Quinazolidinone-based organic probe, synthesis method and application thereof

Abstract

The present invention belongs to organic probe compounds and fields, and in particular to an organic probe based on a quinazolidinone structure, a synthesis method thereof and its application. The present invention uses triphenylamine as an electron donor, quinazolidinone as an electron acceptor, and 2,4-dinitrobenzenesulfonyl as a response site for hydrogen sulfide to synthesize a new type of organic probe based on a quinazolidinone structure, which is synthesized for the first time by the present invention, and the raw materials are easily available, the synthesis steps are simple, and industrialization is possible. And the organic probe has high selectivity and high sensitivity for the detection of hydrogen sulfide, can realize rapid detection of hydrogen sulfide, and is accompanied by the phenomenon of fluorescence on. It has a wide range of value in the detection of environmental hydrogen sulfide and the detection of hydrogen sulfide signal molecules in the biomedical field.

Description

Technical Field

[0001] The invention belongs to the field of organic dye compounds and small molecule fluorescent probes, and specifically relates to an organic probe based on a quinazolidinone structure, a synthesis method thereof and an application thereof. Background Art

[0002] Hydrogen sulfide (H2S) is an important gas signal molecule. It is considered to be the third endogenous gaseous transmitter after NO and CO, and plays a vital role in life systems. Low concentrations of hydrogen sulfide can cause damage to the eyes, respiratory system and central nervous system, affecting human health. Studies have shown that hydrogen sulfide is involved in many physiological and pathological processes, and plays an important role in physiological processes such as myocardial protection, neuromodulation, anti-inflammation, vasodilation, respiratory tract, gastrointestinal tract and endocrine, and is closely related to diseases such as hypertension, Alzheimer's disease, Parkinson's disease, and cancer. Therefore, the development of a highly selective and sensitive hydrogen sulfide detection method, especially the detection of hydrogen sulfide levels in vivo, has great research significance.

[0003] Compared with traditional hydrogen sulfide detection methods, small molecule fluorescent probes have special applications in biological systems due to their high sensitivity, simple operation, and low sample destructiveness. At the same time, fluorescence imaging provides a method to directly observe the performance of probes in vivo and in vitro. The design of organic small molecule fluorescent probes generally uses fluorophores to connect recognition groups. At present, scientific researchers have developed many fluorescent probes for H2S detection, but there are still problems such as long response time, detection is easily interfered by biological thiols, and the synthesis steps are complicated.

[0004] Quinazolidinone organic dyes have attracted extensive attention from scientists since the 21st century due to their excellent photophysical properties. Quinazolidinone dyes have the following advantages: high fluorescence quantum yield, large Stokes shift, high chemical stability, etc. Quinazolidinone dyes with excellent fluorescence properties have the potential to be high-performance fluorophores. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an organic probe based on a quinazolidinone structure, a synthesis method thereof and an application thereof.

[0006] In a first aspect of the present invention, a quinazolidinone-based organic dye is provided, wherein the general structural formula of the organic probe is:

[0007]

[0008] Where: R is H, halogen, C1~C 20 Straight chain alkoxy, C1~C 20 of branched alkoxy.

[0009] Preferably, R is H.

[0010] The second aspect of the present invention provides a method for synthesizing the above-mentioned quinazolidinone structure-based organic probe, comprising the following steps:

[0011] (1) Compound 1 reacts with compound 3 to obtain compound 2;

[0012] The structural formula of the compound 1 is:

[0013]

[0014] The structural formula of the compound 3 is:

[0015]

[0016] The structural formula of the compound 2 is:

[0017]

[0018] (2) Compound 2 is reacted with 2,4-dinitrobenzenesulfonyl chloride to obtain the quinazolidinone structure-based organic probe as claimed in claim 1 or 2.

[0019] Preferably, in step (1), compound 1 and compound 3 are dissolved in anhydrous ethanol and a catalyst is added. The reaction is refluxed for 6 hours under nitrogen protection, and after cooling to room temperature, 2,3-dicyano-5,6-dichlorohydroquinone (DDQ) is added and stirred at room temperature overnight. Compound 2 is obtained by vacuum filtration.

