Diatomic bridged fluorescent dye and application thereof

Abstract

The invention provides a diatom-bridged fluorescent dye and application thereof, the diatom-bridged fluorescent dye is a compound represented by a formula A, R1 to R9 and Y <-> are defined in the description, the maximum ultraviolet absorption wavelength is about 620 nm, and the maximum fluorescence emission wavelength is about 650 nm. The diatom bridged fluorescent dye disclosed by the invention has excellent viscosity responsiveness, high fluorescence intensity and good stability, and has an extremely high application value in biological imaging. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of organic dyes, and particularly relates to a diatomic-bridged fluorescent dye and its application. Background Art

[0002] Xanthene dyes are a class of compounds based on the structures of xanthene and dibenzopyran, and occupy an important position in the field of organic dyes due to their unique chemical structures and excellent optical properties. Common xanthene dyes include rhodamines (such as rhodamine B, rhodamine 6G), fluoresceins (such as sodium fluorescein), and Rhodol, etc.

[0003] Since the German chemist Maurice Ceresole first synthesized rhodamine dyes in 1887, these compounds have gradually become the cornerstone of the fields of fluorescence labeling and bioimaging by virtue of their unique spirolactam / ring-opening equilibrium mechanism and excellent photophysical properties. Early rhodamine dyes (such as rhodamine B, rhodamine 6G) were widely used for organelle labeling and immunofluorescence detection due to their high fluorescence quantum yields and photostabilities. After the 1990s, researchers extended the emission wavelengths of rhodamine dyes from the visible light region (500–570 nm) to the near-infrared edge (600–650 nm) through strategies such as aminoalkylation (such as tetramethylrhodamine) and aromatic ring substitution (such as Texas Red), and developed pH-sensitive and metal ion-responsive probes, promoting their applications in in vivo imaging and dynamic molecular tracking.

[0004] Researchers have been continuously exploring the design and synthesis of novel rhodamine dyes. Common modification strategies include alkylation modification of amino groups, substitution of its xanthene mother nucleus, modification of the benzene ring at the 9-position, hybridization with other fluorophores, and replacement of oxygen atoms with other heteroatoms such as silicon, carbon, sulfur, selenium, etc. Replacing the oxygen atoms of traditional rhodamine dyes with other elements can significantly expand their spectral ranges. However, the replacement of different heteroatoms will affect the molar extinction coefficient (ε) and quantum yield (Φ) of rhodamine dyes. For example, the molar extinction coefficient value of Si-rhodamine is usually lower than that of traditional rhodamine. Therefore, there is an urgent need to develop a novel rhodamine structural mother nucleus to meet the requirements of its biofluorescence labeling and imaging. Summary of the Invention

[0005] Based on the above technical problems, the present invention proposes a compound of formula A. By replacing the oxygen atom with a diatomic atom, the six-membered ring in the rhodamine dye is expanded to a seven-membered ring, such that the maximum ultraviolet absorption wavelength of the compound of formula A is about 620 nm, and the maximum fluorescence emission wavelength is about 650 nm.

[0006] Specifically, the first aspect of the present invention provides a compound of formula A:

[0007]

[0008] In formula A,

[0009] R1, R2, R3 and R4 are each independently selected from substituted or unsubstituted C1-C6 alkyl groups, and the C1-C6 alkyl groups are optionally substituted with 1, 2, 3 or 4 substituents selected from amino, hydroxyl and halogen;

[0010] R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted C1-C6 amide group, a substituted or unsubstituted C1-C6 ester group, and a substituted or unsubstituted C6-C14 aryl group; the C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 amide group, C1-C6 ester group and C6-C14 aryl group are optionally substituted with 1, 2, 3 or 4 substituents selected from amino, hydroxyl and halogen;

[0011] Y - is selected from one or more of sulfate (1 / 2), chloride ion, fluoride ion, bromide ion, nitrate ion, carbonate (1 / 2), bicarbonate ion, acetate ion, oxalate (1 / 2) and trifluoroacetate ion.

[0012] In one or more embodiments, R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted C1-C6 amide group, a substituted or unsubstituted C1-C6 ester group, and a substituted or unsubstituted C6-C14 aryl group; the C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 amide group, C1-C6 ester group and C6-C14 aryl group are optionally substituted with 1, 2, 3 or 4 substituents selected from amino and hydroxyl.

