Dual-response organic fluorescent compound and its preparation method and application

Abstract

A dual-responsive organic fluorescent compound, whose chemical name is (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide, contains H + and ONOO ‑ The two response sites, whose emission colors can change with changes in molecular structure, can be used as fluorescent probes for detection and analysis in a variety of situations, including the absence of analytes, detection and analysis of hydrogen ion concentration alone or peroxynitrite anion concentration, or the coexistence of hydrogen ions and peroxynitrite anions. It can also be used to monitor changes in hydrogen ion concentration and peroxynitrite anion concentration in mitochondria of living cells and living organisms. The preparation method provided by the present invention is simple, has convenient raw material sources, and has a high yield. As a fluorescent probe, it has a wide range of applications, a sensitive response, a fast speed, and can simultaneously detect hydrogen ion concentration and peroxynitrite anion.

Description

Technical Field

[0001] The present invention relates to an organic fluorescent compound and a preparation method and application thereof, in particular to a dual-response organic fluorescent compound and a preparation method and application thereof, belonging to the technical field of preparation and application of organic fluorescent compounds. Background Art

[0002] An organism is a complex living system, in which the occurrence and development of many physiological and pathological processes involve the joint participation of multiple chemical events, rather than being caused and determined by a single factor. Therefore, revealing the intrinsic connections between multiple chemical events is of great significance for in-depth understanding and recognition of the pathology, pathogenesis and influencing factors of diseases, and providing new means and methods for disease prevention and treatment.

[0003] Inside an organism, acid-base balance is the basis for maintaining life activities and is closely related to intracellular behaviors such as cell metabolism, cell apoptosis, and ion transport. A decrease in hydrogen ion concentration in an organism can cause mitochondrial imbalance and hinder cell proliferation, and can be accompanied by the occurrence of various diseases such as inflammation, neurodegenerative diseases, and cancer.

[0004] In terms of inflammation, peroxynitrite has been considered to be a key pathogenic factor that causes inflammation by undergoing nitration reactions with proteins, lipids and DNA. Among them, 3-nitrotyrosine, as a characteristic product of the nitration reaction of peroxynitrite with proteins, lipids or DNA, has been found in many pathological conditions, such as atherosclerosis, chronic rejection of transplanted organs, viral infections and neurodegenerative diseases.

[0005] Through in vitro synthesis, it was found that the nitration reaction between peroxynitrite and protein, lipid or DNA is a highly dynamic process, and its chemical reactivity is closely related to the hydrogen ion concentration index (i.e. pH, the same below) in the body, that is, in the presence of tyrosine and peroxynitrite anion (i.e. ONOO - , the same below) substrate, the decrease in hydrogen ion concentration is conducive to the formation of 3-nitrotyrosine, from which it can be inferred that the change in hydrogen ion concentration in the organism and ONOO - There should be a certain correlation between them and play an important role in physiological and pathological processes; however, due to the lack of appropriate tools in existing technologies, the changes in hydrogen ion concentration index and ONOO in vivo are still unclear. - Therefore, in order to reveal their synergistic or antagonistic effects in related life processes and their changing processes, it is urgent to develop a method that can simultaneously visualize the hydrogen ion concentration index and ONOO - tools to provide new ideas and methods for the treatment of diseases.

[0006] Relying on high-sensitivity and high-resolution fluorescence microscopy technology, small molecule fluorescent sensors, commonly known as fluorescent probes, provide powerful tools for the study of biological systems and have completely changed researchers' understanding of chemical species and microenvironments in biological systems.

[0007] Dual-analyte-responsive probes, also known as dual-responsive probes or dual-responsive fluorescent probes, are also called logic gate probes because they can respond to two analytes (inputs) by reporting one or more detectable signals (outputs), thereby functioning like a logic gate.

[0008] This dual-responsive fluorescent probe is becoming a powerful tool and making great contributions to the study of multiple analytes in complex biological systems because it can effectively avoid a series of problems brought about by the combination of multiple single-responsive probes, such as greater invasiveness, spectral crosstalk, more complex positioning and photobleaching.

[0009] However, although dual-analyte response probes have made great progress with the development of technology and have received increasing attention, their development is still in its infancy. Although existing dual-response probes can respond to two or more analytes, they cannot distinguish between the coexistence of two analytes and the presence of one analyte alone, nor can they determine the mixed response to two analytes.

[0010] An ideal dual-response probe should be able to give a uniquely measurable response output signal for the presence of each analyte alone or the co-presence of two analytes, providing the easiest-to-interpret data and more biological information.

[0011] Due to the importance of the intracellular microenvironment and oxidative substances, fluorescent probes involving hydrogen ions or peroxynitrite anions in organisms have been developed. However, fluorescent sensors that can simultaneously detect the hydrogen ion concentration index and peroxynitrite anions in organisms have not been reported. Summary of the Invention

[0012] To overcome the deficiencies of the prior art, the present invention first provides a dual-responsive organic fluorescent compound and a method for preparing the same. Furthermore, the present invention proposes its application as a fluorescent probe for dual-responsive fluorescence detection of hydrogen ion concentration index and / or peroxynitrite anion in living cells or living bodies, wherein:

[0013] The dual-response organic fluorescent compound has a chemical name:

[0014] (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide, abbreviated as MNIR, has the following chemical structure:

[0015]

[0016] The preparation method of the dual-responsive organic fluorescent compound includes step d, or steps b, c, and d, or steps a to b, c, and d in the following synthesis route:

[0017]

[0018] Furthermore, the above step a includes:

[0019] Under an ice bath, phosphorus tribromide is slowly added to a mixed solution of N,N-dimethylformamide and chloroform, and the mixture is stirred. Cyclohexanone dissolved in chloroform is then added and the mixture is stirred at room temperature. After the reaction is completed, the reaction mixture is poured into ice water, and sodium hydroxide solution is slowly added to adjust the pH to neutral. The aqueous layer is extracted with dichloromethane, and the resulting organic layer is dried over anhydrous sodium sulfate and then filtered. After removing the solvent from the filtrate, a yellow oily compound I, namely 2-bromocyclohexyl-1-ene-1-carbaldehyde, is obtained, wherein:

[0020] The volume ratio of N,N-dimethylformamide to carbon trichloride is 1:(1.5-1.6), the molar ratio of the reactants phosphorus tribromide to cyclohexanone is (3.0-3.5):1, and the compound I is stored at -20°C.

