A d-pi-a-pi-d type fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a D-pi-A-pi-D type fluorescent probe and a preparation method and application thereof, the fluorescent probe can be a compound as shown in the following formula or a salt or solvate thereof, a D-pi-A-pi-D type structure is introduced into the compound in the scheme, and the limitation that the current NO fluorescent probe fluorophore is mostly concentrated in the traditional classic dye category is overcome; the fluorescent probe provided by the application has the characteristics of simple operation, rapid reaction, good selectivity, excellent fluorescence signal anti-photobleaching property, colorimetric and fluorescence / ultraviolet multi-mode detection in NO detection.

Description

Technical Field

[0001] This invention relates to the fields of chemistry and analytical detection, and in particular to a D-π-A-π-D type fluorescent probe, its preparation method, and its application. Background Technology

[0002] Nitric oxide (NO), as an important signaling molecule, plays a crucial role in signal transduction and regulation in numerous physiological systems, including the cardiovascular, immune, respiratory, gastrointestinal, and central nervous systems. NO imbalances in the body can lead to diseases such as atherosclerosis, immune disorders, neurodegenerative diseases, cancer, and acute and chronic inflammation. Furthermore, excessive NO in the environment can combine with hemoglobin in the blood, reducing the blood's oxygen-carrying capacity. The NO2 produced by its oxidation can cause lung and bronchitis, posing a serious threat to human health. Therefore, NO detection is of great significance in biomedical research and environmental monitoring. Currently, NO detection methods mainly include electrochemical methods, chromatographic analysis, and colorimetric methods, but these methods suffer from drawbacks such as slow detection speed, low sensitivity, complex equipment, and high cost. Therefore, the development of novel fluorescent probes with high selectivity, significant effects, low cost, fast response, and high sensitivity is urgently needed.

[0003] Fluorescence analysis is convenient, intuitive, real-time, and rapid, and the research on NO fluorescent probes has attracted much attention. However, most existing fluorescent probes are developed based on classical fluorophores (see references (1) to (11)), such as classic dyes like BODIPY, coumarin, fluorescein, rhodamine, naphthaleneimide, and Nile red. Classical fluorophores exhibit superior fluorescence performance, but they are prone to photobleaching and have insufficient photostability, which limits their application.

[0004] DA-type large π-conjugated nonclassical fluorophores with strong electron donors (D) and acceptors (A) possess significant advantages such as high photostability and tunable photophysical properties. However, reports on constructing D-π-A-π-D type fluorescent probes for NO detection based on this strategy are still very rare. Therefore, developing a novel D-π-A-π-D type fluorescent probe for NO detection is of great significance.

[0005] References:

[0006] (1)J.Am.Chem.Soc.2021,143,7196-7202.

[0007] (2)J.Mater.Chem.B 2018,6,4096-4103.

[0008] (3)Anal.Chem.2017,89,9620-9624.

[0009] (4)J.Am.Chem.Soc.2006,128,14364-14373.

[0010] (5) Chem. Sci. 2016, 7, 5230-5235.

[0011] (6)Sens.Actuators B Chem.2021,329,129147.

[0012] (7)Sens.Actuators B Chem.2023,383,133592.

[0013] (8)Spectrochim.Acta,Part A,2016,169,1-6.

[0014] (9)Anal.Chem.2021,93,4391-4397.

[0015] (10)Anal.Chem.2023,95,7320-7328.

[0016] (11)J.Mater.Chem.B,2019,7,3792-3795. Summary of the Invention

[0017] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a substance that can be used as a fluorescent probe for NO detection, which can achieve highly sensitive, naked-eye detection of NO.

[0018] The present invention also proposes a method for preparing the above-mentioned compounds.

[0019] The present invention also proposes applications of the above-mentioned compounds.

[0020] According to one aspect of the invention, a compound of formula I or a salt or solvate thereof is provided:

[0021]

[0022] In the formula, R is any one of alkyl, alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein Ar1 is heteroaryl or substituted heteroaryl, and Ar2 is any one of aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0023] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: The compounds of the present invention, or their salts or solvates, can be used as NO fluorescent probes, enriching the types of NO fluorescent probes and solving the problem of the scarcity of NO fluorescent probe types. When used as NO fluorescent probes, these substances exhibit high sensitivity, fast response speed, and can be detected by ultraviolet, fluorescence, or colorimetric methods, requiring no complex equipment, making them convenient to use and inexpensive. This provides a highly sensitive fluorescent probe capable of detecting NO with the naked eye, and this probe has promising application prospects in the diagnosis of NO-related diseases. The compounds of the present invention introduce a D-π-A-π-D type structure, overcoming the limitation that the fluorophores of current NO fluorescent probes are mostly concentrated in the category of traditional classical dyes. The fluorescent probe provided by the present invention features simple operation, rapid reaction, good selectivity, excellent photobleaching resistance of the fluorescence signal, and multi-mode detection by colorimetry and fluorescence / ultraviolet.