[0020] Preferably, the molar ratio of compound 1, compound 3, DDQ and catalyst is 1:1.5:1:0.2.

[0021] Preferably, the catalyst is p-toluenesulfonic acid (p-TsOH).

[0022] Preferably, in step (2), anhydrous dichloromethane is used as a solvent, compound 2 and triethylamine are added, and the mixture is stirred at room temperature under the protection of an inert gas, and then a dichloromethane solution containing 2,4-dinitrobenzenesulfonyl chloride is added dropwise. After the addition is completed, the mixture is reacted at room temperature. After the reaction is completed, the mixture is separated and purified to obtain the organic probe based on the quinazolidinone structure as claimed in claim 1 or 2.

[0023] The third aspect of the present invention provides the use of the above-mentioned quinazolidinone structure-based organic probe for preparing a hydrogen sulfide detection reagent.

[0024] The fourth aspect of the present invention provides the use of the above-mentioned quinazolidinone structure-based organic probe for hydrogen sulfide detection, characterized in that: after the quinazolidinone structure-based organic probe reacts with H2S, a fluorescence "turn-on" phenomenon occurs.

[0025] The fifth aspect of the present invention provides the use of the above-mentioned quinazolidinone structure-based organic probe for intracellular H2S detection.

[0026] The beneficial effects of the present invention are embodied in:

[0027] (1) The present invention uses triphenylamine as an electron donor, quinazolinone as an electron acceptor, and 2,4-dinitrobenzenesulfonyl as a reaction site for hydrogen sulfide to synthesize a new type of organic probe based on quinazolinone structure. The organic probe is synthesized for the first time by the present invention, the raw materials are easily available, the process is simple, it can be industrialized, and it has strong commercial value.

[0028] (2) After the organic probe provided by the present invention reacts with hydrogen sulfide in a THF / H2O (v / v=1 / 2) solution, both the UV-visible absorption spectrum and the fluorescence emission spectrum undergo significant changes. Therefore, this type of organic probe can realize visual detection of hydrogen sulfide.

[0029] (3) The organic dye provided by the present invention has good selectivity and anti-interference ability for the detection of hydrogen sulfide. - , Cl - ,Br - , I - , NO3 - ,CO3 2- ,PO4 3- , HSO3 - , S2O5 - , glycine (Gly), leucine (Leu), proline (Pro), glutamic acid (Glu), glutamine (Gln), methionine (Met), arginine (Arg), serine (Ser), tryptophan (Try), phenylalanine (Phe), valine (Val), histidine (His), cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH) and other ions and amino acids will not interfere with the detection of hydrogen sulfide by this organic probe.

[0030] (4) The organic probe provided by the present invention has a high sensitivity to the detection of hydrogen sulfide, reaching the nanomolar level, which provides support for the detection experiment of endogenous hydrogen sulfide in cells.

[0031] (5) The detection of hydrogen sulfide by the organic probe provided by the present invention is based on the nucleophilicity of hydrogen sulfide and the nucleophilic substitution reaction of hydrogen sulfide on the organic dye.

[0032] (6) The organic probe provided by the present invention can quickly enter cells and realize the detection of endogenous hydrogen sulfide in colon cancer cells.

[0033] In summary, the present invention provides a new organic probe based on quinazolinone structure. The organic small molecule fluorescent probe is simple to prepare, has high selectivity and high sensitivity for the detection of hydrogen sulfide, can realize rapid detection of hydrogen sulfide, and is accompanied by the phenomenon of fluorescence on. It has broad value in the detection of environmental hydrogen sulfide and the detection of hydrogen sulfide signal molecules in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0035] Figure 1 The synthetic route diagram of Example 1 is shown in FIG.