[0021] Furthermore, the above step b includes:

[0022] Compound I, 4-(diethylamino)salicylaldehyde and cesium carbonate are added to N,N-dimethylformamide solvent and stirred at room temperature for reaction. After the reaction is completed, the insoluble matter is removed by filtration, and the filtrate is concentrated. The residue is extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure and the crude product is purified by silica gel chromatography to obtain a dark yellow solid compound DXC, i.e., 6-diethylamino-2,3-dihydro-1H-xanthene-4-carboxaldehyde, wherein:

[0023] The molar ratio of the reactants is: compound I: 4-(diethylamine) salicylaldehyde: cesium carbonate = 3:1:3;

[0024] The eluent in the silica gel chromatography method is a mixture of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 10:1.

[0025] Furthermore, the above step c includes:

[0026] 2,3,3-Trimethyllindoleamine and 2-iodine alcohol were dissolved in acetonitrile and refluxed. After the reaction, the solvent was removed and the resulting solid was washed with n-hexane to obtain a purple solid compound II, i.e., 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indole iodide, wherein:

[0027] The molar ratio of the reactants is: 2,3,3-trimethyllindoleamine: 2-iodine alcohol = 1:1.2.

[0028] Furthermore, the above step d includes:

[0029] Compound DXC, potassium carbonate and compound II are co-dissolved in anhydrous acetic anhydride, and the reaction is stirred at room temperature to obtain a dark green solution. The dark green solution is then dissolved in dichloromethane, washed with water, and then dried over anhydrous sodium sulfate. The solvent is removed, and the residue is purified by silica gel chromatography to obtain a dark green solid compound MINIR, i.e., (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide salt, wherein:

[0030] The molar ratio of the reactants is: compound DXC: potassium carbonate: compound II = 1:2:1.2;

[0031] The eluent in the silica gel chromatography purification method is a mixture of dichloroethane and methanol, and the volume ratio of the dichloroethane to the methanol is 25:1.

[0032] An application of the above-mentioned dual-responsive organic fluorescent compound is to use the above-mentioned dual-responsive organic fluorescent compound as a fluorescent probe for dual-responsive fluorescence detection of hydrogen ion concentration index and / or peroxynitrite anion in living cells or living bodies, wherein the living cells are immortalized cells or normal cells, and the immortalized cells are HeLa cells or A549 cells.

[0033] Compared with the prior art, the present invention has the following outstanding beneficial effects and significant improvements:

[0034] 1) The dual-responsive organic fluorescent compound provided by the present invention is (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide, referred to as MNIR, containing H + and ONOO -The two response sites can change their emission color depending on the molecular structure under acidic conditions or after reaction with peroxynitrite anion. Therefore, it can be used as an ideal fluorescent probe for dual-analyte sensing. It can be used for analytical studies in a variety of different situations, including the absence of analytes, changes in the concentration of hydrogen ions or peroxynitrite anion alone, or the coexistence of hydrogen ions and peroxynitrite anion.

[0035] 2) Since mitochondria are the main site of aerobic respiration, electron transfer during respiration is the main source of peroxynitrite anions, and H + / K + Disturbances in the ATP proton pump directly lead to changes in mitochondrial hydrogen ion concentration. The dual-responsive organic fluorescent compound provided by the present invention contains a cationic salt and can exhibit specific targeting to mitochondria. Therefore, the dual-responsive organic fluorescent compound provided by the present invention can be selectively used to monitor changes in mitochondrial hydrogen ion concentration and peroxynitrite anion in living cells and living bodies, allowing changes in both in living cells or living bodies to be well monitored.

[0036] 3) Dynamic studies have shown that during some drug treatments, the concentration of peroxynitrite anions changes exponentially with the hydrogen ion concentration. For example, a decrease in hydrogen ion concentration further promotes the generation of peroxynitrite anions during the action of cisplatin on DNA, resulting in side effects such as toxicity. The dual-responsive organic fluorescent compounds provided by the present invention can provide ideal dual-responsive logical sensing detection for ratiometric imaging and differential imaging of hydrogen ion concentration and peroxynitrite anions, thereby facilitating the discovery of new signaling pathways and understanding interactions between multiple species.

[0037] 4) The dual-responsive organic fluorescent compound provided by the present invention is the first compound to be able to simultaneously and distinguishably detect changes in the hydrogen ion concentration index and peroxynitrite anion concentration in active samples. This overcomes the deficiency of existing probes for hydrogen ion concentration index or peroxynitrite anion detection, which can only identify a single analyte. This eliminates the tedious staining and repeated washing processes caused by the use of different probe combinations in related biological experiments, avoids spectral crosstalk, and increases cytotoxicity. It can visualize the interactions between multiple small molecules in real time, thereby facilitating the discovery of new signaling pathways and understanding the interactions between exogenous and endogenous species, contributing to the research and exploration of biological systems.

[0038] 5) The dual-responsive organic fluorescent compound provided by the present invention, as a new fluorescent probe, has the characteristics of a wide range of applications, sensitive response, fast speed, and the ability to simultaneously distinguish and detect the hydrogen ion concentration index and the peroxynitrite anion. It has broad application prospects, and the preparation method is simple, the raw materials are easily available, and the comprehensive yield is high. It provides new ideas and methods for the preparation of new fluorescent probes, has outstanding substantial characteristics and beneficial effects and significant progress, and therefore has great promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solution of the present invention and the technical effects of implementing the present invention, a brief introduction is given below to the drawings used in the embodiments of the present invention.

[0040] The obvious:

[0041] The drawings described below are only drawings of some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work, but these other drawings also belong to the drawings required for use in the embodiments of the present invention.

[0042] Figure 1 Figure 1 is a collection of absorption spectra of MNIR at a concentration of 10 μM in PBS buffer solutions with a pH range of 9 to 2 and an interval of 0.5.

[0043] Figure 2 : This is a collection of fluorescence emission spectra of MNIR with a concentration of 10 μM in PBS buffer solutions with a pH range of 9 to 2 and an interval of 0.5, wherein the excitation light wavelength is 550 nm.

[0044] Figure 3 MNIR in the presence of different concentrations of ONOO - Absorption spectrum in PBS buffer, where:

[0045] α is ONOO - MNIR absorption spectrum when concentration = 0;

[0046] β is ONOO - MNIR absorption spectrum at concentration = 35 μM, PBS buffer pH = 7.4.