[0013] In one or more embodiments, R1, R2, R3 and R4 are the same, and R1, R2, R3 and R4 are selected from C1-C6 alkyl groups.

[0014] In one or more embodiments, R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C1-C6 amide group, a C1-C6 ester group, and a C6-C14 aryl group.

[0015] In one or more embodiments, R1, R2, R3 and R4 are the same, and R1, R2, R3 and R4 are selected from C1-C2 alkyl groups; R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 amide group, a C1-C4 ester group, and a phenyl group.

[0016] In one or more embodiments, R1, R2, R3, and R4 are the same, and R1, R2, R3, and R4 are selected from C1-C2 alkyl; R5 is a hydrogen atom; R9 is a carboxyl group; R6, R7, and R8 are each independently selected from: a hydrogen atom, a carboxyl group, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 amide, C1-C4 ester, and phenyl.

[0017] In one or more embodiments, R1, R2, R3, and R4 are the same, and R1, R2, R3, and R4 are selected from C1-C2 alkyl.

[0018] In one or more embodiments, R1, R2, R3, and R4 are methyl.

[0019] In one or more embodiments, R5 and R9 are the same, and R5 and R9 are selected from C1-C4 alkyl and a hydrogen atom.

[0020] In one or more embodiments, R6, R7, and R8 are each independently selected from: a hydrogen atom, a carboxyl group, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 amide, C1-C4 ester, and phenyl.

[0021] In one or more embodiments, the compound of formula A is

[0022]

[0023] Another aspect of the present invention provides a fluorescent probe composition, which contains the compound of formula A described in any embodiment of the present invention and a solvent.

[0024] In one or more embodiments, the solvent is a buffer solution.

[0025] In one or more embodiments, the buffer solution is a HEPES buffer solution or a PBS buffer solution.

[0026] Another aspect of the present invention also provides the use of the compound of formula A described in any embodiment of the present invention in the preparation of a reagent for biofluorescence imaging.

[0027] In one or more embodiments, the biofluorescence imaging is fluorescence imaging of a physiological or pathological process related to viscosity change.

[0028] In one or more embodiments, the biofluorescence imaging is directional imaging of mitochondria.

[0029] The compound of formula A of the present invention has a maximum ultraviolet absorption wavelength of about 620 nm, a maximum fluorescence emission wavelength of about 650 nm, and a molar extinction coefficient in dichloromethane up to 188,000 cm -1 ·M-1 , superior to most rhodamine dyes and possessing excellent stability. Due to the introduction of the seven-membered ring, the compound of formula A has a certain degree of structural freedom after entering the excited state. At the same time, since the compound of formula A can be obtained by introducing a benzene ring linking group at the 9-position of the structure of formula I , the structural freedom of the compound of formula A in the excited state is further increased, resulting in a significant decrease in its fluorescence brightness. The high-viscosity environment will limit the free torsion of the excited-state molecules, which makes the compound of formula A of the present invention also have excellent viscosity responsiveness and can be used for fluorescence imaging in biomedicine, especially for mitochondrial imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 are the ultraviolet absorption spectra and fluorescence emission spectra of compound EdC3 in different solvents.

[0031] Figure 2 are the ultraviolet absorption spectra of compound EdC3 and reactive oxygen species in PBS buffer.

[0032] Figure 3 is the viscosity responsiveness test of compound EdC3n.

[0033] Figure 4 is the comparison of the ultraviolet absorption spectra and fluorescence emission spectra of compound EdC3n in different common solvents with the ultraviolet absorption spectra and fluorescence emission spectra of compound EdC3n in PBS buffer solution (10 mM, pH = 7.4) containing 90% by volume of glycerol.

[0034] Figure 5 is the cytotoxicity test of compound EdC3n. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] To enable those skilled in the art to understand the features and effects of the present invention, the following provides a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein shall have the ordinary meaning understood by those skilled in the art for the present invention. In case of conflict, the definition in this specification shall prevail.

[0036] The theories or mechanisms described and disclosed herein, whether correct or incorrect, shall not limit the scope of the present invention in any way, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0037] In this text, terms such as "comprising", "including", "containing" and similar terms encompass the meanings of "consisting essentially of" and "consisting of". For example, when this text discloses that "A comprises B and C", "A consists essentially of B and C" and "A consists of B and C" should be considered to have been disclosed in this text.

