Fluorescent quinoline compounds, methods of making and uses thereof

Abstract

The application discloses a quinoline fluorescent compound, a preparation method and application thereof, and belongs to the field of biological medicines. The application provides a novel quinoline fluorescent compound or a pharmaceutically acceptable salt thereof with a structure shown in a general formula I, which has low toxicity, a long emission wavelength, and is of great significance in the field of biological imaging.

Description

Technical Field

[0001] This invention relates to quinoline fluorescent compounds, their preparation methods, and uses, and belongs to the field of biomedicine. Background Technology

[0002] Imaging is an indispensable tool in the diagnosis, treatment, and research of diseases. Fluorescence imaging technology has also received widespread attention from researchers with the development of modern medicine, with the aim of developing an imaging molecular tool that can help doctors visualize the expression of specific molecules, cells, and biological processes. Biofluorescence imaging, with its high sensitivity, simplicity of implementation, real-time detection, and good compatibility with biological samples, has become one of the newest and most powerful biochemical techniques for monitoring and tracking targets in biological systems.

[0003] Quinoline is a bicyclic heterocyclic system composed of a six-membered benzene ring fused with pyridine. It is a multifunctional pharmacophore, and quinoline and its derivatives possess a wide range of biological activities, such as anti-Alzheimer's disease, anti-diabetic, antibacterial, anti-inflammatory, anticancer, and antimalarial effects. Furthermore, quinoline has a large conjugated system, exhibiting π-π* electronic transition characteristics, making it a potential fluorophore with excellent fluorescence properties. Some of its derivatives have already been used in metal ion detection and biosensors.

[0004] Therefore, exploring design strategies to optimize the fluorescence properties of the quinoline skeleton and obtaining novel fluorescent molecules that can visualize disease development and treatment processes is of great significance. Summary of the Invention

[0005] This invention provides a novel class of quinoline-based fluorescent molecules and applies them to the development of fluorescent probes and biofluorescence imaging technologies.

[0006] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0007] [Compound]

[0008] A class of novel quinoline fluorescent compounds or pharmaceutically acceptable salts thereof with the structure shown in Formula I:

[0009]

[0010] in,

[0011] R1 is selected from halogens (F, Cl, Br, I), C1-4 alkoxy groups, and aryl groups.

[0012] R2 is selected from -CN, -COOR4;

[0013] R3 is selected from H, -OR', -COOR", and R4, R', and R" are each independently selected from H and C1-4 alkyl groups.

[0014] In one embodiment of the present invention, R1 is specifically selected from -Br and -OCH3, and R2 is specifically selected from -CN and -COOH.

[0015] In one embodiment of the present invention, the structure of the novel quinoline fluorescent compound is shown in Formula II:

[0016]

[0017] Among them, R3 is independently selected from -COOMe, -COOH, -H, and -OH, and R2 is independently selected from -CN and -COOH.

[0018] In one embodiment of the present invention, the structure of the novel quinoline fluorescent compound is shown in Formula II:

[0019]

[0020] Among them, R3 is independently selected from -COOMe, -COOH, -H, and -OH, and R2 is independently selected from -CN and -COOH.

[0021] In one embodiment of the present invention, most preferably, the specific structure of a novel quinoline fluorescent compound is as follows:

[0022]

[0023]

[0024] In one embodiment of the present invention, pharmaceutically acceptable salts include inorganic salts or organic salts; wherein, inorganic salts include hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, hydrogen sulfate, nitrate, phosphate, and acid phosphate; and the organic salts are selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, and ascorbate.