[0024] In some embodiments of the present invention, the number of carbon atoms in the alkyl and / or alkoxy groups is independently 6 to 24.

[0025] In some embodiments of the present invention, the alkyl group is -(CH2). n CH3, n = 5 to 23.

[0026] In some embodiments of the present invention, the alkoxy group is -O(CH2). n CH3, n = 5 to 23.

[0027] In some embodiments of the present invention, R is selected from at least one of the following structures:

[0028]

[0029] n = 0 to 23.

[0030] In some embodiments of the present invention, the Ar1 is selected from one of the following structures and the Ar1 forms a fused ring with the adjacent benzene ring:

[0031]

[0032] In some embodiments of the present invention, the Ar2 is selected from one of the following structures:

[0033]

[0034] According to another aspect of the present invention, a method for preparing the compound represented by Formula I above is provided, comprising the following steps:

[0035] S1. React compound a with compound b to obtain compound c;

[0036] S2. React compound c with a reducing agent to obtain the compound shown in Formula I;

[0037] The structural formulas of compounds a, b, and c are as follows:

[0038]

[0039] In the formula, Y is a boric acid group, borate ester group, zinc halide group, magnesium halide group or trialkyltin group; X is a halogen.

[0040] The preparation method according to a preferred embodiment of the present invention has at least the following beneficial effects: the preparation method of the present invention is simple to operate, uses inexpensive and readily available raw materials, is easy to achieve mass production, and has good prospects for industrial application. The compound is prepared by using carbazole donors with different structures, π-bridge groups, and electron acceptors via Ullmann reaction, lithium-ionization reaction, and Stilling coupling reaction. The o-phenylenediamine group with electron-donating ability on the electron acceptor can serve as a highly efficient NO recognition site. In the presence of low concentrations of NO, the o-phenylenediamine group can be converted into a benzotriazole group with stronger electron-withdrawing ability, thereby generating a stronger intramolecular charge transfer process, accompanied by a dual change in the color and fluorescence of the probe solution, thus achieving the purpose of highly sensitive, naked-eye detection of NO.

[0041] In some embodiments of the present invention, if present, the borate group is selected from 1,3,2-dioxaborane-2-yl, 4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl or 5,5-dimethyl-1,3,2-dioxaborane-2-yl; if present, the trialkyltin group is selected from trimethyltinyl, triethyltinyl or tributyltinyl.

[0042] In some embodiments of the present invention, the reaction conditions of compounds a and b are as follows: under alkaline conditions I, catalyzed by palladium catalyst I, the reaction takes place in an organic solvent I, wherein the organic solvent I is anhydrous and oxygen-free. The organic solvent I used must be treated to be anhydrous and oxygen-free.

[0043] In some embodiments of the present invention, the reaction time of compounds a and b is 12 to 24 hours.

[0044] In some embodiments of the present invention, the reaction temperature of compounds a and b is 80–130°C.

[0045] In some embodiments of the present invention, the organic solvent I is toluene, dioxane, or tetrahydrofuran.

[0046] In some embodiments of the present invention, the palladium catalyst I comprises at least one of tetrakis(triphenylphosphine)palladium, palladium acetate, or 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride.

[0047] In some embodiments of the present invention, the pH of the alkaline condition I is 10-13; preferably, it is achieved by adding a strong base carbonate salt.

[0048] In some embodiments of the present invention, the strong base carbonate salt is selected from alkali metal salts, such as potassium, sodium, and cesium salts.

[0049] In some embodiments of the present invention, in step S2, a reducing agent is added to cause a reduction reaction of compound c, wherein the reducing agent includes at least one of iron powder, zinc powder or tin dichloride.

[0050] In some embodiments of the present invention, the reduction reaction takes 6 to 12 hours and / or the reaction temperature is 80-135°C.

