Leukotriene A4 hydrolase activated fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a leukotriene A4 hydrolase activated fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the fluorescent probe, hemicyanine fluorescent dye is introduced into L-aspartic acid-1-benzyl ester to serve as an overexpression enzyme response switch in inflammatory diseases, so that the compound can realize selective fluorescence imaging of the inflammatory bowel disease. The fluorescent probe has a structure as shown in general formula I: # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a leukotriene A4 hydrolase-activated fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] Leukotriene A4 hydrolase (LTA4H) is a monomeric bifunctional zinc metalloprotease that can efficiently catalyze the generation of lipid inflammatory mediators and plays a key role in acute and chronic inflammatory diseases. It has been found that the expression level of LTA4H significantly increases during the occurrence of inflammation and is closely related to its pathological process. Therefore, LTA4H can be used as an effective biomarker for inflammation detection and evaluation of the efficacy of anti-inflammatory drugs.

[0003] Near-infrared fluorescent dyes provide an efficient diagnostic technique for fluorescence imaging of inflammatory tissues due to their good tissue penetration and low background interference, which is crucial for optimizing treatment plans and improving treatment effects. However, there is currently a lack of clinical applications of fluorescent probes for selective imaging diagnosis of inflammatory diseases.

[0004] Therefore, the development of a fluorescent probe that can accurately and sensitively evaluate the severity of inflammatory diseases will bring an important breakthrough to clinical diagnosis and treatment. Summary of the Invention

[0005] In order to solve the defects existing in the prior art, the purpose of the present invention is to provide a leukotriene A4 hydrolase-activated fluorescent probe, a preparation method thereof, and an application thereof.

[0006] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0007] In the first aspect of the present invention, a leukotriene A4 hydrolase (LTA4H)-activated fluorescent probe is provided, and the fluorescent probe has the following structure:

[0008]

[0009] In the second aspect of the present invention, a preparation method of the above-mentioned leukotriene A4 hydrolase (LTA4H)-activated fluorescent probe is provided, and the preparation method includes the following steps:

[0010] S1. Reacting compound 3 with methylated 1,4-dimethylquinolin-1-iodide under the condition of sodium acetate to obtain compound 4, and subjecting compound 4 to a nucleophilic reaction with 3,5-dihydroxybenzyl alcohol to obtain compound 5;

[0011] The synthesis route of step S1 is shown as follows:

[0012]

[0013] S2. L-Aspartic acid 1-benzyl ester (6) and 4-aminobenzyl alcohol (7) are dehydrated and condensed under the action of HATU and DIPEA to form compound 8, which is then brominated with PBr3 to obtain compound 9;

[0014] The synthesis route of step S2 is shown as follows:

[0015]

[0016] S3. Compound 5 and compound 9 are subjected to etherification reaction under alkaline conditions, and then the Boc protection is removed to form compound I.

[0017] The synthesis route of step S3 is shown as follows:

[0018]

[0019] In some embodiments, the preparation method includes the following steps:

[0020] S100. N-[(3-(Anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride, 1,4-dimethylquinolinium iodide and sodium acetate are dissolved in anhydrous ethanol and refluxed under a nitrogen atmosphere to obtain compound 4;

[0021] S200. Compound 4, 3,5-dihydroxybenzyl alcohol and KHCO3 are dissolved in anhydrous DMF and reacted under a nitrogen atmosphere to obtain compound 5;

[0022] S300. N-(tert-Butoxycarbonyl)-L-aspartic acid benzyl ester is dissolved in anhydrous dichloromethane, and DIPEA and HATU are successively added with stirring, and then 4-aminobenzyl alcohol is added, and the reaction obtains compound 8;

[0023] S400. Compound 8 is dissolved in anhydrous dichloromethane, and phosphorus tribromide is slowly added dropwise under an ice bath condition, and the reaction obtains compound 9;

[0024] S500. Compound 5, compound 9, K2CO3 and KI are dissolved in anhydrous DMF, and stirred and reacted at 40-50 °C under nitrogen protection. After extraction and washing with dichloromethane, the organic phase is concentrated to obtain a concentrated solution, and the Boc group is removed by adding HCl methanol solution to obtain the leukotriene A4 activating fluorescent probe.

[0025] In some embodiments, in step S500, the stirring reaction is an overnight reaction; the time for Boc group removal reaction is 1 h.

[0026] In the third aspect of the present invention, there is provided an application of the above-mentioned leukotriene A4 activating fluorescent probe in the preparation of a fluorescent diagnostic reagent for inflammatory diseases.