[0038] In this text, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of a numerical range or percentage range should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions). For example, "containing 1 to 6 carbon atoms" will include containing 1 to 6 carbon atoms, containing 2 to 6 carbon atoms, containing 4 carbon atoms, etc.

[0039] In this text, unless otherwise specified, percentages refer to mass percentages and ratios refer to mass ratios.

[0040] In this text, when describing embodiments or examples, it should be understood that they are not used to limit the present invention to these embodiments or examples. On the contrary, all alternatives, modifications and equivalents of the methods and materials described in the present invention can be covered within the scope defined by the claims.

[0041] In this text, for the sake of concise description, all possible combinations of all technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered to be within the scope described in this specification.

[0042] In this text, the total number of carbon atoms present in certain chemical groups defined herein is represented by a simplified symbol in front of the group. For example, C1-C6 alkyl refers to an alkyl group having a total of 1 to 6 carbon atoms as defined below. The total number of carbon atoms in the simplified symbol does not include the carbon present in the substituents that may be present in the group.

[0043] As used herein, "halogen" refers to F, Cl, Br, and I.

[0044] As used herein, "amino" refers to -NH2.

[0045] As used herein, "amide group" refers to -CONH-.

[0046] As used herein, "ester group" refers to -COO-.

[0047] As used herein, "carboxyl group" refers to -COOH.

[0048] As used herein, "hydroxyl group" refers to -OH.

[0049] As used herein, "sulfate group" refers to SO4 2- .

[0050] As used herein, "nitrate group" refers to NO3 - .

[0051] As used herein, "carbonate group" refers to CO3 2- .

[0052] As used herein, "bicarbonate group" refers to HCO3 - .

[0053] As used herein, "acetate group" refers to CH3COO - .

[0054] As used herein, "oxalate group" refers to C2O4 2- .

[0055] As used herein, "trifluoroacetate group" refers to CF3COO - .

[0056] As used herein, "alkyl group" refers to a straight-chain or branched-chain monovalent saturated hydrocarbon group having a specified number of carbon atoms. Specific alkyl groups are those having 1 to 6 carbon atoms ("C1-C6 alkyl groups"), preferably those containing 1-4 carbon atoms (C1-C4 alkyl groups). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0057] As used herein, "aryl group" refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl), where the fused rings may or may not be aromatic. In one variation, the aryl group contains 6 to 14 ring carbon atoms, preferably a C6-C10 aryl group. An aryl group having more than one ring and at least one non-aromatic ring may be attached to the parent structure at an aromatic ring position or at a non-aromatic ring position. In one variation, an aryl group having more than one ring and at least one non-aromatic ring is attached to the parent structure at an aromatic ring position. Examples of aryl groups include phenyl, naphthyl, phenanthryl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylene, and fluorenyl.

[0058] As used herein, "alkoxy group" refers to alkyl-O-, and preferred alkoxy groups are C1-C6 alkoxy groups, such as C1-C5 alkoxy groups, C1-C4 alkoxy groups, C1-C3 alkoxy groups, C1-C2 alkoxy groups, which include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, and the like.

[0059] As used herein, the term "substituted", whether or not preceded by the term "optionally" (i.e., equivalent to substituted or unsubstituted), means that one or more hydrogens of the designated group or moiety are replaced by a "suitable substituent". In this text, the number of substituents can be one or more, i.e., 1, 2, 3, 4, 5 or 6 or more, depending on the group being substituted and the nature of the substituent. For example, when the substituent of an ethyl group is a halogen, depending on the structure of the group being substituted, this group can be substituted by 1, 2, 3, 4 or 5 substituents, such as trifluoroethyl, pentafluoroethyl, etc. In some embodiments, the number of said substituents is 1, 2 or 3. In some embodiments, the number of said substituents is 1 or 2. In some embodiments, the number of said substituents is 1. It will be understood that "substituted" or "substituted by..." includes the implicit condition that such substitution occurs according to the allowable valences of the substituting atoms and results in a stable or chemically viable compound, e.g., a compound that does not undergo spontaneous transformation, such as by rearrangement, cyclization, elimination, etc. Unless otherwise specified, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from the designated group, the substituents can be the same or different at each position. Those skilled in the art will understand that if appropriate, the substituents themselves can be substituted. Unless specifically indicated as "unsubstituted", chemical moieties mentioned herein should be understood to include substituted variants. For example, mention of an "aryl" group or moiety implicitly includes unsubstituted aryl groups and substituted variants.

