / L) were added to the buffer system of derivative (I) (10 μmol / L), and the fluorescence data were measured after 20 minutes. A working curve was plotted with the Cys concentration on the abscissa and the fluorescence intensity on the ordinate. A good linear relationship was presented between the Cys concentration and the fluorescence intensity. For details, see Figure 10 . According to the detection limit (LOD) calculation formula: LOD = 3 × SD / K (3 is the set confidence parameter, SD is the standard deviation of the fluorescence change without adding Cys, and K is the slope of the linear relationship), the detection limit was calculated to be 158 nmol / L by linearly fitting the experimental data, which is much lower than the concentration of Cys in human plasma (at the μmol / L level). Using the same method, the detection limits of Hcy and GSH were measured to be 163 nmol / L and 182 nmol / L respectively. For the linear fitting graphs, see Figure 11 and Figure 12 .

[0060] (4) Live cell imaging

[0061] Human hepatoma cells (HepG2) in the proliferative phase were taken and cultured in cell culture medium containing 100 U / mL penicillin, 100 mg / mL streptomycin and 10% fetal bovine serum at 37 °C in a 5% CO2 environment. The cells were seeded at a density of 1×10 5 cells / mL in a glass-bottom culture dish with a diameter of 30 mm, and imaged and photographed using a confocal laser scanning microscope (CLSM), which was recorded as the blank. The compound was diluted with PBS using a 10 mmol / L stock solution. The cell imaging was divided into five groups: In the first group, the cells were incubated with 10 μmol / L of derivative (I) at 37 °C for 30 minutes, washed 3 times with PBS, and then photographed with CLSM; in the second group, the cells were first incubated with 300 μmol / L of N-acetylmaleimide (NEM) at 37 °C for 30 minutes to consume the biological thiols in the cells, washed with PBS, and then incubated with 10 μmol / L of derivative (I), and photographed after washing with PBS; in the third group, after repeating the operation of the second group, the cells were incubated with Cys (20 μmol / L) at 37 °C for 30 minutes, washed and photographed; the fourth group and the fifth group were similar to the third group, except that Hcy and GSH were used to replace Cys respectively. The photographing results are shown in Figure 13 .

[0062] Under normal culture conditions, HepG2 cells showed no fluorescence under the microscope and were completely dark; when the cells were incubated with 10 μmol / L of derivative (I), weak red fluorescence could be observed ( Figure 13 a). When the cells were pre-incubated with NEM to consume the biological thiols in the cells, there was no red fluorescence in the cells and it was in a dark state ( Figure 13b); After incubating with 20 μmol / L Cys again, strong red fluorescence can be clearly observed inside HepG2 cells ( Figure 13 c). Replacing Cys with Hcy or GSH can obtain the same results as Figure 13 c, and obvious red fluorescence appears inside the cells ( Figure 13 d and 13e); while replacing Cys with other amino acids, the pictures are similar to Figure 13 b, showing a dark state. It indicates that this derivative can enter cells and detect Cys, Hcy or GSH in living cells, and has no fluorescence response to amino acids without sulfhydryl groups. In addition, when 10 μmol / L of intermediate 3 was co-incubated with cells, it was found that intermediate 3 aggregated outside the cells and could not enter the cells ( Figure 14 ). These results show that derivative (I) first enters the cells and then reacts with biological thiols inside the cells to show red fluorescence. Therefore, derivative (I) can be used as a tool for rapidly and sensitively detecting biological thiols in living cells.

[0063] (5) In vivo imaging of mice

[0064] Compared with visible light emission probes, the biggest advantage of near-infrared emission probes is that their emission wavelength is longer, the penetration is deeper, the interference of spontaneous fluorescence of organisms is smaller, and they are more suitable for in vivo imaging. Healthy BALB / c experimental mice were anesthetized and depilated. Glucose (50 μL) was subcutaneously injected into the left hind limb as a control, and derivative (I) (50 μL, 200 μmol / L, the solvent was a dimethyl sulfoxide / glucose mixture with a volume ratio of 1:9) was subcutaneously injected into the right hind limb. Fluorescence images were collected at different times using an in vivo imaging system (IVIS), and the results are shown in Figure 15 . It can be seen from the figure that no fluorescence signal was detected in the left hind limb of the mouse, while a fluorescence signal quickly appeared in the right hind limb, and the fluorescence signal increased significantly within 30 min, indicating that derivative (I) can effectively detect endogenous biological thiols in mice.

[0065] As a biological thiol blocker, NEM can consume biological thiols in the body. It was injected into the peritoneal cavity of BALB / c mice to construct a biological thiol deficiency model. The model mice and normal mice were taken, anesthetized and depilated, and then 50 μL of the above derivative (I) solution was intraperitoneally injected respectively, and then imaged and photographed using IVIS. The results are shown in Figure 16 . It can be seen that there is basically no fluorescence signal in the abdomen of the model mice, while the fluorescence intensity in the abdomen of normal mice increased significantly at 10 minutes. This result indicates that derivative (I) can detect changes in the concentration of biological thiols in vivo and can provide visual signals observed by IVIS.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention are equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An (E)-3-(6-(dimethylamino)naphthalen-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivative, characterized in that, The chemical structure of the said derivative is shown in formula (I): 。 2. The preparation method of the derivative according to claim 1, characterized in that, The synthetic route is as follows: The specific preparation steps are as follows: (1) Mix compound 1, compound 2 and an organic solvent, add a base, stir the reaction at room temperature, concentrate the organic phase after the reaction, neutralize with an acid, and purify to obtain intermediate 3; (2) Mix intermediate 3, compound 4 and an organic solvent, add a base, stir the reaction at room temperature, after the reaction is completed, concentrate the organic phase, and purify to obtain the said derivative (I).

3. The preparation method of the derivative according to claim 2, characterized in that: The organic solvent in the said step (1) and step (2) is methanol, ethanol, acetonitrile, dichloromethane, chloroform, tetrahydrofuran or N,N-dimethylformamide.

4. The preparation method of the derivative according to claim 2, characterized in that: In the said step (1), the molar ratio of compound 1, compound 2 and the base is 1:(0.8~1.2):(0.05~1); the base is pyrrolidine, piperidine, sodium hydroxide or potassium hydroxide.

5. The preparation method of the derivative according to claim 2, characterized in that: The acid in the said step (1) is an inorganic acid or an organic acid. The inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid, and the organic acid is selected from citric acid, malic acid or ascorbic acid.

6. The preparation method of the derivative according to claim 2, characterized in that: In the said step (2), the molar ratio of compound 3, compound 4 and the base is 1:(1~2):(1~5); the base is sodium carbonate, potassium carbonate, triethylamine, pyrrolidine, piperidine or N,N-diisopropylethylamine (DIPEA).

7. The preparation method of the derivative according to claim 2, characterized in that: The said purification includes washing, recrystallization, or column chromatography, and the solvent used is selected from any single-component solvent or a mixed solvent of two or more solvents among ethanol, methanol, ethyl acetate, ether, chloroform, dichloromethane, petroleum ether and n-hexane.

8. Use of the derivative according to claim 1 in the preparation of a fluorescent probe for detecting thiol content, characterized in that: The fluorescent probe for detecting the thiol content for non-diagnostic and non-therapeutic purposes refers to the fluorescent detection, visual qualitative detection, cell imaging and in vivo imaging of mice for detecting biological thiols.

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