[0027] In some embodiments, the inflammatory disease is inflammatory bowel disease.

[0028] The leukotriene A4 hydrolase-activated fluorescent probe provided by the embodiments of the present invention, its preparation method and application have the following advantages compared with the prior art:

[0029] (1) The fluorescence intensity of the leukotriene A4 hydrolase-activated fluorescent probe of the present invention is significantly enhanced at 762 nm after adding LTA4H, and has specific response characteristics.

[0030] (2) The leukotriene A4 hydrolase-activated fluorescent probe of the present invention can effectively monitor the activity change of LTA4H in living cells, provides a powerful tool for the real-time diagnosis of inflammatory diseases, provides a new strategy for the precise diagnosis and treatment of inflammatory diseases such as inflammatory bowel disease (IBD), and has important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The technical solutions of the present invention will be further specifically described below through examples in combination with the drawings. The description of the embodiments of the present invention with reference to the drawings is intended to explain the overall inventive concept of the present invention, and should not be construed as a limitation to the present invention. In the drawings:

[0032] Figure 1 is the fluorescence spectrum diagram obtained in Example 7 of the present invention;

[0033] Figure 2 is the test for the specific response ability of Compound I of the present invention to LTA4H;

[0034] Figure 3 is the representative confocal fluorescence image of RAW264.7 cells treated with different groups. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present application belong to the scope of protection of the present application.

[0036] For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0037] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0038] Example 1: Preparation of 1,4-dimethylquinolin-1-ium iodide (2)

[0039] Compound 1 (720 mg, 5 mmol) and CH3I (710 mg, 5 mmol) were dissolved in acetonitrile (5 mL), and the reaction was carried out at 80 °C for 2 h. After the reaction was completed, the mixture (crude product) was cooled to room temperature and filtered. The residue was washed with ethyl acetate and dried to obtain a yellow solid, which was compound 2 with a yield of 98%.

[0040] Analysis data of compound 2: 1 H NMR (DMSO-d6, 400 MHz) δ 9.44 (d, J = 6.0 Hz, 1H, ArH), 8.59 (m, 2H, ArH), 8.28 (ddd, J = 8.8, 7.0, 1.4 Hz, 1H, ArH), 8.14 (m, 2H, 2ArH), 4.62 (s, 3H, CH3), 3.02 (s, 3H, CH3).

[0041] Example 2: 4-((E)-2-((E)-6-chloro-5-(2-((Z)-1-methylquinolin-4(1H)-ylidene)ethylidene)cyclohex-1-en-1-yl))vinyl)-1-methylquinolin-1-ium iodide (4)

[0042] 4-Methyl-N-methylquinoline (432 mg, 2.5 mmol), N-((3-(phenyliminomethyl)-2-chloro-1-cyclohexen-1-yl)methylene)aniline hydrochloride (338 mg, 1.0 mmol) and anhydrous sodium acetate (205 mg, 2.5 mmol) were dissolved in 15 mL of ethanol, and the reaction was carried out at 80 °C for 1 h under nitrogen protection. After cooling, the solvent was removed by rotary evaporation, and then the mixture was stirred in chloroform / sodium acetate solution for 1 h, filtered, dried in vacuo, and separated by column chromatography (the eluent was methanol / dichloromethane, V / V, 1:19) to obtain 286 mg of a brownish-red solid (compound 4) with a yield of 47.2%.

[0043] Analysis data of compound 4: 11H NMR (DMSO-d6, 400 MHz) δ 9.85 (d, 1H, ArH), 8.12 (m, 6H, 6ArH), 6.72 (m, 5H, 3ArH, 2CH=C), 6.63 (s, 1H, CH=C), 6.52 (d, 1H, CH=C), 6.15 (s, 1H, CH=C), 5.45 (d, 1H, CH), 4.85 (q, 2H, 2CH=C), 2.76 (m, 4H, 2CH2), 1.52 (t, 3H, CH3), 1.38 (t, 3H, CH3).

[0044] Example 3: (E)-2-(2-(6-Hydroxy-7-(hydroxymethyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-methylquinolin-1-ium iodide (5)

[0045] Under N2 protection, compound 4 (778 mg, 1.332 mmol), 4-(hydroxymethyl)benzene-1,2-diol (1.29 g, 9.214 mmol), and KHCO3 (411.5 mg, 4.115 mmol) were dissolved in DMF (4 mL), heated at 75 °C and stirred for 4 h. After the reaction was completed, it was diluted with ethyl acetate (10 mL) and extracted with brine (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (DCM:MeOH = 50:1, v / v) to obtain a blue solid (compound 5) with a yield of 47%.