[0060] Unless otherwise specified, the structural formulas described in this invention are intended to include all isomeric forms (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, the R and S configurations containing an asymmetric center, the (Z) and (E) isomers of a double bond, etc. Therefore, individual stereochemical isomers of the compounds of this invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformational isomers) are all within the scope of this invention.

[0061] General synthetic route for compounds of formula A:

[0062]

[0063] Step 1: N-alkylation of aromatic amines. The method used in this invention is, under normal temperature or heating (40 - 60 °C) conditions, using N,N-dimethylformamide or acetonitrile as the solvent, adding compound of formula A1, R1X and R2X (X is a halogen, such as iodine), adding an inorganic base, reacting for 12 - 24 h, and the inorganic base includes but is not limited to potassium carbonate and cesium carbonate.

[0064] Step 2: Under anaerobic conditions, connect the Br in Formula A2 to trimethylsilylacetylene through Sonogashira coupling reaction. The reaction solvents include but are not limited to N,N-dimethylformamide, tetrahydrofuran, acetonitrile, and organic bases. The added organic bases include but are not limited to triethylamine and diethylamine, and the added inorganic bases include but are not limited to cesium carbonate and sodium bicarbonate. The added palladium catalysts include but are not limited to tetrakis(triphenylphosphine)palladium, dichlorotetrakis(triphenylphosphine)palladium, and palladium chloride. The reaction is carried out under heating conditions (60 - 80 °C).

[0065] Step 3: Removal of silane. Under basic conditions, use a fluorine reagent to remove the trimethylsilyl group in Formula A3. The reaction solvents include but are not limited to methanol and tetrahydrofuran, the added inorganic bases include but are not limited to potassium carbonate and cesium carbonate, and the added fluorine reagents include but are not limited to tetrabutylammonium fluoride (TBAF) and ammonium fluoride.

[0066] Step 4: Connect Formula A4 to Formula A2 through Sonogashira coupling reaction.

[0067] Step 5: Hydrogenation reaction. The method used in the present invention is to react with hydrogen under the action of a metal catalyst to reduce the carbon-carbon triple bond in Formula A5 to a carbon-carbon single bond. The added metal catalysts include but are not limited to palladium, nickel, and platinum, and the added reaction solvents include but are not limited to methanol, ethanol, and tetrahydrofuran. The reaction is carried out at room temperature.

[0068] Step 6: Achieve formylation on the aromatic ring at the para position of one N in Formula A6. Introduce an aldehyde group on the aromatic ring through Vilsmeier-Haack reaction. The reaction solvent is a halogenated hydrocarbon reagent with a relatively high boiling point, such as 1,2-dichloroethane. The commonly used catalyst is phosphorus oxychloride, the commonly used acylating agent is N-substituted formamide, and the commonly used reaction solvent is N,N-dimethylformamide. The reaction is carried out under heating (60 - 80 °C).

[0069] Step 7: Carry out a nucleophilic addition reaction on the carbonyl group of Formula 7 under inert gas conditions for Formula B1. M in Formula B1 represents a lithium atom. The reaction solvent is an ether solvent, including but not limited to anhydrous tetrahydrofuran, anhydrous diethyl ether, and anhydrous dioxane, etc. The reaction temperature is 0 °C or room temperature.

[0070] Step 8: Carry out an electrophilic substitution reaction to close the ring by attacking benzyl alcohol at the para position of N in Formula A8. The reaction solvent is a halogenated hydrocarbon, including but not limited to dichloromethane and chloroform. The acid is commonly a protonic acid, such as methanesulfonic acid, trifluoroacetic acid, etc. The reaction is carried out at room temperature.

[0071] Step 9: Dehydrogenation reaction. The hydrogen atom in Formula A9 is oxidized. The oxidants include but are not limited to iodine, dichlorodicyanobenzoquinone, and tetrachlorobenzoquinone, etc. The solvent can be a halogenated hydrocarbon, such as dichloromethane.