[0051] In some embodiments of the present invention, step S2 is carried out in acetic acid. In some embodiments of the present invention, Ar2 is aryl or substituted aryl, then the preparation method of compound a includes the following steps: Using [a specific ingredient] as a raw material, the compound a is prepared by reacting it in an organic solvent II under alkaline conditions II and catalyzed by palladium catalyst II, wherein the organic solvent is anhydrous and oxygen-free. The organic solvent used must be treated to be anhydrous and oxygen-free.

[0052] In some embodiments of the present invention, the preparation conditions of compound a include a reaction time of 12 to 24 hours.

[0053] In some embodiments of the present invention, the preparation conditions of compound a include a reaction temperature of 80–130°C.

[0054] In some embodiments of the present invention, the preparation conditions of compound a include: the organic solvent being toluene, dioxane, or tetrahydrofuran.

[0055] In some embodiments of the present invention, the palladium catalyst comprises at least one of tetra(triphenylphosphine)palladium, palladium acetate, or 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride.

[0056] In some embodiments of the present invention, the pH of the alkaline condition II is 7.5-9.0, preferably achieved by adding a strong base salt of acetate.

[0057] In some embodiments of the present invention, the strong base salt of acetate is selected from alkali metal salts, such as potassium, sodium, and cesium salts.

[0058] In some embodiments of the present invention, Ar2 is a heteroaryl or substituted heteroaryl, and the preparation method of compound a includes the following steps: using Using tetrahydrofuran (THF) as a solvent, n-butyllithium and SnBu3Cl were added at -70 to -80°C and reacted for 0.2 to 1 h. Then, the reaction was carried out at room temperature for 10 to 15 h to prepare compound a.

[0059] In some embodiments of the present invention, Ar2 is a heteroaryl or substituted heteroaryl, and the preparation method of compound a includes the following steps: using Using tetrahydrofuran (THF) as a solvent, n-butyllithium and SnBu3Cl were added at -78℃ and reacted for 0.5 h, followed by 12 h at room temperature to prepare compound a.

[0060] According to another aspect of the invention, a kit is provided comprising the above-described compound or its salt or solvate.

[0061] In some embodiments of the present invention, the kit further includes a solvent.

[0062] In some embodiments of the present invention, the kit further includes a nitric oxide donor.

[0063] According to another aspect of the invention, the use of the above-described compound or its salt or solvate in the preparation of reagents for the detection of nitric oxide is proposed.

[0064] According to another aspect of the present invention, a method for detecting nitric oxide in a sample is provided, the method comprising the steps of contacting the sample with the above-mentioned compound or its salt or solvate.

[0065] In some embodiments of the present invention, the sample is a biological sample or an environmental sample. For example, a biological sample may be a bodily fluid such as blood.

[0066] This method has practical application value in the field of NO detection research and can be used for in vitro and in vivo detection, environmental monitoring, etc.

[0067] In some embodiments of the present invention, the detection method further includes a step of detection and analysis by ultraviolet, fluorescence spectroscopy or colorimetry.

[0068] The fluorescent probe provided by this invention has the characteristics of colorimetric and fluorescence / ultraviolet multi-mode detection in NO detection. It can achieve qualitative or quantitative detection through ultraviolet and fluorescence spectroscopy, and can also achieve qualitative or semi-quantitative detection through naked-eye colorimetry.

[0069] In some embodiments of the present invention, the detection method includes contacting a sample with the above-mentioned compound or its salt or solvate in a solvent, said solvent being a mixture of dimethyl sulfoxide (DMSO) and phosphate (PBS) buffer.

[0070] In some embodiments of the present invention, the concentration of the above-mentioned compound or its salt or solvate in the solvent is 10. -7 ~10 -2 mol / L.

[0071] In some embodiments of the present invention, the contact time is 2 to 25 minutes. The reaction between the compound of the present invention or its salt or solvate and NO is rapid, and detection can be completed within 30 minutes, resulting in high detection efficiency.

[0072] In some embodiments of the present invention, the detection limit of NO during the detection process is 100 μmol / L.

[0073] In some embodiments of the present invention, the detection concentration range of NO during the detection process is 0.0768 to 100 μmol / L.

[0074] According to another aspect of the invention, the use of the above-described compound or its salt or solvate in the preparation of a reagent for diagnosing a target disease or evaluating a treatment for a target disease, the disease affecting the content of nitric oxide.