[0046] Analytical data of compound 5: 1 1H NMR (DMSO-d6, 400 MHz) δ 8.50 (d, J = 8.5 Hz, 1H, ArH), 8.21 (d, J = 14.0 Hz, 2H, 2ArH), 7.86 (d, J = 6.8 Hz, 1H, ArH), 7.60 (d, J = 7.2 Hz, 2H, 2ArH), 7.18 (d, J = 9.2 Hz, 2H, 2ArH), 6.90 (d, J = 14.1 Hz, 1H, CH=C), 6.53 (s, 1H, CH=C), 6.46 (dd, J = 8.7, 2.1 Hz, 1H, CH=C), 4.73 (s, 2H, CH2), 4.58 (s, 1H, OH), 4.12 (s, 1H, OH), 4.03 (s, 3H, CH3), 2.70 (t, J = 6.1 Hz, 2H, CH2), 2.59 (t, J = 6.0 Hz, 2H, CH2), 1.79 (dd, J = 12.7, 6.6 Hz, 2H, CH2).

[0047] Example 4: Benzyl (R)-2-((tert-butoxycarbonyl)amino)-3-((4-(hydroxymethyl)phenyl)amino)-3-oxopropionate (8)

[0048] Dissolve N-(tert-butoxycarbonyl)-L-aspartic acid benzyl ester (2.02 g, 6 mmol) in anhydrous DCM (15 mL). While stirring, sequentially add DIPEA (1.551 g, 12 mmol) and HATU (1.62 g, 12 mmol). Then continue stirring for 30 minutes, add 4-aminobenzyl alcohol (0.739 g, 6 mmol), and react for 24 h. After the reaction is completed, wash the reaction solution with saturated NaCl solution (3 × 200 mL), extract with ethyl acetate (3 × 150 mL), and concentrate the organic layer under reduced pressure over Na2SO4. Use petroleum ether / ethyl acetate (1:1, v / v) as the eluent for column chromatography to purify and obtain a yellow solid (Compound 8) with a yield of 78%.

[0049] Analytical data of Compound 8: 1 H NMR (DMSO-d6, 400 MHz) δ 9.98 (s, 1H, COOH), 7.59 (m, 2H, 2ArH), 7.39 (m, 5H, 5ArH), 7.25 (t, J = 8.5 Hz, 3H, 2ArH, NH), 5.17 (m, 3H, CH, CH2), 4.52 (s, 1H, OH), 4.44 (d, J = 5.7 Hz, 2H, CH2), 2.84 (dd, J = 16.2, 5.5 Hz, 1H, CH), 2.52 (s, 1H, CH), 1.39 (s, 9H, 3CH3).

[0050] Example 5: Benzyl (R)-3-((4-(bromomethyl)phenyl)amino)-2-((tert-butoxycarbonyl)amino)-3-oxopropionate (9)

[0051] Dissolve Compound 8 (1240 mg, 3 mmol) in CH2Cl2 (30 mL). After stirring in an ice bath for 10 min, slowly add phosphorus tribromide (812 mg, 3 mmol) dropwise and react for 20 min. After the reaction is completed, adjust the pH to 7 with 2 mol / L sodium bicarbonate solution, then extract with CH2Cl2. Dry the organic layer and concentrate under reduced pressure to obtain a white solid (Compound 9) with a yield of 79%.

[0052] Analytical data of Compound 9: 11H NMR (DMSO-d6, 400 MHz) δ 9.98 (s, 1H, COOH), 7.59 (m, 2H, 2ArH), 7.39 (m, 5H, 5ArH), 7.25 (t, J = 8.5 Hz, 3H, 2ArH, NH), 5.17 (m, 3H, CH, CH2), 4.44 (d, J = 5.7 Hz, 2H, CH2), 2.84 (dd, J = 16.2, 5.5 Hz, 1H, CH), 2.52 (s, 1H, CH), 1.39 (s, 9H, 3CH3).