[0047] Figure 4 MNIR in the presence of 0-35 μM concentration of ONOO - The absorption spectrum of the PBS buffer solution with a pH of 7.4 is shown in FIG.

[0048] ONOO - When the concentration ranges from 1 to 20 μM, the concentration interval is 2 μM; ONOO -When the concentration ranges from 20 to 35 μM, the concentration interval is μM; the excitation wavelength of short-wavelength fluorescence is 360 nm; the excitation wavelength of long-wavelength fluorescence is 700 nm.

[0049] Figure 5 The co-localization images of MNIR and commercial probes MTG and LTG in HeLa cells and the corresponding fluorescence intensity spectra along the white arrows are shown;

[0050] MTG is a commercially available mitochondrial green fluorescent probe; LTG is a commercially available lysosomal red fluorescent probe; the R value is the Pearson colocalization coefficient; the excitation wavelength of MNIR is 633 nm, and the emission band is 700-800 nm; the excitation wavelengths of MTG and LTG are 488 nm, and the emission bands are 500-600 nm, respectively; the scale bar is 20 μm.

[0051] Figure 6 This is a collection of confocal fluorescence images and ratiometric imaging images of HeLa cells stained with MNIR under different pH conditions;

[0052] The confocal fluorescence image of the red channel has an excitation light wavelength of 633 nm and an emission band of 700-800 nm; the confocal fluorescence image of the green channel has an excitation light wavelength of 561 nm and an emission band of 590-690 nm; the scale bar is 20 μm.

[0053] Figure 7 This is a collection of confocal fluorescence images of HeLa cells stained with MNIR after treatment with different drugs; among them:

[0054] The SIN-1 group is a HeLa cell population treated with lindoxycycline; the SIN-1+MC group is a HeLa cell population treated with lindoxycycline and minocycline; the SIN-1+pH2 group is a HeLa cell population treated with lindoxycycline and continuously incubated in PBS buffer at pH = 2; and

[0055] The excitation wavelength of the red channel is 633 nm, and the emission band is 700-800 nm; the excitation wavelength of the blue channel is 405 nm, and the emission band is 450-550 nm; the scale bar is 20 μm.

[0056] Figure 8 To correspond Figure 7 The confocal fluorescence image data of HeLa cells treated with different drugs and stained with MNIR were processed by Matlab software to draw a confocal fluorescence relative intensity bar graph.

[0057] Figure 9This is a collection of images of ICR mice after intraperitoneal injection of different drugs, stained with MNIR and imaged using the indocyanine green dye channel for near-infrared imaging;

[0058] The SIN-1 group consisted of ICR mice injected with lindoxycycline; the SIN-1+MC group consisted of ICR mice injected with lindoxycycline and minocycline.

[0059] Figure 10 This is a collection of bright field and confocal fluorescence images of A549 cells treated with 500 μM cisplatin and stained with MNIR; among them:

[0060] The excitation light wavelength of the blue channel is 405nm, and the emission band is 450-550nm; the excitation light wavelength of the red channel 1 is 633nm, and the emission band is 700-800nm; the excitation light wavelength of the green channel is 561nm, and the emission band is 590-690nm; the excitation light wavelength of the red channel 2 is 561nm, and the emission band is 700-800nm. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions, beneficial effects and significant improvements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the structural formulas or reaction formulas provided in the embodiments of the present invention. Obviously, all the described embodiments are only basic embodiments of the present invention, rather than all embodiments.

[0062] Based on the embodiments provided by the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0063] It should be noted that:

[0064] The terms "first", "second", etc. in the specification and claims of the present invention and the embodiments of the present invention are only used to distinguish different objects, rather than to describe a specific order; in addition, the term "comprise" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units and unrestricted listed steps or units, and optionally also includes unlisted steps or units, or optionally also includes other operating steps or units inherent to these processes, methods, products or devices.

[0065] It should be understood that in the description of the embodiments of the present invention, some basic operating terms commonly used in the art are used, such as "heating", "stirring", "mixing", "dissolving", "washing", "filtering" and "drying", etc. These terms should be understood in a broad sense, that is, they can be conventional operations performed using various conventional equipment and instruments in the art, or they can be program-controlled operations, unmanned automatic operations, etc. performed using the latest equipment. Unless otherwise clearly defined, ordinary technicians in this field can understand the specific meanings of the above terms in the present invention according to the specific circumstances, and adopt specific operating methods to achieve their operating purposes.

[0066] It should also be noted that:

[0067] The following specific embodiments may be combined with each other, and the same or similar concepts or processes therein may not be described in detail in some embodiments;

[0068] In addition, the raw materials, auxiliary materials, reaction equipment and facilities involved in the following specific examples are all commercially available or can be prepared according to existing technologies.

[0069] The technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0070] Example 1

[0071] This embodiment is used to provide an organic fluorescent compound with dual-response characteristics.

[0072] The dual-responsive organic fluorescent compound provided in this embodiment has a chemical name:

[0073] (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide, abbreviated as MNIR, has the following chemical structure:

[0074]

[0075] Structural analysis and experimental testing of the above compounds revealed that:

[0076] The dual-responsive organic fluorescent compound provided in this embodiment, (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide (MNIR), contains H + and ONOO - Two response sites, under acidic conditions or with peroxynitrite anion (i.e. ONOO -) reaction, its emission color can change with the change of molecular structure, so it can be used as a fluorescent probe, where:

[0077] In the absence of analytes, the probe has a large D-π-A structure, and the maximum emission wavelength of 740nm is located in the near-infrared biological imaging window, which can greatly reduce cell damage and light scattering effects;

[0078] Under acidic conditions, the nitrogen on the dimethylamino group is protonated to form an A-π-A structure, the emission peak is blue-shifted, and the maximum emission wavelength is 650nm, thereby achieving a proportional response to the hydrogen ion concentration index;

[0079] When ONOO - When it exists alone, a new blue-green fluorescence will appear at 480nm based on the dual reaction sites of ester group and double bond;

[0080] When H + and ONOO - When they coexist, there is no fluorescent signal;

[0081] Therefore, the dual-responsive organic fluorescent compound MNIR provided in this embodiment can be used as an ideal fluorescent probe for dual-analyte sensing and can be used for analysis in a variety of different situations, including analysis in the absence of analytes, analysis of the hydrogen ion concentration index alone or in combination with the concentration change of peroxynitrite anion, or analysis of the coexistence of hydrogen ions and peroxynitrite anions.