[0075] Such as hypertension-related diseases, cancer, diabetes, cardiovascular diseases, viral diseases (such as immune, respiratory, gastrointestinal, acute and chronic inflammation, etc.), impotence, or nerve damage.

[0076] According to another aspect of the invention, the use of the above-described compound or its salt or solvate in the preparation of reagents for cell imaging is proposed.

[0077] According to another aspect of the invention, the use of the above-mentioned compound or its salt or solvate in the preparation of a reagent for evaluating the therapeutic effect of a drug used to diagnose a target disease or to evaluate a treatment for a target disease, the disease affecting nitric oxide levels, such as hypertension-related diseases, cancer, diabetes, cardiovascular diseases, viral diseases (such as immune, respiratory, gastrointestinal, acute and chronic inflammation, etc.), impotence, or nerve damage, etc.

[0078] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0079] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0080] Figure 1 This is the high-resolution mass spectrum of compound 2 in Example 2 of the present invention;

[0081] Figure 2 This is a high-resolution mass spectrum of the probe Car-NO-1 in Example 2 of the present invention;

[0082] Figure 3 This is the high-resolution mass spectrum of compound 4 in Example 3 of the present invention;

[0083] Figure 4 This is a high-resolution mass spectrum of the probe Car-NO-2 in Example 3 of the present invention;

[0084] Figure 5 The UV absorption spectra of the solutions before and after the reaction of probe Car-NO-1 with NO in Example 4 of this invention are shown.

[0085] Figure 6 The fluorescence spectrum of the solution after the reaction of probe Car-NO-1 with NO in Example 5 of this invention is shown.

[0086] Figure 7 The fluorescence intensity of the solution after the reaction of probe Car-NO-1 with NO in Example 5 of this invention changes over time;

[0087] Figure 8 This shows the linear relationship between the fluorescence intensity and NO concentration of the probe Car-NO-1 in Example 6 of this invention. Detailed Implementation

[0088] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0089] In the description of this invention, if I and II are mentioned, they are only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or the order of the technical features indicated. They may be the same or different.

[0090] In the description of this invention, the term "alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group. Examples of alkyl groups include, but are not limited to, hexyl, heptyl, octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecyl, tridecyl, and tetradecyl.

[0091] In the description of this invention, the term "alkoxy" refers to a group of the formula "-O-alkyl", including but not limited to hexoxy, heptoxy, octoxy, decoxy, undecoxy, dodecoxy, tridecoxy, tetradecoxy, pentadecoxy, hexadecoxy, heptadecanoxy, octadecoxy, nonadecanoxy, eicosoxy, dodecoxy, tridecoxy, tridecoxy, and tetradecoxy.

[0092] In the description of this invention, the term "aryl" represents an aromatic hydrocarbon group comprising one or more phenyl groups, preferably an aromatic hydrocarbon group with 6 to 10 carbon atoms, and more preferably phenyl.

[0093] In the description of this invention, the term "heteroaryl" represents a heteroatom aromatic group containing one to four heteroatom aromatic groups selected from Se, Te, O, N and S, preferably a five-membered ring heteroatom aromatic group, including but not limited to thiophene groups.

[0094] In the description of this invention, the term "substitution" means that a group may or may not be further substituted by one or more groups selected from the following: alkyl, alkenyl, alkynyl, aryl, halogen, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxyl, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, heteroaryl, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclic, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, phenylamino, diphenylamino, benzylamino, dibenzylamino, hydrazyl, acyl, acylamino, diacylamino, acyloxy, heterocyclic, heterocyclic oxy, heterocyclic amino, haloheterocyclic, carboxyl ester, carboxyl, carboxylamide, mercapto, alkylthio, benzylthio, acylthio, and phosphorus-containing groups.

[0095] In the description of this invention, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0096] In the description of this invention, the term "compound" refers to all stereoisomers, geometric isomers, tautomers, and isotopes including the described structure. Unless otherwise stated, compounds identified herein as a particular tautomer by name or structure are intended to include other tautomers. It should be understood that some compounds provided herein may contain one or more asymmetric centers and can therefore be prepared and isolated as mixtures of isomers, such as racemic mixtures, or in enantiomeric purity.

[0097] In the description of this invention, the term "tautomer" refers to a compound whose structure is significantly different in atomic arrangement but exists in a simple and rapid equilibrium, and it should be understood that the compounds provided herein can be described as different tautomers, and when a compound has a tautomer form, all tautomer forms are within the scope of this invention, and the naming of the compound does not exclude any tautomer.