[0025] [Synthesis Method]

[0026] This invention also provides methods for preparing the above-mentioned novel quinoline fluorescent compounds of general formulas I and II (the method for preparing quinoline fluorescent compounds of general formula III is the same as that for general formula II), and the methods are implemented by the following reaction scheme:

[0027] Synthesis Scheme 1

[0028]

[0029] Reagents and conditions: a) Phosphorus oxychloride, N,N-dimethylformamide, 90℃, 16h; b) 70% acetic acid, 90℃, 12h; c) Anhydrous ethanol, pyridine, 75℃, 4h

[0030] Phosphorus oxychloride was added dropwise to DMF under ice bath conditions and stirred at room temperature for 30 minutes. Then, compound 1-1 was added to the mixed solution and refluxed in an oil bath at 90°C for 16 hours. After the reaction was complete, the reaction solution was added dropwise to ice water, and a solid precipitated. The solid was filtered, washed with water, collected, and dried under vacuum to obtain compound 1-2. Compound 1-2 was dissolved in 70% acetic acid solution, and the reaction solution was heated under reflux in an oil bath at 90°C for 14 hours. After the reaction was complete, the solid was filtered, washed with water, collected, and dried under vacuum to obtain compound 1-3. Compound 1-3 was dissolved in anhydrous ethanol, 3 drops of pyridine were added, and then malononitrile was added. The solution was refluxed in an oil bath at 75°C for 4 hours. After the reaction was complete, the solid was filtered, washed with anhydrous ethanol, collected, and dried under vacuum to obtain compound I-4.

[0031] Synthesis Scheme 2

[0032]

[0033] Reagents and conditions: a) chloroform, pyridine, 60℃, 48h;

[0034] Dissolve compound 1-3 in chloroform, add 3 drops of pyridine, then add cyanoacetic acid. Reflux the solution in an oil bath at 60°C for 48 hours. After monitoring the reaction to ensure complete reaction, filter the solid, wash with anhydrous ethanol, collect the solid, and dry under vacuum to obtain compound 2-1.

[0035] Synthesis Scheme 3

[0036]

[0037] Reagents and conditions: a) 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, potassium carbonate, N,N-dimethylformamide (DMF), H2O, 80℃, 4h; b) phosphorus oxychloride, N,N-dimethylformamide (DMF), 8h; c) acetic acid, 90℃, 16h; d) lithium hydroxide, tetrahydrofuran (THF), H2O, 24h;

[0038] Compounds 3-1, 3-2, potassium carbonate, and 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride were dissolved in a mixed solution of DMF and H2O (20:1). N2 was replaced three times. The solution was refluxed in an oil bath at 80°C for 4 hours. After the reaction was complete, ethyl acetate and saturated brine were added for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. Compound 3-3 was purified by column chromatography. Phosphorus oxychloride was added dropwise to DMF under ice bath conditions, and the mixture was stirred at room temperature for 30 minutes. Then, 3-3 was added to the mixed solution, and the solution was refluxed in an oil bath at 90°C for 8 hours. After the reaction was complete, The reaction solution was dropped into ice water, and a solid precipitated. The solid was filtered, washed with water, and collected. The solid was purified by column chromatography to obtain compound 3-4. Compound 3-4 was dissolved in acetic acid solution, and the solution was heated under reflux in an oil bath at 90°C for 16 h. After monitoring the completeness of the reaction, the solid was filtered, washed with water, collected, and dried under vacuum to obtain compound 3-5. Compound 3-5 was dissolved in THF, and lithium hydroxide aqueous solution was added under ice bath conditions. The mixture was stirred at room temperature for 24 h. After monitoring the completeness of the reaction, the solvent was removed by vacuum drying to obtain a solid. The solid was dissolved in water, and 1M hydrochloric acid solution was added to adjust the pH to 1-2. The solid was filtered, freeze-dried, and obtained compound 3-6.

[0039] Synthesis Scheme 4

[0040]

[0041] Same as step c in synthesis scheme 1.

[0042] Synthesis Scheme 5

[0043]

[0044] Same as step a in synthesis scheme 2.

[0045] [use]

[0046] The present invention also provides the application of the above-mentioned novel quinoline fluorescent compounds or their pharmaceutically acceptable salts in the field of fluorescent probe preparation.

[0047] The present invention also provides the application of the above-mentioned quinoline fluorescent compounds or their pharmaceutically acceptable salts in the field of bioimaging.