[0082] In addition, since mitochondria are the main site of aerobic respiration, electron transport in respiration is ONOO - The main source of H + / K + The disorder of ATP proton pump will directly lead to the change of hydrogen ion concentration index. Since the MNIR provided in this embodiment contains cationic salts, it shows specific targeting to mitochondria, so it can regulate the hydrogen ion concentration index and ONOO in mitochondria. - The changes in living cells and in vivo are well monitored, wherein the living cells are preferably immortalized cells or normal cells, and the immortalized cells are preferably HeLa cells or A549 cells; and

[0083] Dynamic studies have shown that during some drug treatments, the concentration of peroxynitrite anions changes exponentially with the hydrogen ion concentration. For example, a decrease in hydrogen ion concentration will further promote the generation of peroxynitrite anions during the action of cisplatin on DNA, thereby producing side effects such as toxicity. The dual-responsive organic fluorescent compound provided in this embodiment is used as an ideal dual-responsive logic sensor for ratiometric imaging and differential imaging of hydrogen ion concentration and peroxynitrite anions, which helps to discover new signal pathways and understand the interactions between multiple species, revealing the relationship between hydrogen ion concentration and ONOO in chemotherapy with drugs such as cisplatin. - The changes in and the relationship between the two can provide a solution to reduce the side effects induced by drugs such as cisplatin;

[0084] The search revealed that the dual-responsive organic fluorescent compound provided in this embodiment is the first compound obtained that can simultaneously and distinguishably detect changes in the hydrogen ion concentration index and the peroxynitrite anion concentration in active samples. It overcomes the deficiency in the prior art that probes for hydrogen ion concentration index or peroxynitrite anion detection can only identify a single analyte, thereby eliminating the tedious staining and repeated washing processes caused by the use of different probe combinations in related biological experiments, avoiding spectral crosstalk and increased cytotoxicity, and can visualize the interactions between multiple small molecules in real time, thereby facilitating the discovery of new signaling pathways and understanding the interactions between exogenous and endogenous species, and contributing to the research and exploration of biological systems.

[0085] From the above we can see that:

[0086] The dual-responsive organic fluorescent compound provided in this embodiment can be used as a new logical fluorescent probe for dual-responsive fluorescence detection of hydrogen ion concentration index and / or peroxynitrite anion in living cells or living bodies, and has the characteristics of wide application range, sensitive response, and fast speed, and has broad application prospects.

[0087] Example 2

[0088] This embodiment provides a method for preparing the dual-responsive organic fluorescent compound described in the above embodiment 1.

[0089] A method for preparing the dual-responsive organic fluorescent compound described in Example 1 above includes step d, or steps b, c, and d, or steps a to b, c, and d in the following synthetic route:

[0090]

[0091] Specifically, the above step a includes:

[0092] Under an ice bath, phosphorus tribromide is slowly added to a mixed solution of N,N-dimethylformamide and chloroform, and the mixture is stirred. Cyclohexanone dissolved in chloroform is then added and the mixture is stirred at room temperature. After the reaction is completed, the reaction mixture is poured into ice water, and sodium hydroxide solution is slowly added to adjust the pH to neutral. The aqueous layer is extracted with dichloromethane, and the resulting organic layer is dried over anhydrous sodium sulfate and then filtered. After removing the solvent from the filtrate, a yellow oily compound I, namely 2-bromocyclohexyl-1-ene-1-carbaldehyde, is obtained, wherein:

[0093] The volume ratio of N,N-dimethylformamide to carbon trichloride is 1:(1.5-1.6), the molar ratio of the reactants phosphorus tribromide to cyclohexanone is (3.0-3.5):1; and compound I is stored at -20°C;

[0094] Step b includes:

[0095] Compound I, 4-(diethylamino)salicylaldehyde and cesium carbonate are added to N,N-dimethylformamide solvent and stirred at room temperature for reaction. After the reaction is completed, the insoluble matter is removed by filtration, and the filtrate is concentrated. The residue is extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure and the crude product is purified by silica gel chromatography to obtain a dark yellow solid compound DXC, i.e., 6-diethylamino-2,3-dihydro-1H-xanthene-4-carboxaldehyde, wherein:

[0096] The molar ratio of the reactants is: compound I: 4-(diethylamine) salicylaldehyde: cesium carbonate = 3:1:3;

[0097] Silica gel chromatography method wherein the eluent is a mixture of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate mixture is 10:1;

[0098] Step c includes:

[0099] 2,3,3-Trimethyllindoleamine and 2-iodine alcohol were dissolved in acetonitrile and refluxed. After the reaction, the solvent was removed and the resulting solid was washed with n-hexane to obtain a purple solid compound II, i.e., 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indole iodide, wherein:

[0100] The molar ratio of the reactants is: 2,3,3-trimethyllindoleamine: 2-iodine alcohol = 1:1.2;

[0101] Step d includes:

[0102] Compound DXC, potassium carbonate and compound II are co-dissolved in anhydrous acetic anhydride, and the reaction is stirred at room temperature to obtain a dark green solution. The dark green solution is then dissolved in dichloromethane, washed with water, and then dried over anhydrous sodium sulfate. The solvent is removed, and the residue is purified by silica gel chromatography to obtain a dark green solid compound MINIR, i.e., (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide salt, wherein:

[0103] The molar ratio of the reactants is: compound DXC: potassium carbonate: compound II = 1:2:1.2;

[0104] The silica gel chromatography purification method uses a mixture of dichloroethane and methanol as the eluent, and the volume ratio of dichloroethane to methanol is 25:1.

[0105] To further help understand the technical solution provided by this embodiment, as well as the specific operation process and obtainable effects of this embodiment, the preparation method and test results provided by this embodiment are further described below through specific examples.

[0106] Of course, those skilled in the art should understand that the examples described below are merely illustrative and not restrictive, and should not be used to limit the scope of protection claimed by the present invention.