[0098] In the description of this invention, the Formula I compound includes its salt, which can be used, for example, as an intermediate for the preparation and / or purification of Formula I compound and / or the isolation of Formula I compound. Non-limiting examples of salts include chlorides, bromides, or fluorides of Formula I compound, etc. In one or more embodiments, the Formula I compound includes Formula II compound and its stereoisomers, salts, and solvates.

[0099] The compounds of Formula I or their salts described in this invention can be isolated as solvates, and therefore any such solvates are included within the scope of this invention. For example, compounds of Formula I and their salts can exist in both unsolvated and solvated forms with solvents (e.g., water, ethanol, etc.).

[0100] The compounds of Formula I of this invention can cross cell membranes and / or organelle membranes. In some embodiments, the compounds of Formula I can enter organelles. "Organelles" are substructures or microorganisms in the cytoplasm that have specific morphological structures and functions. Organelles include, but are not limited to, mitochondria, endoplasmic reticulum, centrosomes, chloroplasts, Golgi apparatus, ribosomes, etc. In some embodiments, the compounds of Formula I target mitochondria.

[0101] In the description of this invention, the term "treating" includes treating a disease state in mammals, particularly humans, and includes: (a) preventing or delaying the onset of a disease state in a mammal, specifically when the mammal is susceptible to the disease state but has not yet been diagnosed with the disease state; (b) suppressing the disease state, i.e., preventing its development; and / or (c) achieving a complete or partial reduction of symptoms or disease state, and / or alleviating, improving, reducing or curing the disease or condition and / or its symptoms.

[0102] Used in the structural formula of the present invention Represents the connection site.

[0103] In the description of this invention, "room temperature" refers to any temperature between 25 and 5°C, and specifically 25°C in the embodiments.

[0104] Example 1

[0105] This invention provides a compound that can be used as a fluorescent probe and has the structure shown in Formula I:

[0106]

[0107] In the formula, R is any one of alkyl, alkoxy, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, where Ar1 is a heteroaryl or substituted heteroaryl, and Ar2 is a benzene ring or a five-membered aromatic heterocycle.

[0108] (I) For probe molecules where the π-bridge of the Ar2 structure is a five-membered heterocyclic structure, the synthetic route for preparing such probes is shown in "Preparation Method I". This method specifically includes the following steps:

[0109]

[0110] Preparation Method 1

[0111] (1) The compound shown in formula i reacts with n-butyllithium and SnBu3Cl in tetrahydrofuran at -78°C for 0.5 h, and then reacts at room temperature for 12 h to obtain the compound shown in formula ii. Wherein, A represents S, Se, and Te atoms.

[0112] (2) Under alkaline conditions, the compounds shown in formula ii and formula iii above are reacted in an organic solvent with a palladium catalyst for 12-24 h to obtain a class of molecules as shown in formula iv. Here, A represents S, Se, or Te atoms; the organic solvent is toluene or tetrahydrofuran, and the organic solvent used must be anhydrous and oxygen-free; the palladium catalyst is tetra(triphenylphosphine)palladium, palladium acetate, or dichlorotriphenylphosphine palladium, etc.; the base can be potassium carbonate, sodium carbonate, or cesium carbonate, etc.; the reaction temperature is 80-130℃.

[0113] (3) The compound shown in formula iv reacts with a reducing agent in an organic solvent for 6-12 hours to obtain a class of molecules as shown in general formula I-1. The reducing agent is iron powder, zinc powder, or tin dichloride, etc., and the organic solvent is acetic acid, etc.

[0114] (II) For probe molecules where the π-bridge of the Ar2 structure is a benzene ring structure, the synthetic route for preparing such probes is shown in "Preparation Method II". This method specifically includes the following steps:

[0115]

[0116] Preparation Method 2

[0117] (4) Under alkaline conditions, the compound shown in formula v above reacts in an organic solvent with a palladium catalyst for 12-24 hours to obtain a class of molecules shown in formula vi. The organic solvent can be toluene, dioxane, or tetrahydrofuran, and must be anhydrous and oxygen-free. The palladium catalyst is tetra(triphenylphosphine)palladium, palladium acetate, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, etc. The reaction temperature is 80-130℃. The base can be potassium acetate.

