Preparation method and application of probe Cu-HClO for detecting peroxynitrous acid

Abstract

The invention aims to disclose a preparation method and application of a probe Cu-HClO for detecting peroxynitrous acid. Peroxynitrous acid is used as an important physiological activation factor and signal molecule, and can regulate dynamic oxidation-reduction dynamic balance in a living body. Fluorescence sensing and fluorescence imaging can mark and track molecules with wide biological activity, and have good temporal and spatial resolution. Coumarin has unique fluorescence, and the fluorescence characteristic of coumarin depends on the structure of coumarin to a great extent, so that the modification of coumarin becomes a basis for constructing an efficient fluorescent probe. The novel fluorescent probe for detecting peroxynitrous acid is obtained on the basis of modifying a coumarin structure.

Description

Technical Field

[0001] The present invention relates to the field of chemical medicine, and in particular to a preparation method and application of a peroxynitrous acid probe Cou-HClO. Background Art

[0002] Aerobic organisms produce various reactive nitrogen species (RNS) during their life processes, including nitric oxide (NO), nitrogen dioxide (NO2), peroxynitrite (ONOO - ) and nitroxyl (COU-HCLONO). NO is produced by different subtypes of nitric oxide synthase in different cells, and peroxynitrite is produced by the non-enzymatic reaction of NO and O2. Under physiological conditions, ONOO - In equilibrium with its protonated form (ONOOCOU-HCLO), ONOOCOU-HCLO rapidly decomposes to form ·OCOU-HCLO and NO2. NO is involved in regulating physiological processes such as cardiovascular, immune and neural processes, and plays an important role in pathological processes such as cancer, neurodegenerative damage and inflammation. ONOO oxidizes carbohydrates, lipids, proteins and DNA in various physiological and pathological processes, thus leading to many pathological phenomena, including cardiovascular, neurodegenerative diseases and cancer. Peroxynitrite is an essential cell signaling pathway regulator and physiological activation molecule in biological processes. ONOO - It helps regulate the dynamic redox balance in organisms. It can affect the transcription factors of redox in cells, subsequently regulate gene expression, and can help humans resist the invasion of exogenous harmful substances. In addition, ONOO - It can kill cancer cells and has the effect of assisting in the treatment of cancer. Based on this, the present invention discloses a probe activated by peroxynitrite to achieve the purpose of targeted cancer cell therapy. Summary of the invention

[0003] Based on the current status of the above-mentioned prior art research, the present invention provides a method for preparing a fluorescent probe Cou-HClO for detecting peroxynitrite and its use. The peroxynitrite probe Cou-HClO of the present invention can detect ONOO in cells. - . BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 The probe Cou-HClO and ONOO of Example 1 - Time-dependent fluorescence spectra after action.

[0005] Figure 2 The probe Cou-HClO and ONOO of Example 1 - The fluorescence spectrum after action is concentration-dependent.

[0006] Figure 3 The probe Cou-HClO and ONOO of Example 1 - Fluorescence intensity under different pH conditions.

[0007] Figure 4 The fluorescence intensity of the probe Cou-HClO in Example 1 was selectively tested. Example 1

[0008] The present invention discloses a peroxynitrite probe Cou-HClO, the structure of which is shown in the following formula:

[0009]

[0010] The specific preparation method of the peroxynitrite probe Cou-HClO of the present invention is as follows:

[0011]

[0012] The specific preparation method of Cou-HClO of the present invention is as follows:

[0013] Synthesis of compound Cou-1: Weigh compound a (140.11 mg, 1 mmol, 1 equiv.) and compound b (71.12 mg, 1 mmol, 1 equiv.) respectively, mix and dissolve in 15 mL of ultra-dry DMF under Ar gas protection. Stir at room temperature for 0.5 h and then add TEA (101.12 mg, 1 equiv.). Heat to 95 °C and stir under reflux for 24 h. After the reaction is completed, add 150 mL of distilled water and 20 mL × 3 of ethyl acetate for extraction. Combine the organic phases and dry them over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, purify using a silica gel column (PE: EA = 20: 1). After purification, spin dry the solvent to obtain compound Cou-1. Yield: 85%. 1H NMR (400MHz, Chloroform-d) δ11.70 (s, 1H), 9.50 (s, 1H), 7.28 (s, 1H), 6.17 (dd, J=8.7, 2.3Hz, 1H), 5.97 (d, J=2.3Hz, 1H), 3.40-3.36 (m, 4H), 2.06-2.01 (m, 4H).

[0014] Synthesis of compound Cou-2: Weigh diethyl malonate (320.34 mg, 2 equiv.), hexahydropyridine (340.6 mg, 4 equiv.) and 40 mL of anhydrous ethanol respectively, mix under Ar gas protection and stir at room temperature for 0.5 h. Weigh compound Cou-1 (191.01 mg, 1 mmol, 1 equiv.) and add it to the previously mixed reaction solution. Heat to 90°C and stir and reflux for 6 h. Spin dry the liquid and add 40 mL of 6 mol / L hydrochloric acid solution. Stir and reflux at 130°C for 6 h. After the solution is cooled to room temperature, slowly add 5 mol / L NaOH solution in an ice water bath to adjust to pH = 6. At this time, solids precipitate. Filter the reaction solution and wash with anhydrous ethanol. Scrape out the solid and dry it in a vacuum drying oven. Yield: 71%. 1 H NMR (600MHz, Chloroform-d) δ7.54 (d, J=9.3Hz, 1H), 7.25 (d, J=8.7Hz, 1H), 6.47 (dd, J=8.6, 2 .3Hz, 1H), 6.38 (d, J=2.3Hz, 1H), 6.04 (d, J=9.3Hz, 1H), 3.37-3.34 (m, 4H), 2.07-2.04 (m, 4H).