[0107] Example 1. Preparation of Compound I, 2-bromocyclohexyl-1-ene-1-carbaldehyde

[0108] Reaction formula:

[0109]

[0110] Preparation process:

[0111] Under an ice bath, 2.9 mL, i.e. 9.5 mmol, of phosphorus tribromide (PBr3) was slowly added to a mixed solution consisting of 1.2 mL, i.e. 15.5 mmol, of N,N-dimethylformamide (DMF) and 20 mL of chloroform (CHCl3). After stirring for 0.5 hour, 0.4 mL, i.e. 3 mmol, of cyclohexanone dissolved in 10 mL of chloroform (CHCl3) was added. Stirring was continued at room temperature for 15 hours, and the mixture was then poured into ice water. Sodium hydroxide solution was slowly added to adjust the pH to 7. The aqueous layer was extracted with dichloromethane (CH2Cl2), and the organic layer was dried over anhydrous sodium sulfate (Na2SO4). After filtration, the solvent was removed by evaporation under reduced pressure to obtain 2.49 g of compound I, i.e. 2-bromocyclohexyl-1-ene-1-carbaldehyde, as a yellow oil with a weight ratio yield of 85%.

[0112] 2-Bromocyclohexyl-1-ene-1-carbaldehyde is unstable at room temperature and usually needs to be stored at -20°C.

[0113] Example 2: Preparation of Compound DXC, 6-diethylamino-2,3-dihydro-1H-xanthene-4-carboxaldehyde

[0114] Reaction formula:

[0115]

[0116] Preparation process:

[0117] 1.5 g, i.e. 8.0 mmol, of compound I, 0.78 g, i.e. 4.0 mmol, of 4-(diethylamino)salicylaldehyde, and 3.9 g, i.e. 12.0 mmol, of cesium carbonate (Cs2CO3) were added to 30 mL of N,N-dimethylformamide (DMF) solvent and stirred at room temperature for 12 hours. The insoluble matter was removed by filtration, and the filtrate was concentrated. The residue was first extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using a mixed solvent of petroleum ether:ethyl acetate = 10:1 by volume as the eluent to obtain 0.68 g of dark yellow solid DXC, i.e. 6-diethylamino-2,3-dihydro-1H-xanthene-4-carboxaldehyde, with a weight ratio yield of 60%.

[0118] Test results:

[0119] 1 H NMR (600MHz, CD3CN) δ (ppm) 10.17 (s, 1H), 6.97 (d, J = 8.5Hz, 1H), 6.62 (s, 1H), 6.46-6.30 (m, 2H), 3.31 (q, J = 7.1Hz, 4H), 2 .45 (ddd, J=7.6, 5.0, 1.5Hz, 2H), 2.25 (t, J=6.1Hz, 2H), 1.58 (p, J=6.1Hz, 2H), 1.21-1.17 (m, 1H), 1.07 (t, J=7.1Hz, 6H).

[0120] Example 3. Preparation of Compound II, 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indole iodide

[0121] Reaction formula:

[0122]

[0123] Preparation process:

[0124] 0.32 g, i.e. 2.0 mmol, of 2,3,3-trimethyllindoleamine and 0.41 g, i.e. 2.4 mmol, of 2-iodine alcohol were dissolved in 15 mL of acetonitrile (CH3CN), refluxed and stirred overnight, and then the solvent was removed under reduced pressure. The resulting solid was washed with n-hexane to obtain 0.59 g of purple solid compound II, i.e. 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indole iodide, with a weight yield of 90%.

[0125] Test results:

[0126] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 8.05-7.77 (m, 2H), 7.63 (dt, J=5.6, 2.6Hz, 2H), 4.6 0 (t, J=5.3Hz, 2H), 3.89 (d, J=5.5Hz, 2H), 3.73 (s, 1H), 2.82 (s, 3H), 1.55 (s, 6H).

[0127] Example 4. Preparation of the compound MINIR, i.e., (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide

[0128] Reaction formula:

[0129]

[0130] Preparation process:

[0131] 0.28 g, i.e., 1.0 mmol, of compound DXC, 0.28 g, i.e., 2.0 mmol, of potassium carbonate (K2CO3), and 0.40 g, i.e., 1.2 mmol, of compound II were dissolved in 5 mL of anhydrous acetic anhydride (Ac2O) and stirred at room temperature for 15 hours to obtain a dark green solution. This solution was then dissolved in dichloromethane (CH2Cl2), washed twice with water, and dried over anhydrous sodium sulfate (Na2SO4). The solvent was evaporated and the residue was purified by silica gel chromatography using a mixed solution of dichloroethane:methanol in a volume ratio of 25:1 as eluent to obtain 0.37 g of dark green solid compound MINIR, i.e., (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide salt, in a weight yield of 58%.

[0132] Test results:

[0133] 1H NMR (400MHz, CD3CN): δ (ppm) 8.53 (d, J = 14.1Hz, 1H), 7.54 (s, 1H), 7.48 (d, J = 7.50Hz, 1H), 7.45-7 .35 (m, 2H), 7.24 (t, J=7.9Hz, 2H), 6.88 (d, J=8.9Hz, 1H), 6.68 (s, 1H), 6.17 (d, J=14.1Hz, 1H), 4. 50-4.40 (m, 2H), 4.37-4.27 (m, 2H), 3.63-3.42 (m, 4H), 2.78-2.69 (m, 2H), 2.66 (t, J=6.3Hz, 2H), 1.86 (t, J=6.1Hz, 2H), 1.80 (d, J=2.0Hz, 3H), 1.71 (d, J=2.0Hz, 6H), 1.21 (dt, J=8.2, 4.2Hz, 6H);

[0134] 13 C NMR (150MHz, CD3CN): δ (ppm) 174.40, 171.09, 164.60, 157.13, 153.09, 143.12, 142.41, 141.48, 139.90, 130.24, 129.07, 125.50, 123.90 , 122.87, 117.89, 115.99, 113.30, 111.47, 99.94, 96.49, 60.89, 49.80, 45.40, 43.80, 28.81, 28.17, 27.15, 24.76, 21.05, 20.49, 12.30;

[0135] HR-MS calculated for C 33 H 39 N2O3 + m / z 511.2956, found 511.2957.

[0136] From the above description, we can see that:

[0137] The preparation method provided in this embodiment has a simple process, convenient raw material sources, and a high comprehensive yield, and provides a new idea and method for preparing a new type of fluorescent probe, which is of great promotion and application value.

[0138] Example 3

[0139] This embodiment is used to provide an application of the dual-responsive organic fluorescent compound provided in the above-mentioned embodiment 1.