[0048] The present invention also provides a bioimaging reagent containing the above-mentioned fluorescent compound or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

[0049] Effective effects:

[0050] This invention contains novel quinoline-based fluorescent compounds with a maximum excitation wavelength between 350-450 nm and a maximum emission wavelength between 550-630 nm, exhibiting excellent fluorescence properties and successfully applied to tumor cell fluorescence imaging. Furthermore, cytotoxicity experiments show low cytotoxicity, providing a new molecular tool for biofluorescence imaging and integrated diagnosis and treatment, which is of significant importance for the diagnosis, treatment, and research of related tumor diseases. Attached Figure Description

[0051] Figure 1 The image shows the results of the test on the activity of the compound in Example 3 in inhibiting human peripheral blood mononuclear cells (PBMCs).

[0052] Figure 2 This is a diagram showing the cell imaging results of the compound in Example 4 in U2OS cells. Detailed Implementation

[0053] The synthesis process involved in this application includes the following steps:

[0054] Unless otherwise specified, the methods and instruments used in this invention are technologies known in the art.

[0055] Example 1

[0056]

[0057] Reagents and conditions: a) Phosphorus oxychloride, N,N-dimethylformamide (DMF), 90℃, 16h; b) 70% acetic acid, 90℃, 12h; c) Anhydrous ethanol, pyridine, 75℃, 4h;

[0058] Phosphorus oxychloride (32 g, 0.210 mol) was added dropwise to DMF (4.3 g, 0.058 mol) under ice bath conditions, and stirred at room temperature for 30 minutes. Then, 3-bromo-N-acetanilide 1-1 (5 g, 0.023 mol) was added to the mixed solution, and the mixture was refluxed in an oil bath at 90 °C for 16 h. After monitoring the reaction to ensure complete reaction, the reaction solution was added dropwise to ice water, and a solid precipitated. The solid was filtered, washed with water, collected, and dried under vacuum to give compound 1-2 (2.2 g, 35% yield).

[0059] Weigh 1-2 (2.0 g, 0.007 mol) and dissolve it in 20 ml of 70% acetic acid solution. Place the reaction solution in a 90℃ oil bath and heat under reflux for 14 h. After monitoring the reaction to be complete, filter the solid, wash it with water, collect the solid, and dry it under vacuum to obtain compound 1-3 (1.3 g, yield 68%).

[0060] Dissolve 1-3 (1.0 g, 0.004 mol) in 10 mL of anhydrous ethanol, add 3 drops of pyridine, and then add malononitrile (0.5 g, 0.008 mol). Place the solution in an oil bath at 75 °C and reflux for 4 h. After monitoring the reaction to ensure it is complete, filter the solid, wash it with anhydrous ethanol, collect the solid, and dry it under vacuum to give yellow solid I-2 (0.9 g, yield 73%).

[0061] 1 H NMR (400MHz, DMSO-d6) δ12.43(s,1H),8.71(s,1H),8.34(s,1H),7.80(d,J=8.4Hz,1H),7.52(d,J=1.6Hz,1H),7.44(dd,J=8.4,1.6Hz,1H).MS(ESI):m / z calcd forC 13 H7BrN3O[M+H] + 299.97, found 299.85.

[0062] Example 2

[0063]

[0064] Reagents and conditions: a) chloroform, pyridine, 60℃, 48h;

[0065] Dissolve compound 1-3 (1.0 g, 0.004 mol) in 10 mL of chloroform, add 3 drops of pyridine, and then add cyanoacetic acid (0.7 g, 0.008 mol). Reflux the solution in an oil bath at 60 °C for 48 h. After monitoring the reaction to ensure it is complete, filter the solid, wash with anhydrous ethanol, collect the solid, and dry it under vacuum to give compound I-4 (0.773 g, yield 62%).

[0066] 1 H NMR (600MHz, DMSO-d6) δ12.34(s,1H),8.79(s,1H),8.41(s,1H),7.78(d,J=8.4Hz,1H),7.52(d,J=1.8Hz,1H),7.42(dd,J=8.4,1.8Hz,1H).MS(ESI):m / z calcd forC 13 H8BrN2O3[M+H] + 318.96, found 319.00.