[0053] Example 6: (R,E)-2-(2-(6-((4-(3-(Benzyloxy)-2-amino-3-oxopropanamido)benzyl)oxy)-7-(hydroxymethyl))-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-methylquinolin-1-ium iodide (I)

[0054] Compound 5 (591 mg, 1 mmol), compound 9 (476 mg, 1 mmol), KI (142.3 mg, 0.857 mmol), and potassium carbonate (42.7 mg, 0.427 mmol) were dissolved in anhydrous DMF (3 ml), protected by N2, and stirred at room temperature for 8 h. After the reaction was completed, the reaction solution was diluted with DCM (10 mL), washed with brine (3 x 10 mL), and the organic layer was dried over Na2SO4 and concentrated. After adding 1N HCl in methanol solution and stirring for 1 h, it was neutralized with triethylamine and concentrated, and purified by silica gel column chromatography (DCM:MeOH = 30:1, v / v) to obtain a blue solid (compound I) with a yield of 45%.

[0055] Analytical data of compound I: 1 1H NMR (CDCl3, 400 MHz) δ 8.51 (d, J = 14.6 Hz, 2H, 2ArH), 8.11 (d, J = 8.5 Hz, 1H, ArH), 7.86 (m, 2H, 2NH), 7.55 (m, 1H, ArH), 7.41 - 7.56 (m, 4H, 4ArH), 7.33 (d, J = 8.4 Hz, 1H, ArH), 7.15 - 7.22 (m, 6H, ArH), 6.91 (m, 2H, ArH), 6.18 (d, J = 14.9 Hz, 1H, CH=), 5.75 (d, J = 8.6 Hz, 1H, CH=), 5.06 (m, 3H, CH=, CH2), 4.72 (s, 2H, CH2), 3.81 (s, 2H, CH2), 2.95 (m, 1H, CH), 2.84 (m, 2H, CH2), 2.53 (m, 4H, 2CH2), 1.78 (t, J = 6.1 Hz, 2H, CH2), 1.22 (m, 3H, CH3).

[0056] Example 7: Fluorescence Spectral Properties of Compound I

[0057] The experiment was divided into the following groups:

[0058] Group I: Compound I was dissolved in PBS (pH 7.4, 5% DMSO) solution without adding LTA4H.

[0059] Group I + LTA4H: Compound I was dissolved in PBS (pH 7.4, 5% DMSO) solution and LTA4H (100 U / L) was added.

[0060] Group I + LTA4H + Ubenimex: Compound I was dissolved in PBS (pH 7.4, 5% DMSO) solution, Ubenimex (LTA4H inhibitor) was added, and then LTA4H (100 U / L) was added.

[0061] Using 721 nm as the excitation wavelength, a fluorescence spectrometer was used to collect the fluorescence emission spectral data of the three groups of samples at 721 - 900 nm. The results are as Figure 1 shown.

[0062] According to Figure 1 it can be seen that the fluorescence intensity of the compound I of the present invention was significantly enhanced after incubation with LTA4H, and the fluorescence emission was at 762 nm; after treatment with the LTA4H inhibitor Ubenimex, the fluorescence intensity was significantly weakened, proving that the compound of the present invention can respond to LTA4H.

[0063] Example 8: Test on the Specific Response Ability of Compound I of the Present Invention to LTA4H

[0064] The fluorescence spectrometry was used to detect the specific response ability of the compound LTA4H of the present invention, and the selectivity of the compound of the present invention to potential interferents such as cations, amino acids, redox agents and other enzymes was evaluated. At room temperature, the compound of the present invention was added to the solutions (1 μM) of the corresponding bioanalytes (such as KCl, ZnCl2, MgCl2, CaCl2, FeCl3, LAP, L-Phe, L-Ser, NQO1, VcNa, H2O2, GST1 and LTA4H 100 U / L) respectively, and PBS (pH 7.4, 5% DMSO) solution was used at 37 °C for 0.5 h. All emission spectra were excited at 720 nm and recorded at 720 - 800 nm.

[0065] Among them, Figure 2 is the fluorescence intensity of the compound of the present invention at 762 nm in the presence of different species. The results show ( Figure 2) When a variety of interfering substances are introduced into the solution of the compound of the present invention, the effects of these interfering substances on its fluorescence signal are negligible. It is noteworthy that the introduction of LTA4H has caused a significant increase in fluorescence intensity, which confirms that the compound of the present invention has a highly specific response characteristic to LTA4H.

[0066] Example 9: Selective imaging ability of Compound I for LTA4H in an inflammation model

[0067] The selective imaging ability of Compound I in an inflammation model was evaluated by confocal fluorescence imaging.

[0068] The experiment was divided into four groups.

[0069] PBS control group: RAW264.7 cells without any treatment were used as a control.