[0072] The present invention also provides a fluorescent dye or a dye composition, which contains the compound of formula A of the present invention and an optional solvent. Solvents applicable to the present invention include, but are not limited to, buffers, preferably HEPES buffer or PBS buffer. The fluorescent dye or dye composition provided by the present invention has excellent viscosity responsiveness and can be used for targeting mitochondria.

[0073] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. Unless otherwise specified, percentages and parts are calculated by weight.

[0074] Example 1: Synthesis of Compound EdC3

[0075]

[0076] (1) Synthesis of Compound 2: Add Compound 1 (12.66 mL, 116.3 mmol) and potassium carbonate (24 g) into a 500 mL flask, dissolve them in 200 mL of acetonitrile, then add methyl iodide (15.9 mL, 255.8 mmol) under stirring, and react at 40 °C under nitrogen protection for 24 h. After completion, add ammonia water to quench the reaction. After spinning off the acetonitrile, add dichloromethane for extraction and spin dry, and purify by silica gel column chromatography (PE:EA = 100:2, v / v) to obtain 20 g of a yellow oil, namely Compound 2, with a yield of 87%. 1 H NMR (400 MHz, Chloroform-d) δ 7.05 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 8.4 Hz, 2H), 6.58 (dd, J = 8.2, 4.0 Hz, 1H), 2.92 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 133.37, 126.51, 121.41, 116.29, 114.15, 42.31

[0077] (2) Synthesis of Compound 3: Dissolve Compound 2 (1 g, 4.99 mmol) in 60 mL of triethylamine. After deoxygenation, under nitrogen protection, add palladium acetate (110 mg, 0.5 mmol), triphenylphosphine (0.4 g, 1.5 mmol), copper(I) iodide (0.3 g, 1.5 mmol), and trimethylsilyl azide (TMSA) (1.1 g, 11 mmol) to the reaction system. React at 80 °C for 24 h. Then remove triethylamine by rotary evaporator and purify by silica gel column chromatography (PE:EA = 100:3, v / v) to obtain 0.9 g of yellow oil, which is Compound 3, with a yield of 82%. 1 H NMR (400 MHz, Chloroform-d) δ 7.24 (t, J = 8.0 Hz, 1H), 6.66 (d, J = 7.8 Hz, 2H), 6.68 (d, J = 8.4 Hz, 1H), 2.84 (s, 6H), 0.26 (s, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 152.49, 128.23, 126.85, 122.86, 118.45, 114.79, 106.58, 92.87, 40.45, 0.24.

[0078] (3) Synthesis of Compound 4: Dissolve Compound 3 (1.35 g, 6.21 mmol) in 15 mL of methanol and 15 mL of tetrahydrofuran, add potassium carbonate (5 g), react at room temperature for 2 h, add saturated ammonium chloride solution, extract with dichloromethane, and after drying the solvent by rotary evaporation, purify by silica gel column chromatography (PE:EA = 100:3, v / v) to obtain 0.87 g of light yellow oil, which is Compound 4, with a yield of 96%. 1 H NMR (400 MHz, Chloroform-d) δ 7.21 (t, J = 8.2 Hz, 1H), 6.76 (d, J = 7.5 Hz, 2H), 6.78 (d, J = 9.4 Hz, 1H), 3.01 (s, 1H), 2.78 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 150.56, 129.12, 122.76, 120.34, 116.32, 113.65, 87.74, 77.13, 39.87.

[0079] (4) Synthesis of Compound 5: Dissolve Compound 4 (0.75 g, 5.15 mmol) and Compound 2 (1.1 g, 0.55 mmol) in 40 mL of triethylamine. After deoxygenation, add tetrakis(triphenylphosphine)palladium (0.6 g, 0.5 mmol) and copper(I) iodide (0.3 g, 1.55 mmol) under nitrogen protection, and then react at 80 °C for 8 h. Then remove triethylamine by rotary evaporator, and purify by silica gel column chromatography (PE:EA = 100:4, v / v) to obtain 1 g of light yellow solid, which is Compound 5, with a yield of 76%. 1 H NMR (400 MHz, Chloroform-d) δ 7.32 (t, J = 8.2 Hz, 2H), 6.78 (d, J = 6.8 Hz, 4H), 6.67 (d, J = 8.4 Hz, 2H), 2.89 (s, 12H). 13 C NMR (101 MHz, Chloroform-d) δ 146.39, 128.13, 123.05, 120.68, 115.37, 112.27, 89.47, 39.82.