[0118] (5) Under alkaline conditions, the compound shown in formula vi reacts with a palladium catalyst in an organic solvent for 12-24 hours to yield a class of molecules shown in formula vii. The organic solvent is toluene, dioxane, or tetrahydrofuran, and must be anhydrous and oxygen-free. The palladium catalyst is tetra(triphenylphosphine)palladium, palladium acetate, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, etc. The reaction temperature is 80-130℃. The base can be potassium carbonate, sodium carbonate, or cesium carbonate, etc.

[0119] (6) The compound shown in formula vii reacts with a reducing agent under an organic solvent for 6-12 hours to obtain a class of molecules as shown in general formula I-2. The reducing agent is iron powder, zinc powder, or tin dichloride, etc., and the organic solvent is acetic acid, etc.

[0120] The compound obtained through the above operations, along with a mixed solution of DMSO and PBS, were used to prepare an NO detection kit. This kit can be used for the diagnosis of NO-related diseases and for the detection of environmental samples. Qualitative or semi-quantitative detection can be achieved through visual observation, or quantitative detection can be performed using fluorescence spectroscopy. It features high sensitivity, rapid response, and high detection efficiency.

[0121] Example 2

[0122] This embodiment prepared a compound, Car-NO-1, which can be used as a fluorescent probe. The molecular structure of the probe molecule Car-NO-1 prepared in this embodiment is as follows:

[0123]

[0124] Its synthetic route is as follows:

[0125]

[0126] The specific synthesis steps are as follows:

[0127] (1) Compound 1 was prepared according to the method disclosed in the literature H.Li, T.Koh, Y.Hao, F.Zhou, Y.Abe, H.Su, A.Hagfeldt, ACGrimsdale, ChemSusChem 7 (2014) 3396-3406.

[0128] (2) Synthesis of Compound 2

[0129] Compound 1 (507.5 mg, 0.78 mmol), 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (100 mg, 0.26 mmol), and bis(triphenylphosphine)palladium dichloride (18.2 mg, 0.026 mmol) were weighed into a 100 mL double-necked flask. Redistilled tetrahydrofuran was added, the mixture was evacuated, purged with nitrogen, and the atmosphere was refluxed at 125 °C overnight. The reaction was monitored by TLC. After the reaction was terminated, the mixture was cooled to room temperature, saturated potassium fluoride solution was added, and the mixture was filtered through diatomaceous earth. The filter cake was washed with dichloromethane. The mixture was then extracted three times with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain 122.9 mg of purple powder, compound 2, in 59% yield. 1 H NMR (500MHz, Chloroform-d) δ 8.12 (d, J = 1.9 Hz, 4H), 7.65–7.61 (m, 6H), 7.56 (dd, J = 8.7, 1.9 Hz, 4H), 7.32 (d, J = 4.0 Hz, 2H), 1.48 (s, 36H), its high-resolution mass spectrum is as follows: Figure 1 As shown.

[0130] (3) Synthesis of probe Car-NO-1

[0131] Compound 2 (140 mg, 0.15 mmol) was weighed and placed in a 100 mL double-necked flask. HAc (12 mL) was added, followed by iron powder (248.2 mg, 4.5 mmol). The mixture was evacuated, purged with nitrogen, and stirred at 100 °C. The reaction was monitored by TLC. After the reaction was terminated, the iron powder was removed by filtration. Saturated sodium bicarbonate aqueous solution was added to the filtrate, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain 86.6 mg of orange-yellow powder, with a yield of 66%. 1 ¹H NMR (500MHz, Chloroform-d) δ 8.13 (d, J = 1.9 Hz, 4H), 7.62 (d, J = 8.6 Hz, 4H), 7.53 (dd, J = 8.7, 2.0 Hz, 4H), 7.49 (d, J = 3.8 Hz, 2H), 7.33 (d, J = 3.7 Hz, 2H), 4.62 (s, 4H), 1.48 (s, 36H), and its high-resolution mass spectrum is as follows: Figure 2 As shown.