[0036] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the probe synthesized in Example 1;

[0037] Figure 3 (a) is the UV-visible absorption spectrum of the probe synthesized in Example 1 before and after the reaction with H2S (100 μM) in THF / H2O (v / v=1 / 2) solvent (probe concentration: 10 μM), Figure 3 (b) is the fluorescence emission spectrum of the probe before and after the reaction with H2S (100 μM) in THF / H2O (v / v=1 / 2) solvent (probe concentration: 10 μM);

[0038] Figure 4 (a) is a histogram of the fluorescence intensity of the probe synthesized in Example 1 after reacting with the test sample (concentration: 100 μM) in THF / H2O (v / v=1 / 2) solvent (concentration: 10 μM), Figure 4 (b) is a histogram of the fluorescence intensity of the probe after the reaction in THF / H2O (v / v=1 / 1) solvent (concentration: 10μM) with H2S (100μM) and other test samples; test samples: 1. Free, 2. Hcy, 3. Cys, 4. GSH, 5. Arg, 6. Glu, 7. Gly, 8. His, 9. Leu, 10. Pro, 11. Phe, 12. Ser, 13. Try, 14. Val, 15. Gln, 16. Met, 17. Br - , 18.Cl -, 19.NO3 - , 20.CO3 2- , 21.HSO 3- , 22.PO4 3- , 23.I - , 24.S2O5 2- , 25.F - , 26.H2S;

[0039] Figure 5 The fluorescence graph of the probe synthesized in Example 1 in THF / H2O (v / v=1 / 2) solvent (concentration: 10 μM) and H2S (100 μM) response time;

[0040] Figure 6 (a) is a graph showing the fluorescence spectrum changes of the probe (10 μM) synthesized in Example 1 after reacting with different concentrations of H2S (2, 4, 6, 8, 10, 15, 18, 20, 30, 40, 50, 60, 70, 80, 90, 95, 100 μM). Figure 6 (b) is a linear relationship diagram of the fluorescence intensity of the synthetic probe (10 μM) and the H2S concentration (1-10 μM) in Example 1;

[0041] Figure 7 The probe synthesized in Example 1, compound 2, and the high performance liquid chromatogram after the probe responds to H2S;

[0042] Figure 8 Fluorescence confocal images of the probe synthesized in Example 1 after reacting with endogenous and exogenous H2S in colon cancer cells (HCT116), (A1, A5, A9) HCT116 cells were cultured with AOAA (1mM) for 1h, and then Probe (10μM) was added for 2h; (A2, A6, A10) HCT116 cells were cultured with Probe (10μM) for 2h; (A3, A7, A11) HCT116 cells were cultured with Cys (1mM) for 1h, and then Probe (10μM) was added for 2h; (A4, A8, A12) HCT116 cells were cultured with AOAA (1mM) for 1h, then cultured with H2S (50μM) for 30min, and then Probe (10μM) was added for 2h; A1-A4 are DAPI images corresponding to A5-A8; A9-A12 are bright field images corresponding to A5-A8. DETAILED DESCRIPTION

[0043] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0044] Example 1

[0045] Synthesis of Quinazolinone-based Organic Probe HTPQ-HS

[0046] (1) Synthesis of Compound 2

[0047]

[0048] Under argon protection, 150 mg (0.41 mmol) of compound 1, 84 mg of o-aminobenzamide (0.615 mmol), 15 mL of anhydrous ethanol, and finally 15 mg (0.081 mmol) of p-toluenesulfonic acid (p-TsOH) catalyst were added to a 50 mL two-necked round-bottom flask. The reaction liquid temperature was raised to 85 ° C and stirred for 6 hours. After the reaction liquid was cooled to room temperature, 93 mg (0.41 mmol) of 2,3-dicyano-5,6-dichlorohydroquinone (DDQ) dissolved in anhydrous ethanol was added and stirred at room temperature overnight. 100 mg (0.2 mmol) of compound 2 was obtained by vacuum filtration with a yield of 50%. 1 H NMR (400MHz, DMSO-d6) δ14.06 (s, 1H), 12.52 (s, 1H), 8.33 (d, J = 9.0Hz, 1H) ,8.18(d,J=8.2Hz,1H),7.93–7.85(m,1H),7.80(d,J=7.9Hz,1H),7.74(d,J =8.7Hz,2H),7.60–7.54(m,1H),7.41–7.35(m,4H),7.33–7.27(m,2H),7.12 (ddd,J=8.6,7.5,2.2Hz,6H),7.05(d,J=8.7Hz,2H).HRMS(ESI,m / z):[M+H] + cacld for C 32 H 23 N3O2:482.1869,found:482.1853.