[0140] The application provided in this embodiment is to use the dual-responsive organic fluorescent compound provided in the above embodiment 1 as a fluorescent probe for dual-responsive fluorescence detection of hydrogen ion concentration index and / or peroxynitrite anion in living cells or living bodies, wherein the living cells are immortalized cells or normal cells, and the immortalized cells are HeLa cells or A549 cells.

[0141] To further help understand the technical effects that can be achieved by the technical solution provided in this embodiment, the application effects of the dual-responsive organic fluorescent compound provided in Example 1 of the present invention, that is, the application effects as a dual-responsive fluorescent probe, are further explained below through specific effect examples.

[0142] Effect Example 1: MNIR response to hydrogen ion concentration

[0143] from Figure 1 The absorption spectrum of MNIR with a concentration of 10 μM in PBS buffer solution with a pH range of 9 to 2 and an interval of 0.5 is shown in the figure. Figure 2 The fluorescence emission spectrum of MNIR at a concentration of 10 μM in PBS buffer solutions with a pH range of 9 to 2 and an interval of 0.5 can be seen in the following figure:

[0144] As the pH value of the PBS buffer solution increased from 9 to 2, a new absorption peak of the probe MNIR gradually appeared at 550 nm, while the absorption at 710 nm weakened; accordingly, the fluorescence of MNIR itself at 740 nm gradually decreased, while the new luminescence of the diethylamine protonation product at 652 nm gradually increased.

[0145] By plotting the 740nm / 652nm intensity ratio versus pH, we can use the formula:

[0146] log[(I max -I) / (II min )] = pH - pKa;

[0147] Among them: I max , I min and I are the maximum, minimum, and observed intensity ratios at a given pH value, respectively. The pKa of MNIR can be calculated to be 4.75, thus confirming that MNIR can sense acidified environments through the ratiometric method.

[0148] Effect Example 2: MNIR response to peroxynitrite anion

[0149] ONOO was prepared by adding 0.6M NaNO2, 0.6M HCl and 0.7M H2O2 into 1.5M NaOH solution at 0℃. -The absorbance of the mother solution was measured at 302 nm, and its extinction coefficient was 1670 M -1 cm -1 According to the Lambert-Beer law, ONOO - Concentration of the mother liquor.

[0150] Under normal physiological conditions of pH = 7.4, the effects of MNIR on the absorption of different concentrations of ONOO were tested. - The response can be obtained Figure 3 MNIR contains different concentrations of ONOO - Absorption spectrum in PBS buffer, and Figure 4 MNIR contains 0~35μM concentration of ONOO - Collection of absorption spectra in PBS buffer at pH = 7.4.

[0151] from Figure 3 and Figure 4 In it can be seen that:

[0152] PBS buffer does not contain ONOO - When , the maximum absorption wavelength of MNIR is 710nm and the maximum emission wavelength is 740nm;

[0153] Add ONOO to PBS buffer - After that, the absorption of MNIR at 710nm disappears, and a new absorption wavelength appears at 360nm; accordingly, the fluorescence intensity at 740nm decreases, and the new blue-green fluorescence at 482nm gradually increases; and in ONOO - Its concentration range is 0~8μM. - The response showed a good linear relationship, and the detection limit was as low as 273nM (3σ / slope).

[0154] Effect Example 3: Colocalization effect of MNIR in HeLa cells

[0155] 1) Cultivation of immortalized HeLa cancer cells

[0156] HeLa cells were inoculated into a cell culture medium containing 10% fetal bovine serum and 1% double-antibody H-DMEM and cultured in a CO2 incubator at 37°C and 5% CO2. After the HeLa cells grew to the logarithmic phase, they were plated and cultured: that is, after counting the cells, cells of appropriate density were left, and the culture medium was added to the required volume (controlling the final cell concentration to be 1×10 5 ), inoculated into confocal microplates, and placed in a CO2 incubator to allow the cells to adhere to the wall and grow.

[0157] 2) Colocalization experiment of MNIR probe in HeLa cells

[0158] In the co-staining experiment, HeLa cells were stained with 4 μM MNIR for 20 minutes, washed twice with PBS buffer solution, and then stained with 200 nM commercial probes MTG and LTG for 30 minutes respectively, and then washed twice with PBS buffer solution. Cell imaging was then performed under a confocal fluorescence microscope (Leica TCS SP8) to obtain the following results: Figure 5 The co-localization images of MNIR and commercial probes MTG and LTG in HeLa cells are shown, along with the corresponding fluorescence intensity spectra along the white arrows.

[0159] from Figure 5 It can be seen that:

[0160] The overlap coefficient between MNIR and MTG is very high, reaching 89%, while the overlap coefficient with LTG is lower, only 39%, indicating that MNIR has the ability to target mitochondria in biological systems.

[0161] Effect Example 4: MNIR's response to hydrogen ion concentration in living cells

[0162] HeLa cells were pretreated with a medium containing MNIR (4 μM) at 37°C for 20 min, washed twice with PBS buffer, and then incubated with a high K buffer containing 10.0 μM nigericin at pH 4-8. + HeLa cells were cultured in a buffer solution, wherein high K + The buffer solution consisted of 30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES, and 20 mM NaOAc. After 30 minutes, images of the green and red channels were recorded using a confocal microscope, and ratio imaging (green / red) was constructed to obtain Figure 6 The confocal fluorescence images and ratiometric imaging images of HeLa cells stained with MNIR under different pH conditions are shown, where the red channel is the emission of MNIR and the green channel is the fluorescence after MNIR protonation.

[0163] from Figure 6 In it, you can find:

[0164] As the pH value decreases, the fluorescence of the red channel weakens, while the fluorescence of the green channel increases, and the green / red ratio gradually increases.

[0165] It is worth noting that when the pH value is lower than 3, MNIR begins to migrate from the mitochondria to the nucleolus. When the pH value is further reduced, this phenomenon is more obvious. This may be due to the synergistic effect of amino groups and dications leading to strong binding with RNA. Therefore, this MNIR can monitor pH changes by ratiometric imaging and evaluate the mitochondrial acidification process by simple target conversion.