[0080] (5) Synthesis of Compound 6: Dissolve Compound 5 (1 g, 3.75 mmol) in 20 mL of methanol, add 0.1 g of palladium on carbon, and react under a hydrogen atmosphere for 12 h, then filter off the palladium on carbon. Purify by silica gel column chromatography (PE:EA = 100:3, v / v) to obtain 0.95 g of white solid, which is Compound 6, with a yield of 95%. 1 H NMR (600 MHz, Methanol-d4) δ 7.09 (t, J = 7.8 Hz, 2H), 6.61 (dd, J = 8.2, 2.4 Hz, 2H), 6.57 (d, J = 7.5 Hz, 2H), 6.55 (s, 2H), 2.85 (s, 12H), 2.83 (s, 4H). 13 C NMR (151 MHz, Methanol-d4) δ 152.36, 143.89, 129.82, 118.85, 115.04, 112.21, 41.31, 39.65. HRMS (ESI) m / z C 18 H 24 N2[M + H] + , calculated value: 269.2018; measured value: 269.2017.

[0081] (6) Synthesis of Compound 7: Add N,N-dimethylformamide (DMF) (0.28 mL, 3.7 mmol) to a 50 mL round-bottom flask and stir it in an ice bath. While stirring, slowly add phosphorus oxychloride (0.35 mL, 3.7 mmol) drop by drop. After the addition, continue the reaction for 20 min. Then add 9.5 mL of 1,2-dichloroethane to dissolve the reactants (DMF and phosphorus oxychloride will form a solid). Take out 2 mL and add it to a (20 mL) 1,2-dichloroethane solution (1,2-DCE) containing Compound 6 (0.1 g, 0.37 mmol). Gradually heat the mixture to 70 °C and continue stirring for 8 h. Quench the reaction with saturated aqueous sodium carbonate solution. Extract with dichloromethane, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a crude product. Purify it by silica gel column chromatography (PE:EA = 100:5, v / v) to obtain 50 mg of a light yellow solid, which is Compound 7, with a yield of 41%. 1 H NMR (400 MHz, Chloroform-d) δ 10.02 (s, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.32 (t, J = 7.4 Hz, 1H), 6.72–6.54 (m, 4H), 6.43 (d, J = 2.5 Hz, 1H), 3.32 - 3.23 (m, 2H), 3.02 (s, 6H), 2.88 (s, 6H), 2.83–2.85 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 195.14, 152.85, 149.82, 147.85, 141.97, 134.96, 123.43, 121.63, 118.34, 114.31, 113.24, 110.88, 109.45, 40.74, 40.14, 39.15, 35.73. HRMS (ESI) m / z C 19 H 25 N2O [M + H] + , calculated value: 297.1967; measured value: 297.1968.

[0082] (7) Synthesis of Compound 8: In a dried Schlenk flask, dissolve 2,6-dimethylbromobenzene (200 mg, 1.67 mmol) in 20 mL of anhydrous tetrahydrofuran and cool to -78 °C. Add n-butyllithium (0.42 mL, 1.50 mmol) and react for 30 min. Dissolve Compound 7 (25 mg, 0.08 mmol) in 10 mL of anhydrous tetrahydrofuran in a Schlenk flask and cool to 0 °C. Add the prepared phenyllithium reagent to the above reaction system. After reacting for 1 h, quench the reaction with saturated ammonium chloride solution, extract with dichloromethane, rotary evaporate the solvent, and then purify by silica gel column chromatography (PE:EA = 100:15, v / v) to obtain 31 mg of a light yellow solid, namely Compound 6, with a yield of 93%. 1 H NMR (400 MHz, Methanol-d4) δ 7.13–7.02 (m, 3H), 6.89 (d, J = 7.3 Hz, 2H), 6.62–6.57 (m, 2H), 6.53 (t, J = 7.7 Hz, 2H), 6.47 (s, 1H), 6.32 (s, 1H), 3.02–2.94 (m, 1H), 2.85 (dd, J = 6.7, 1.8 Hz, 12H), 2.78–2.65 (m, 2H), 2.61–2.49 (m, 1H), 2.25 (s, 6H). 13 C NMR (101 MHz, Methanol-d4) δ 152.26, 151.74, 150.21, 144.29, 142.04, 140.87, 138.15, 130.37, 129.84, 129.38, 127.12, 118.96, 116.29, 114.96, 112.14, 111.28, 71.07, 41.59, 41.22, 39.05, 36.07, 21.65.