[0049] (2) Synthesis of organic probe HTPQ-HS

[0050]

[0051] Under the protection of argon, 40 mg (0.0832 mmol) of compound 2, 22 mg of 2,4-dinitrobenzenesulfonyl chloride (0.0832 mmol), 15 mL of dichloromethane and 0.1 mL of triethylamine were added to a 50 mL two-necked round-bottom flask. The reaction mixture was stirred at room temperature for 3 hours. After the reaction, 20 mL of distilled water was added to the reaction solution, and the mixture was extracted three times with dichloromethane. The combined organic phase was dried over anhydrous Na2SO4. The organic solvent was removed by rotary evaporator, and the crude product was separated and purified by silica gel column chromatography, using petroleum ether: dichloromethane: ethyl acetate (4:2:1) as the mobile phase to obtain (15 mg) of brown solid dye HTPQ-HS with a yield of 23%. 1 H NMR (400MHz, CDCl3) δ8.32(d,J=1.2Hz,1H),8.15–8.08(m,3H),7.96(d,J=8.2Hz,1H),7.79–7.73(m,3H),7.70(d,J=8.1Hz,1H),7.5 4(d,J=8.5Hz,2H),7.48(t,J=7.4Hz,1H),7.32(t,J=7.8Hz,4H),7.17(d,J=8.0Hz,6H),7.11(t,J=7.3Hz,2H).HRMS(ESI,m / z):[M+H] + cacld for C 38 H 26 N 45 NaO7S:712.1502,found:712.1489.

[0052] Example 2

[0053] The organic probe HTPQ-HS of Example 1 was tested for UV and fluorescence emission spectra after responding to hydrogen sulfide. Figure 3 As shown in a. The maximum absorption wavelength of HTPQ-HS is 365nm. After reacting with H2S, the maximum absorption peak of the probe red-shifts to 380nm. Figure 3 As shown in Figure b, the fluorescence intensity of the dye HTPQ-HS before and after the reaction with H2S has changed significantly. The fluorescence emission signal of the probe HTPQ-HS is very weak before the addition of H2S, but after the addition of H2S, the probe solution has an obvious fluorescence "turn-on" phenomenon, and a strong fluorescence signal peak appears at 525nm. This shows that under ultraviolet light, we can observe the detection results of the dye HTPQ-HS for H2S with the naked eye.

[0054] Example 3

[0055] The organic dye HTPQ-HS of Example 1 was subjected to a selectivity test experiment. Figure 4As shown in a, the addition of amino acids and representative anions other than H2S does not cause significant changes in the fluorescence emission of the dye HTPQ-HS. Figure 4 As shown in b, in the presence of other competitive samples, H2S still triggers the dye probe HTPQ-HS to appear Figure 4 a Similar fluorescence response phenomenon. The selectivity experiment shows that the organic probe HTPQ-HS has good selectivity for H2S.

[0056] Example 4

[0057] The organic probe HTPQ-HS of Example 1 was tested for its response time to H2S. Figure 5 As shown in a, after H2S was added, the fluorescence emission intensity of the probe HTPQ-HS at 525 nm gradually increased with time until it gradually approached the reaction plateau after 4 min. Figure 5 As shown in b, the fluorescence emission intensity of the probe HTPQ-HS that responds to H2S basically does not change after 4 minutes. This result shows that the probe HTPQ-HS has a very fast response speed to H2S.