[0015] Synthesis of compound Cou-3: Weigh phosphorus oxychloride (383.32 mg, 2.5 equiv.) and ultra-dry DMF (365.47 mg, 5 equiv.) respectively, mix and stir in an ice-salt bath under Ar protection for 0.5 h. Weigh compound Cou-2 (215.25 mg, 1 mmol, 1 equiv.) and dissolve it in 15 mL ultra-dry DMF, then add it to the reaction solution, mix and stir in an ice-salt bath for 0.5 h. Heat to 60 ° C and react for 12 h. Pour the reaction solution into ice water, and add saturated sodium bicarbonate solution to quench the reaction. Add 150 mL of distilled water and 20 mL × 3 of ethyl acetate to extract. Combine the organic phases and dry them over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, purify it using a silica gel column (PE: EA = 5: 1). After purification, the solvent is dried by rotary evaporation to obtain compound Cou-3. Yield: 61%. 1 H NMR (400MHz, DMSO-d6) δ9.89 (s, 1H), 8.43 (s, 1H), 7.70 (d, J=8.9Hz, 1H), 6.72 -6.69 (m, 1H), 6.48 (d, J = 2.3Hz, 1H), 3.45-3.42 (m, 4H), 1.99 (q, J = 3.4Hz, 4H).

[0016] Synthesis of compound Cou-HClO: Weigh malononitrile (132.04 mg, 2 equiv.), hexahydropyridine (340.6 mg, 4 equiv.) and 50 mL of anhydrous ethanol, mix under Ar gas protection and stir at room temperature for 0.5 h. Weigh compound Cou-3 (243.26 mg, 1 mmol, 1 equiv.) and add it to the previously mixed reaction solution. Stir at room temperature for 6 h. After removing the solvent by rotary evaporation, purify using a silica gel column (PE: EA = 3: 1). After purification, spin dry the solvent to obtain compound Cou-HClO. Yield: 43%. 1 H NMR (400 MHz, Chloroform-d) δ 8.07 (s, 1H), 6.35 (s, 1H), 3.63 (s, 2H), 3.47 (s, 6H).

[0017] Example 2: Fluorescence spectrum of probe Cou-HClO after reaction with peroxynitrous acid (time dependence)

[0018] Experimental method: To study the effect of probe Cou-HClO on ONOO - The response of the probe Cou-HClO was tested by fluorescence spectroscopy and time dependence test.

[0019] Experimental results: Figure 1 As shown, Cou-HClO and ONOO - After the reaction, the fluorescence intensity at 495 nm gradually increased with time and reached equilibrium at 18 min.

[0020] Example 2: Fluorescence spectrum of probe Cou-HClO after reaction with peroxynitrous acid (concentration dependence)

[0021] Experimental method: To study the effect of probe Cou-HClO on ONOO - The response of the probe Cou-HClO was tested by fluorescence spectroscopy and concentration dependence test.

[0022] Experimental results: Figure 2 As shown, Cou-HClo and ONOO - After the reaction, the fluorescence intensity at 495 nm increased with the increase of ONOO - It gradually increases with the increase of concentration.

[0023] Example 3: pCOU-HCLO test of probe Cou-HClO

[0024] Experimental method: In order to study the fluorescence intensity of the probe Cou-HClO under different pCOU-HCLO, the probe Cou-HClO was subjected to pCOU-HCLO test.

[0025] Experimental results: Figure 3 As shown, Cou-HClO responded at a pCOU-HCLO of about 7.4, indicating that the probe can be used under physiological conditions.

[0026] Example 4: Selectivity test of probe Cou-HClO

[0027] Experimental method: To verify whether the probe Cou-HClO can be well applied to biological systems, we further evaluated the effect of the probe Cou-HClO on ONOO - Selectivity. 1: blank, 2: Asp, 3: Glu, 4: Ala, 5: Cys, 6: Gly, 7: Ile, 8: Leu, 9: PCou-HClOe, 10: Pro 11: Ser, 12: TCou-HClOr, 13: Val, 14: COU-HCLOis, 15: Lys

[0028] Experimental results: Figure 4 As shown, the probe Cou-HClO reacts with ONOO - There is a certain degree of selectivity.

Claims

1. The present invention discloses a method for preparing a probe Cou-HClO for detecting peroxynitrous acid and its use. The structure of the Cou-HClO of the present invention is shown in the following formula (I): （Ⅰ）。 2. The probe Cou-HClO is characterized by: Its structure is shown in the following formula (I): （Ⅰ）。 3. Cou-HClO according to claim 2, characterized in that The compound has the following structure: （Ⅰ）。 4. A use of the peroxynitrite probe Cou-HClO as claimed in claim 2 or 3, characterized in that Peroxynitrite can be selectively detected.

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