[0067] Example 3

[0068]

[0069] Reagents and conditions: a) 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, potassium carbonate, N,N-dimethylformamide (DMF), H2O, 80℃, 4h; b) phosphorus oxychloride, N,N-dimethylformamide (DMF), 8h; c) acetic acid, 90℃, 16h; d) lithium hydroxide, tetrahydrofuran (THF), H2O, 24h;

[0070] 3.0 g (0.0112 mol) of 4-methoxyformylphenylboronic acid pinacol ester 3-1, 2.0 g (0.009 mol) of p-bromoacetanilide 3-2, 2.6 g (0.011 mol) of potassium carbonate, and 0.7 g (0.0009 mol) of 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride were dissolved in a mixed solution of 20 mL DMF and 1 mL H2O. The solution was refluxed three times with N2 and placed in an oil bath at 80 °C for 4 h. After monitoring the reaction to completion, ethyl acetate and saturated brine were added for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain compound 3-3 (1.96 g, 77% yield).

[0071] Phosphorus oxychloride (10.3 g, 0.067 mol) was added dropwise to DMF (1.4 g, 0.017 mol) under ice bath conditions, and stirred at room temperature for 30 minutes. Then, compound 3-3 (2.0 g, 0.007 mol) was added to the mixed solution. The solution was refluxed in a 90°C oil bath for 8 hours. After monitoring the reaction to ensure complete reaction, the reaction solution was added dropwise to ice water, and a solid precipitated. The solid was filtered, washed with water, collected, and purified by column chromatography to obtain compound 3-4 (0.489 g, 20% yield).

[0072] Dissolve compound 3-4 (1.0 g, 0.003 mol) in 10 mL of acetic acid solution. Heat the solution in a 90 °C oil bath under reflux for 16 h. After monitoring the reaction to ensure complete reaction, filter the solid, wash with water, collect the solid, and dry it under vacuum to give compound 3-5 (0.831 g, yield 88%).

[0073] Dissolve 3-5 (1.0 g, 0.003 mol) in 5 mL of tetrahydrofuran, add 5 mL of lithium hydroxide aqueous solution (2 M) under ice bath conditions, stir at room temperature for 24 h, monitor the reaction until complete, remove the solvent by vacuum drying to obtain a solid, dissolve in water, adjust the pH to 1-2 with dilute hydrochloric acid, filter the solid, freeze dry to obtain compound 3-6 (8.9 g, yield 93%).

[0074] Example 4

[0075]

[0076] Reagents and conditions: a) Anhydrous ethanol, pyridine, 75℃, 4h;

[0077] Dissolve 3-5 (0.500 g, 0.002 mol) in 5 mL of anhydrous ethanol, add 3 drops of pyridine, and then add malononitrile (0.213 g, 0.003 mol). Place the solution in an oil bath at 75 °C and reflux for 4 h. After monitoring the reaction to be complete, filter the solid, wash with anhydrous ethanol, collect the solid, and dry it under vacuum to give compound II-1 (0.399 g, yield 69%).

[0078] 1 H NMR (600MHz, DMSO-d6) δ12.53(s,1H),8.82(s,1H),8.39(s,1H),8.31(d,J=1.8Hz,1H),8.11(dd,J=8.4,1 .8Hz,2H),8.05(d,J=8.4Hz,2H),7.93(d,J=8.4Hz,2H),7.46(d,J=8.4Hz,1H),3.88(s,3H).MS(ESI):m / z calcd for C 21 H 12 N3O3[MH] - 355.10, found 354.05.

[0079] Example 5

[0080]

[0081] Reagents and conditions: a) chloroform, pyridine, 60℃, 48h;

[0082] Dissolve 3-5 (0.500 g, 0.002 mol) in 5 mL of chloroform, add 3 drops of pyridine, and then add cyanoacetic acid (0.265 g, 0.003 mol). Reflux the solution in an oil bath at 60 °C for 48 h. After monitoring the reaction to ensure it is complete, filter the solid, wash with anhydrous ethanol, collect the solid, and dry it under vacuum to give compound II-4 (0, 387 g, yield 67%).

[0083] 1 H NMR(600MHz,DMSO-d6)δ12.42(s,1H),8.90(s,1H),8.47-8.44(m,1H),8.25(d,J=1.8Hz,1 H),8.07-8.04(m,4H),7.93-7.91(m,2H),7.45(d,J=9.0Hz,1H),3.88(s,3H).MS(ESI):m / z calcd for C 21 H 13 N2O5[MH] - 373.09, found 373.00.