[0166] Effect Example 5: MNIR response to peroxynitrite anion in cells and in vivo

[0167] 5.1) MNIR response to peroxynitrite anion in cells:

[0168] HeLa cells were first stained with 4 μM MNIR for 20 minutes. Then, the stained HeLa cells were treated with different drugs in groups to stimulate the cells to produce ONOO - Then, the blue and red channel images were recorded using a confocal microscope and the overlay images of the blue and red channels were constructed to obtain Figure 7 That is, a collection of confocal fluorescence images of HeLa cells stained with MNIR after treatment with different drugs, and Figure 8 That is, corresponding Figure 7 The obtained confocal fluorescence image data of HeLa cells treated with different drugs and stained with MNIR were processed by Matlab software to draw the obtained confocal fluorescence relative intensity histogram, where:

[0169] The control group consisted of HeLa cell populations stained with 4 μM MNIR for 20 min;

[0170] The SIN-1 group was treated with 10 mM ONOO - HeLa cell populations treated with the production stimulator linxidamine (i.e., SIN-1) for 30 minutes;

[0171] The SIN-1+MC group consisted of HeLa cells treated with 10 mM SIN-1 for 30 minutes and then with 10 μM peroxynitrite anion scavenger minocycline (MC) for 30 minutes;

[0172] The SIN-1+pH2 group consisted of HeLa cells treated with 10 mM SIN-1 and then incubated in a pH=2 PBS buffer for 30 minutes.

[0173] 5.2) Response of MNIR to peroxynitrite anion in vivo:

[0174] This experiment used ICR mice as experimental subjects, which were purchased from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. During the experiment, the subjects were divided into groups and pretreated with different drugs by intraperitoneal injection, and then incubated with MNIR. Then, each experimental group was placed in an imaging chamber and imaged in the ICG staining channel using a small animal optical in vivo imaging system (VISQUE InVivo Smart-LF). Figure 9 That is, an image collection of ICR mice in vivo after intraperitoneal injection of different drugs, stained with MNIR and imaged using the indocyanine green dye channel for near-infrared imaging;

[0175] in:

[0176] The PBS group consisted of experimental subjects injected with 200 μL of PBS buffer solution within 3 h;

[0177] The probe group consisted of experimental living bodies injected with 200 μL, or a total amount of 10 μM, of MNIR within 30 minutes;

[0178] The SIN-1 group was injected with 200 μL of SIN-1 (a total of 10 mM) within 3 hours and 200 μL of MNIR (a total of 10 μM) within 30 minutes.

[0179] The SIN-1+MC group was injected with 200 μL of SIN-1 and MC (total amount of 10 mM) within 3 hours, and then injected with 200 μL of MNIR (total amount of 10 μM) within 30 minutes.

[0180] In this case, SIN-1, lindoxetine, stimulates ONOO - The formation of MC, namely minocycline, is ONOO - Cleaner.

[0181] from Figure 7 In it can be seen that:

[0182] The control group stained with MNIR alone showed bright red fluorescence and weak blue fluorescence; after SIN-1 treatment, ONOO - The red fluorescence almost disappeared, while the blue fluorescence became quite obvious; in cells treated with SIN-1 and MC continuously, the red fluorescence recovered and the blue fluorescence had almost no signal, which was because MC inhibited ONOO - These results indicate that MNIR can be a good indicator of ONOO by dual-channel imaging. - changes, avoiding false positive information errors;

[0183] In addition, from Figure 7As can be seen in the experimental group treated with SIN-1 and exposed to acidic environment, MNIR can identify ONOO by disappearance of fluorescence signal. - and H + The ability to coexist.

[0184] Combine Figure 6 That is, the results of MNIR response to hydrogen ion concentration shown in the confocal fluorescence images and ratio imaging images of HeLa cells stained with MNIR under different pH conditions are not difficult to find. MNIR can well distinguish between no analyte, only hydrogen ion concentration change, and ONOO - and hydrogen ions and ONOO - Coexistence situation.

[0185] Since near-infrared emission has inherent advantages in in vivo imaging, Figure 9 As shown in the image atlas of the four groups of ICR mice after intraperitoneal injection of different drugs, the following images were obtained after MNIR staining using the indocyanine green dye channel for near-infrared imaging:

[0186] The untreated PBS group experimental living body did not emit fluorescent signals;

[0187] Only the probe group stained with MNIR emitted strong near-infrared fluorescence in vivo;

[0188] Induction of exogenous ONOO by SIN-1 - After the experimental organism is generated, due to the interaction between MNIR and ONOO - The reaction dissipates and the fluorescence becomes very weak

[0189] After adding SIN-1 and MC to the experimental living body, its near-infrared fluorescence was restored.

[0190] The above results show that MNIR can effectively inhibit ONOO in living cells and in vivo. - Be responsive.

[0191] It should be noted that:

[0192] Although this effect example only provides the experimental results of the MNIR provided in Example 1 of the present invention on living organisms represented by ICR mice, those skilled in the art can conduct similar experiments on other living organisms according to the above experimental methods and obtain similar results. Therefore, it can be considered that the MNIR provided by the present invention has the ability to respond to peroxynitrite anions in living organisms.

[0193] Effect Example 6: Relationship between MNIR, hydrogen ion concentration, and peroxynitrite anion during cisplatin treatment

[0194] A549 cells were inoculated into a cell culture medium containing 10% fetal bovine serum and 1% double-antibody H-DMEM and cultured in a CO2 incubator at 37°C and 5% CO2. After A549 cells grew to the logarithmic phase, they were plated and cultured: after counting the cells, cells of appropriate density were left and the culture medium was added to the required volume (control the final cell concentration to be 1×10 5 ), inoculated into confocal microplates, and placed in a CO2 incubator to allow the cells to adhere to the wall and grow.

[0195] The cultured A549 cells were first stained with 4 μM MNIR, then incubated at room temperature for 30 minutes, and then treated with 500 μM cisplatin for different time periods. After treatment, the cells were washed twice with PBS buffer solution and then observed under a confocal microscope. Figure 10 , a collection of bright field and confocal fluorescence images of A549 cells treated with 500 μM cisplatin and stained with MNIR.

[0196] from Figure 10 It can be seen that:

[0197] As the duration of cisplatin treatment prolonged, the cell count decreased significantly, which was due to cisplatin causing cell death by interfering with human cell DNA replication;

[0198] At the same time, it can be observed that the blue fluorescence and green fluorescence gradually increase and the red fluorescence decreases, which indicates that ONOO - The pH value of the cells decreased after 24 h of treatment, and the pH value of the cells gradually became acidic, reaching as low as 3.

[0199] The above results suggest that artificial intervention of hydrogen ion concentration may be effective in inhibiting ONOO - The production of cisplatin can reduce the side effects of cisplatin treatment.