[0058] Example 5

[0059] A fluorescence titration experiment was performed on the organic probe HTPQ-HS of Example 1. Figure 6 As shown in a, the fluorescence of the probe HTPQ-HS itself is very weak. With the addition of H2S, the fluorescence emission intensity of the dye at 525 nm gradually increases. Figure 6 b is the fluorescence titration experiment of probe HTPQ-HS in the range of H2S concentration of 1-10μM. The detection limit of H2S by HTPQ-HS is calculated to be 175nM through linear fitting. This result shows that HTPQ-HS has high sensitivity to H2S.

[0060] Example 6

[0061] The reaction mechanism of the organic probe HTPQ-HS in Example 1 was verified by HPLC. Figure 7 a shows the peak position of probe HTPQ-HS in HPLC. Figure 7 b shows the peak position of compound 2 in HPLC. Figure 7 c shows the peak position of the probe HTPQ-HS in HPLC after the reaction with H2S. By comparison, it can be determined that the product of the organic probe HTPQ-HS after the reaction with H2S is compound 2. At the same time, it is shown that the effect of the probe HTPQ-HS and H2S is caused by the nucleophilic attack reaction of H2S.

[0062] Example 7

[0063] The organic probe HTPQ-HS of Example 1 was used to image and detect endogenous H2S in colon cancer cells (HCT116). Figure 8 As shown. Cys can induce an increase in endogenous H2S levels. In contrast, AOAA reduces endogenous H2S levels by inhibiting CBS expression. Figure 8 As shown in Figure 6, HCT116 cells incubated with HTPQ-HS showed green fluorescence. Figure 8 -A7 HCT116 cells incubated with Cys and HTPQ-HS showed stronger fluorescence. Figure 8 As shown in Figure 8, HCT116 cells incubated with AOAA and then with exogenous H2S and HTPQ-HS also showed obvious green fluorescence. This experiment shows that the organic probe HCT116 cells can light up and detect exogenous hydrogen sulfide in colon cancer cells.

[0064] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. An organic probe based on a quinazolidinone structure, characterized in that: The general structural formula of the organic probe is: Among them: R is H.

2. The method for synthesizing an organic probe based on a quinazolidinone structure as claimed in claim 1, characterized in that The following steps are involved: (1) Compound 1 reacts with compound 3 to obtain compound 2; The structural formula of the compound 1 is: The structural formula of the compound 3 is: The structural formula of the compound 2 is: (2) reacting compound 2 with 2,4-dinitrobenzenesulfonyl chloride to obtain the quinazolidinone structure-based organic probe as claimed in claim 1; In step (1), compound 1 and compound 3 are dissolved in anhydrous ethanol and a catalyst is added. The reaction is refluxed under nitrogen protection for 6 hours. After cooling to room temperature, 2,3-dicyano-5,6-dichlorohydroquinone (DDQ) is added, and the mixture is stirred at room temperature overnight. Finally, compound 2 is obtained by suction filtration.

3. The method for synthesizing an organic probe having a quinazolidinone structure according to claim 2, wherein: The molar ratio of compound 1, compound 3, DDQ and catalyst is 1:1.5:1:0.

2.

4. The method for synthesizing a quinazolidinone structure organic probe according to claim 2, characterized in that: The catalyst is p-toluenesulfonic acid (p-TsOH).

5. The method for synthesizing a quinazolidinone structure organic probe according to claim 2, characterized in that: In step (2), anhydrous dichloromethane is used as a solvent, compound 2 and triethylamine are added, and under the protection of an inert gas nitrogen, the mixture is stirred at room temperature, and then a dichloromethane solution containing 2,4-dinitrobenzenesulfonyl chloride is added dropwise. After the addition is completed, the mixture is reacted at room temperature. After the reaction is completed, the mixture is separated and purified to obtain the organic probe based on the quinazolidinone structure as claimed in claim 1.

6. Use of the quinazolidinone structure-based organic probe as claimed in claim 1 for preparing a hydrogen sulfide detection reagent.

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