[0084] Except for appropriate substitution of the corresponding reaction compounds, the following compounds were prepared by the method described above, and different compounds were obtained, as shown in Table 1.

[0085] Table 1 Characterization data of different quinoline fluorescent compounds

[0086]

[0087]

[0088]

[0089] Example 6: Spectroscopic testing of quinoline fluorescent compounds

[0090] 1) Ultraviolet absorption spectroscopy experiment

[0091] Weigh 1 mg of the compound and prepare a 1 M stock solution with DMSO. Pipette 20 μL of the stock solution into a cuvette, add 180 μL of DMSO, mix well, and measure the ultraviolet absorption spectrum.

[0092] 2) Fluorescence emission spectrum

[0093] Weigh 1 mg of the compound and prepare a 1 M stock solution with DMSO. Transfer 20 μL of the stock solution into a cuvette using a pipette, add 180 μL of DMSO, mix well, and use the UV absorption characteristic peak as the excitation wavelength to determine the fluorescence emission spectrum.

[0094] The test results obtained in Example 2 are shown in Table 2.

[0095] Table 2 Spectral data of novel quinoline fluorescent compounds

[0096]

[0097]

[0098] a: Measurement of fluorescence quantum yield using quinine sulfate as a standard compound

[0099] Example 7: Assay for the inhibitory activity of compounds on human peripheral blood mononuclear cells (PBMCs)

[0100] 1) Materials:

[0101] Cell line: PBMC

[0102] Reagents: 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, 96-well white plates;

[0103] 2) Procedure: Cells were seeded at a density of 10,000 cells / well in clear 96-well cell culture plates, with a cell suspension volume of 80 μL per well. The culture plates were then incubated at 37°C for 2 hours to equilibrate. The culture conditions were 37°C + 5% CO2. Then, graded concentrations of compounds II-3 and II-4 were added. Three days later, 20 μL of CTG solution was added, and after incubation at room temperature for 20 minutes, data were recorded using an Envision microplate reader (Perkin Elmer) to calculate relative cell viability.

[0104] 3) Sample preparation: Dissolve the sample in DMSO and store at -20℃. The concentration of DMSO in the final system should be controlled within the range that does not affect the detection activity.

[0105] The test results are shown below Figure 1 As can be seen from the figure, compounds II-3 and II-4 exhibit low cytotoxicity and have potential applications in cell imaging.

[0106] Example 8: Cell imaging assay of the compound in U2OS cells

[0107] Confocal microscope.

[0108] U2OS cells: U2OS cells were cultured on glass culture dishes for 24 hours before treatment. They were then incubated with 20 μM compounds II-3 and II-4 for 16 hours each, washed three times with PBS, fixed in 4% paraformaldehyde solution for 15 minutes, and washed again with PBS. Cell images were obtained using a Nikon Ti2-E+A1 confocal microscope. Figure 2 As shown.

[0109] The results analysis showed that in U2OS cell fluorescence imaging, compound II-3 emitted stronger blue light than II-4.

Claims

1. A class of quinoline fluorescent compounds of general formula II or pharmaceutically acceptable salts thereof. ， in, R3 is independently selected from -COOMe and -COOH, and R2 is independently selected from -CN and -COOH.

2. The quinoline fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The structure of the quinoline-based fluorescent compound is shown below: 。 3. The quinoline fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Pharmaceutically acceptable salts include inorganic or organic salts; wherein, inorganic salts are selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, nitrate, and phosphate; and the organic salts are selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, and ascorbate.

4. The quinoline fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, The sulfate is a hydrogen sulfate; the phosphate is an acid phosphate.

5. The quinoline fluorescent compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The quinoline fluorescent compounds are excited at wavelengths of 200-800 nm and emit fluorescence at wavelengths of 400-800 nm.

6. The use of the fluorescent compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the field of preparing fluorescent probes.

7. The use of the fluorescent compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of bioimaging reagents.

8. A bioimaging reagent, characterized in that, It contains the fluorescent compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

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