[0083] (9) Synthesis of compound EdC3: Dissolve compound 8 (15 mg, 0.037 mmol) in 10 mL of dichloromethane, add 0.1 mL of trifluoroacetic acid, react for 30 min, then add saturated aqueous sodium bicarbonate, extract and evaporate to dryness. Redissolve the crude product in 10 mL of dichloromethane, add iodine (14 mg, 0.06 mmol), stir at room temperature for 30 min, add saturated sodium carbonate solution, extract, and after evaporating the solvent, purify by silica gel column chromatography (DCM:MeOH = 100:3, v / v) to obtain 11 mg of purple solid, namely compound EdC3, with a yield of 78%. 1H NMR (400 MHz, Chloroform-d) δ 7.29 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 7.6 Hz, 2H), 6.93 (d, J = 9.6 Hz, 2H), 6.85 (d, J = 2.5 Hz, 2H), 6.46 (dd, J = 9.6, 2.7 Hz, 2H), 3.34 (s, 12H), 3.32–2.96 (m, 4H), 1.88 (s, 6H). 13C NMR (101 MHz, Chloroform-d) δ 171.16, 156.69, 152.60, 141.66, 141.28, 135.71, 128.88, 127.87, 125.79, 113.35, 112.22, 41.45, 35.58, 19.75. HRMS (ESI) m / z C 27 H 31 N2 + [M] + , calculated value: 383.2482; measured value: 383.2488.

[0084] Example 2: Synthesis of compound EdC3n

[0085]

[0086] (1) Synthesis of compound 8n: In a dried schlenk flask, dissolve bromobenzene (200 mg, 1.27 mmol) in 20 mL of anhydrous tetrahydrofuran, and cool to -78 °C. Add n-butyllithium (0.46 mL, 1.15 mmol), and react for 30 min. Dissolve compound 7 (25 mg, 0.08 mmol) in a schlenk flask with 10 mL of anhydrous tetrahydrofuran, cool to 0 °C, add the prepared phenyllithium reagent, after reacting for 1 h, quench the reaction with saturated ammonium chloride solution, and separate by extraction with dichloromethane solvent. After purification by silica gel column chromatography (PE:EA = 100:15, v / v), 28 mg of light yellow solid is obtained, namely compound 8n, with a yield of 94%. HRMS (ESI) m / zC 25 H 29 N2+ [M-OH] + , Calculated value: 357.2325; Measured value: 357.2332.

[0087] (2) Synthesis of compound EdC3n: Dissolve compound 8n (15 mg, 0.037 mmol) in 10 mL of dichloromethane, add 0.1 mL of trifluoroacetic acid, react for 30 min, add saturated aqueous sodium bicarbonate solution, extract and evaporate to dryness. Redissolve the crude product in 10 mL of dichloromethane, add DDQ (8 mg, 0.04 mmol), stir at room temperature for 30 min, add saturated sodium carbonate solution for extraction, and purify by silica gel column chromatography (DCM:MeOH = 100:3, v / v) to obtain 10 mg of purple solid, which is compound EdC3n, with a yield of 82%. HRMS(ESI) m / z 355.2169 [M] + , Calculated value: 355.2169; Measured value: 355.2177.

[0088] Test Example 1

[0089] Dissolve compound EdC3 in dichloromethane (CH2Cl2), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), methanol (MeOH) and water (H2O) respectively to prepare a solution with a concentration of 5 μM. At room temperature, use a Shimadzu UV-2600i instrument to detect its corresponding ultraviolet-visible absorption and fluorescence emission spectra. The spectral data results are as Figure 1 and shown in Table 1; and calculate its molar extinction coefficient, fluorescence quantum yield, fluorescence brightness and fluorescence lifetime respectively. The calculation results are shown in Table 1.