[0200] In summary, we can see that:

[0201] The dual-response organic fluorescent compound provided by the present invention, (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodine salt, contains H + and ONOO -The two response sites can change their emission color with the change of molecular structure under acidic conditions or after reacting with peroxynitrite anion. Therefore, it can be used as an ideal fluorescent probe for dual analyte sensing, and can be used for analytical research in a variety of different situations, including no analyte, changes in hydrogen ion concentration or peroxynitrite anion concentration alone, or the coexistence of hydrogen ion and peroxynitrite anion. It can also be selectively used to monitor changes in mitochondrial hydrogen ion concentration and peroxynitrite anion in living cells and living bodies, so that changes in both in living cells or in vivo can be well monitored. It can also be used as a hydrogen ion The ideal dual-response logic sensor for ratiometric and differential imaging of concentration and peroxynitrite anion overcomes the deficiency of existing probes for hydrogen ion concentration index or peroxynitrite anion detection that can only identify a single analyte, thereby eliminating the tedious staining and repeated washing processes caused by the use of different probe combinations in related biological experiments, avoiding spectral crosstalk and increased cytotoxicity, and can visualize the interactions between multiple small molecules in real time, thereby facilitating the discovery of new signaling pathways and understanding the interactions between exogenous and endogenous species, contributing to the research and exploration of biological systems; and

[0202] The preparation method provided by the present invention has simple process, convenient raw material source, high comprehensive yield, and provides a new idea and method for preparing new fluorescent probes;

[0203] As a new type of fluorescent probe, it has the characteristics of wide application range, sensitive response, fast speed, and the ability to simultaneously distinguish and detect hydrogen ion concentration and peroxynitrite anion. It has broad application prospects. Therefore, compared with existing technologies, it has outstanding substantial characteristics, beneficial effects and significant progress. Therefore, it is of great promotion and application value.

[0204] In the description of the above manual:

[0205] The terms "this embodiment", "an embodiment of the present invention", "as shown in", "further", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined or combined in any appropriate manner in any one or more embodiments or examples.

[0206] In addition, those skilled in the art may combine or combine different embodiments or examples and features of different embodiments or examples described in this specification without causing any contradiction.

[0207] Finally, it should be noted that:

[0208] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments or examples, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments or examples, or make equivalent replacements for groups or all of the technical features therein, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments or examples of the present invention. Non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A dual-response organic fluorescent compound, characterized in that: Its chemical name is: (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide, abbreviated as MNIR, has the following chemical structure:

2. A method for preparing the dual-responsive organic fluorescent compound according to claim 1, characterized in that: The method comprises steps a to b, c and d in the following synthetic route:

3. The preparation method according to claim 2, characterized in that The step a comprises: Under an ice bath, phosphorus tribromide is slowly added to a mixed solution of N,N-dimethylformamide and chloroform, and the mixture is stirred. Cyclohexanone dissolved in chloroform is then added, and the mixture is stirred and reacted at room temperature. After the reaction is completed, the reaction mixture is poured into ice water, and sodium hydroxide solution is slowly added to adjust the pH to neutral. The aqueous layer is extracted with dichloromethane, and the resulting organic layer is dried over anhydrous sodium sulfate and then filtered. After removing the solvent from the filtrate, a yellow oily compound I, i.e., 2-bromocyclohexyl-1-ene-1-carbaldehyde, is obtained.

4. The preparation method according to claim 3, characterized in that In the step a: The volume ratio of N,N-dimethylformamide to carbon trichloride is 1:(1.5-1.6), the molar ratio of the reactants phosphorus tribromide to cyclohexanone is (3.0-3.5):1, and the compound I is stored at -20°C.

5. The preparation method according to claim 2, characterized in that The step b comprises: Compound I, 4-(diethylamino)salicylaldehyde and cesium carbonate are added to N,N-dimethylformamide solvent and stirred at room temperature for reaction. After the reaction is completed, the insoluble matter is removed by filtration, and the filtrate is concentrated. The residue is extracted with ethyl acetate, washed with water, and dried over anhydrous sodium sulfate. The organic phase is concentrated under reduced pressure and the crude product is purified by silica gel chromatography to obtain a dark yellow solid compound DXC, i.e., 6-diethylamino-2,3-dihydro-1H-xanthene-4-carboxaldehyde.

6. The preparation method according to claim 5, characterized in that In the step b: The molar ratio of the reactants is: compound I: 4-(diethylamino) salicylaldehyde: cesium carbonate = 3:1:3; The eluent in the silica gel chromatography method is a mixture of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate mixture is 10:

1.

7. The preparation method according to claim 2, characterized in that The step c comprises: 2,3,3-Trimethyllindoleamine and 2-iodine alcohol are co-dissolved in acetonitrile and subjected to reflux reaction. After the reaction, the solvent is removed and the resulting solid is washed with n-hexane to obtain a purple solid compound II, i.e., 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indole iodide, wherein: The molar ratio of the reactants is: 2,3,3-trimethyllindoleamine: 2-iodine alcohol = 1:1.

2.

8. The preparation method according to claim 2, characterized in that The step d comprises: Compound DXC, potassium carbonate and compound II are co-dissolved in anhydrous acetic anhydride, and the reaction is stirred at room temperature to obtain a dark green solution. The dark green solution is then dissolved in dichloromethane, washed with water, and then dried over anhydrous sodium sulfate. The solvent is removed, and the residue is purified by silica gel chromatography to obtain a dark green solid compound MINIR, i.e., (E)-1-(2-acetoxyethyl)-2-(2-(6-(diethylamino)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indole-1-iodide salt.

9. The preparation method according to claim 8, characterized in that In the step d: The molar ratio of the reactants is: compound DXC: potassium carbonate: compound II = 1:2:1.2; The eluent in the silica gel chromatography purification method is a mixture of dichloroethane and methanol, and the volume ratio of the dichloroethane to the methanol is 25:

1.

10. Use of the dual-responsive organic fluorescent compound according to claim 1, characterized in that: The application is to use the dual-responsive organic fluorescent compound according to claim 1 as a fluorescent probe for dual-responsive fluorescence detection of hydrogen ion concentration index and / or peroxynitrite anion in living cells or living bodies, wherein: The living cells are immortalized cells or normal cells, and the immortalized cells are HeLa cells or A549 cells; This application is not for the purpose of treating or diagnosing diseases.

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