[0132] Example 3

[0133] In this embodiment, the compound Car-NO-2, which can be used as a fluorescent probe, was prepared. The molecular structure of the probe molecule Car-NO-2 prepared in this embodiment is as follows:

[0134]

[0135] Its synthetic route is as follows:

[0136]

[0137]

[0138] The specific synthesis steps are as follows:

[0139] (1) Synthesis of compound 4

[0140] Compound 3 (498.42 mg, 1.04 mmol), 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (181.6 mg, 0.473 mmol), potassium carbonate (392.2 mg, 2.838 mmol), and Pd(PPh3)4 (54.7 mg, 0.0473 mmol) were weighed into a 100 mL double-necked flask. Toluene:water (v / v, 4:1) was added, the mixture was evacuated, purged with nitrogen, and the atmosphere was purged three times. The reaction was carried out at 95 °C, and the reaction was monitored by TLC. After the reaction was terminated, the mixture was cooled to room temperature, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain 154.2 mg of orange-red powder, with a yield of 37%. 1 ¹H NMR (500MHz, Chloroform-d) δ 8.17 (d, J=1.8Hz, 4H), 7.84 (s, 6H), 7.57–7.51 (m, 8H), 7.26 (s, 2H), 1.50 (s, 36H), and its high-resolution mass spectrum is as follows: Figure 3 As shown.

[0141] (2) Synthesis of compound Car-NO-2

[0142] Compound 4 (100 mg, 0.107 mmol) was weighed into a 50 mL double-necked flask, HAc (8.6 mL) was added, followed by iron powder (239.4 mg, 4.29 mmol). The mixture was evacuated, purged with nitrogen, and stirred at 100 °C. The reaction was monitored by TLC. After the reaction was terminated, the iron powder was removed by filtration, and a saturated sodium bicarbonate aqueous solution was added. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain a yellow powder compound Car-NO-2 in 42% yield. 1¹H NMR (500MHz, Chloroform-d) δ 8.18 (d, J = 1.9 Hz, 4H), 7.89–7.81 (m, 8H), 7.56 (d, J = 8.6 Hz, 4H), 7.51 (dd, J = 8.7, 2.0 Hz, 4H), 4.26 (s, 4H), 1.50 (s, 36H), and its high-resolution mass spectrum is as follows: Figure 4 As shown.

[0143] Example 4

[0144] This embodiment provides a method for NO detection. Specifically, the compound Car-NO-1 obtained in Example 2 is used as a probe for NO detection, and the detection and analysis are performed by naked-eye observation or ultraviolet spectroscopy.

[0145] Car-NO-1 was dissolved in a mixed solution of PBS (0.1 mol / L, pH 7.4, containing 90% DMSO) to obtain the probe stock solution (10 -3 mol / L), then diluted with a mixture of PBS (0.1 mol / L, pH 7.4, containing 90% DMSO) to obtain 10 -5 A mol / L Car-NO-1 solution was added with NO solution (30 × 10⁻⁶ mol / L). -5 The UV spectrum of the sample (mol / L) was tested after 25 minutes, and the results are as follows: Figure 5 As shown. From Figure 5 It can be seen that a new absorption peak was generated at 650nm, and the solution color changed from yellow to blue. This can also be used for qualitative or semi-quantitative determination by visual observation.

[0146] Example 5

[0147] This embodiment provides a method for NO detection. Specifically, the compound Car-NO-1 obtained in Example 2 is used as a probe for NO detection, and the detection and analysis are performed by fluorescence spectroscopy.

[0148] Car-NO was dissolved in PBS (0.1 mol / L, pH 7.4, containing 90% DMSO) to obtain the probe stock solution (10... -3 mol / L), then added PBS (0.1 mol / L, pH 7.4, containing 90% DMSO) to obtain 10 -5 Add 30 × 10⁻⁶ mol / L Car-NO solution to NO solution. -5 The spectrum and fluorescence intensity at 650 nm were measured using a mol / L solution excited at 450 nm. The results are as follows: Figure 6 and 7 As shown. From Figure 6 It can be seen that the fluorescence is quenched, and from Figure 7 As can be seen, nearly 80% of the fluorescence was quenched within 55 seconds of the reaction.

[0149] Example 6

[0150] This embodiment provides a method for NO detection. Specifically, the compound Car-NO-1 obtained in Example 2 is used as a probe for NO detection, and the detection and analysis are performed by fluorescence spectroscopy.

[0151] Car-NO was dissolved in PBS (0.1 mol / L, pH 7.4, containing 90% DMSO) to obtain the probe stock solution (10... -3 mol / L), then added PBS (0.1 mol / L, pH 7.4, containing 90% DMSO) to obtain 10 -5 The spectra and fluorescence intensity changes at 650 nm were measured for mol / L Car-NO solution and NO solutions of different concentrations under 450 nm excitation. The results are as follows: Figure 8 As shown. From Figure 8 It can be seen that there is a good linear relationship between the emission peak intensity at 652 nm and the NO concentration. Based on this linear relationship, the detection limit of the probe can be calculated to be as low as 76.8 nM, which shows that the compound of the present invention has extremely high sensitivity.