[0070] Compound I group: RAW264.7 cells were incubated with Compound I for 4 h.

[0071] LPS + Compound I group: RAW264.7 cells were incubated with LPS (lipopolysaccharide, 1 μg / mL) for 24 h, and then treated with Compound I for 4 h.

[0072] LPS + Compound I + Ubenimex group: RAW264.7 cells were incubated with LPS (1 μg / mL) for 24 h, then treated with the LTA4H inhibitor Ubenimex for 24 h, subsequently treated with Compound I for 4 h, washed with PBS, and then the cell nuclei were stained with DAPI.

[0073] Images were taken with a confocal microscope, Figure 3 which are representative confocal fluorescence images of RAW264.7 cells treated with different groups.

[0074] The results showed ( Figure 3 ) that negligible red fluorescence was observed in normal and Compound I-treated RAW264.7 cells. In contrast, LPS + Compound I-treated cells showed obvious red fluorescence. The addition of the LTA4H inhibitor Ubenimex resulted in minimal fluorescence, emphasizing the specific cleavage induced by LTA4H. It was demonstrated that the change in the fluorescence signal of Compound I was caused by the selective cleavage of intracellular LTA4H, thus verifying the effectiveness of Compound I in monitoring the activity of intracellular LTA4H. Overall, the cell fluorescence imaging experiment showed that the change in the fluorescence signal of the compound of the present invention was selectively triggered by intracellular LTA4H, and the compound of the present invention could effectively monitor the activity of LTA4H in living cells.

Claims

1. A leukotriene A4 hydrolase-activated fluorescent probe, characterized in that, The leukotriene A4 hydrolase-activated fluorescent probe has the structure shown by the following formula:

2. A method for preparing a leukotriene A4 hydrolase-activated fluorescent probe as described in claim 1, characterized in that, The preparation method includes the following steps: S1. Compound 3 and methylated 1,4-dimethylquinolin-1-ium iodide are reacted under the condition of sodium acetate to obtain compound 4, and compound 4 undergoes a nucleophilic reaction with 3,5-dihydroxybenzyl alcohol to obtain compound 5; The synthetic route of step S1 is shown by the following formula: S2. L-Aspartic acid 1-benzyl ester and 4-aminobenzyl alcohol are dehydrated and condensed under the action of HATU and DIPEA to generate compound 8, and then brominated with PBr3 to obtain compound 9; The synthetic route of step S2 is shown by the following formula: S3. Compound 5 and compound 9 are subjected to an etherification reaction under alkaline conditions, and then the Boc protection is removed to generate compound I, and compound I is the leukotriene A4 hydrolase-activated fluorescent probe; The synthetic route of step S3 is shown by the following formula:

3. The preparation method according to claim 2, characterized in that, The preparation method includes the following steps: S100. N-[(3-(Anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride, 1,4-dimethylquinolin-1-ium iodide and sodium acetate are dissolved in absolute ethanol, and refluxed under a nitrogen atmosphere to obtain compound 4; S200. Compound 4, 3,5-dihydroxybenzyl alcohol and KHCO3 are dissolved in anhydrous DMF, and reacted under a nitrogen atmosphere to obtain compound 5; S300. N-(tert-Butoxycarbonyl)-L-aspartic acid benzyl ester is dissolved in anhydrous dichloromethane, DIPEA and HATU are added sequentially with stirring, and then 4-aminobenzyl alcohol is added, and the reaction is carried out to obtain compound 8; S400. Compound 8 is dissolved in anhydrous dichloromethane, and phosphorus tribromide is slowly added dropwise under an ice bath condition, and the reaction is carried out to obtain compound 9; S500. Compound 5, compound 9, K2CO3 and KI are dissolved in anhydrous DMF, and stirred and reacted at 40-50 °C under nitrogen protection. After extraction and washing with dichloromethane, the organic phase is concentrated to obtain a concentrated solution, and the Boc group is removed by adding HCl methanol solution to obtain the leukotriene A4 hydrolase-activated fluorescent probe.

4. According to the preparation method described in claim 3, characterized in that In step S500, the stirring reaction is an overnight reaction; the time for the Boc deprotection reaction is 1 h.

5. Use of a leukotriene A4 hydrolase-activated fluorescent probe as described in claim 1 in the preparation of a fluorescent diagnostic reagent for inflammatory diseases.

6. The application according to claim 5, characterized in that The inflammatory disease is inflammatory bowel disease.

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