[0152] When the compound obtained in Example 3 or its salt or solvate or the compound obtained in other structures in Example 1 is used for NO detection, similar results can be obtained. To avoid redundancy, they are not shown one by one.

[0153] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A compound or a salt thereof, characterized by: The structure of the compound is shown in the following formula I: I wherein R is an alkyl group, wherein Ar1is selected from one of the following structures: , and Ar2is selected from one of the following structures: ; The alkyl group is a tert-butyl group or a C6-C24 alkyl group.

2. A process for the preparation of a compound as claimed in claim 1, characterized in that: The preparation method comprises the following steps: S1, reacting compound a with compound b to obtain compound c; S2, reacting compound c with a reducing agent to obtain the compound shown in formula I; The structures of compound a, compound b and compound c are as follows: In the formula, Y is a boronic acid group, a borate group, a zinc halide group, a magnesium halide group or a trialkyltin group; X is halogen; the trialkyltin group is selected from trimethyltin, triethyltin or tributyltin.

3. The process of claim 2, wherein: The boronic acid group is selected from 1,3,2-dioxaborinan-2-yl, 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl or 5,5-dimethyl-1,3,2-dioxaborinan-2-yl.

4. The process of claim 2, wherein: The preparation method comprises one or more of the following features: (1) the reaction conditions of compound a and b are as follows: under the catalysis of a palladium catalyst I, in an organic solvent I, wherein the organic solvent I is free of water and oxygen; (2) in step S2, the reducing agent is at least one of iron powder, zinc powder or tin dichloride; (3) step S2 is carried out in acetic acid.

5. The process of claim 4, wherein: The preparation method comprises one or more of the following features: (1) the reaction time of compound a and b is 12-24 h; and / or, the reaction temperature of compound a and b is 80-130°C; and / or, the organic solvent I is toluene, dioxane or tetrahydrofuran; and / or, the palladium catalyst I comprises at least one of tetrakis(triphenylphosphine)palladium, palladium acetate or 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride; and / or, the pH of the basic condition I is 10-13; (2) the time of the reduction reaction is 6-12 h; and / or, the temperature of the reduction reaction is 80-135°C.

6. The process of claim 4, wherein: The basic condition is achieved by adding a strong alkali carbonate salt.

7. A kit characterized in that: The kit further comprises a solvent.

8. The kit of claim 7, wherein: The kit further comprises a nitric oxide donor.

9. The kit of claim 7, wherein:

10. Use of the compound or salt thereof according to claim 1 in the preparation of a reagent for detecting nitric oxide. The detection method comprises the following steps: contacting a sample with the compound or salt thereof according to claim 1, and the detection method is not for the purpose of disease diagnosis or treatment.

11. A method for detecting nitric oxide in a sample, characterized by: The detection method further comprises one or more of the following features:

12. The detection method of claim 11, wherein: (1) the sample is a biological sample or an environmental sample; (2) the detection method further comprises the step of detecting and analyzing by ultraviolet, fluorescence spectroscopy or colorimetry; (3) the detection method comprises contacting the sample with the compound or salt thereof in a solvent, and the solvent is a mixture of dimethyl sulfoxide and phosphate buffer; (4) the contacting time is 2-25 minutes; (5) the detection concentration range of NO in the detection process of the detection method is 0.0768-100 μmol / L. The reagent is used for diagnosing a disease of a subject or evaluating a therapy for treating a disease of a subject, and the disease affects the content of nitric oxide.

13. The method of claim 12, wherein: The concentration of the compound or salt thereof in the solvent is 10 -7 ~ 10 -2 mol / L.

14. Use of the compound or salt thereof according to claim 1 in the manufacture of a reagent, characterized by: ​ 15. Use according to claim 14, characterized in that: The agent is used for cell imaging.

16. Use of the compound or salt thereof according to claim 1 in the manufacture of a reagent for evaluating the therapeutic effect of a drug. The medicament is used for diagnosing a disease or for evaluating a therapy for treating a disease in a subject, the disease affecting the amount of nitric oxide.

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