[0090] Among them, the fluorescence quantum yield is the relative fluorescence quantum yield. Φ reference sample is the absolute fluorescence quantum yield of compound Cy5 in dichloromethane, which is 38.3%,, and the specific calculation formula is as follows:

[0091]

[0092] Among them, Φ is the fluorescence quantum yield, I is the fluorescence intensity, A is the absorbance at the excitation wavelength, and n is the refractive index of the solvent.

[0093] From Figure 1 it can be seen that the ultraviolet absorption wavelength of compound EdC3 is about 620 nm; in DMSO, the maximum emission wavelength of the fluorescence emission of compound EdC3 is about 650 nm, and with the change of the dye dissolution solvent, the maximum emission wavelength of the corresponding fluorescence emission will undergo a blue shift within 20 nm.

[0094] Table 1: Photophysical properties of compound EdC3

[0095]

[0096] As can be seen from Table 1, the compound EdC3 of the present invention has a relatively high molar extinction coefficient and can be used as a dye for bioimaging.

[0097] Test Example 2

[0098] The compound EdC3 was dissolved in a PBS buffer solution (10 mM, pH = 7.4) to prepare a solution with a concentration of 5 μM. At room temperature, using a Shimadzu UV-2600i instrument and setting the excitation light wavelength to 610 nm, the ultraviolet absorption spectra and their maximum ultraviolet absorption values of the compound EdC3 mixed with different concentrations of reactive oxygen species (ROS) (H2O2, ClO - or ONOO - ) were detected respectively. The spectral data results are as Figure 2 shown.

[0099] As can be seen from Figure 2 , ROS has little effect on the ultraviolet absorption signal of the compound EdC3. 2000 μM of H2O2 can only cause a 2.2% attenuation of the maximum ultraviolet absorption of the compound EdC3, and 200 μM of ClO - can only cause a 5.3% attenuation of the maximum ultraviolet absorption of the compound EdC3, and 20 μM of ONOO - can only cause a 12.5% attenuation of the maximum ultraviolet absorption of the compound EdC3, indicating that the compound EdC3 is not easily oxidized by ROS and has relatively high stability, further verifying the possibility of its use in stable bioimaging.

[0100] Test Example 3

[0101] The compound EdC3n was dissolved in a PBS buffer solution (10 mM, pH = 7.4) containing different proportions of glycerol (volume fraction 10% - 90%) to prepare a solution with a concentration of 5 μM. At room temperature, using a Shimadzu UV-2600i instrument and setting the excitation light wavelength to 610 nm, the corresponding ultraviolet-visible absorption and fluorescence emission spectra were detected respectively. The spectral data results are as Figure 3 shown.

[0102] The compound EdC3 was separately dissolved in N,N-dimethylformamide (DMF), toluene, tetrahydrofuran (THF), PBS buffer solution (10 mM, pH = 7.4), dichloromethane (DCM), methanol (MeOH), water (H2O), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), and PBS buffer solution (10 mM, pH = 7.4) containing 90% by volume of glycerol to prepare solutions with a concentration of 5 μM. At room temperature, a Shimadzu UV-2600i instrument was used to detect their corresponding ultraviolet-visible absorption and fluorescence emission spectra, and the spectral data results are as Figure 4 shown

[0103] As can be seen from Figure 3 , as the volume fraction of glycerol in the buffer solution increases (i.e., the higher the system viscosity), the absorption intensity of the maximum ultraviolet absorption peak of the compound EdC3n also increases. At the same time, the intensity of its fluorescence emission has a very significant increase. When the volume fraction of glycerol in the PBS buffer solution is 90%, compared with when the volume fraction of glycerol in the PBS buffer solution is 60%, the corresponding fluorescence emission intensity is enhanced by more than five times; as can be further found from Figure 4 , when the volume fraction of glycerol in the PBS buffer solution is 90%, compared with other common solvents in the art, the fluorescence emission intensity of the compound EdC3n is enhanced by more than ten times, which makes the compound EdC3n potentially useful as a sensitive and specific viscosity fluorescence probe for biological imaging and other fields.

[0104] Test Example 4

[0105] The compound EdC3n was dissolved in DMSO to prepare a 1 mM solution. Different volumes of the above solution were added to the pre-prepared adherent HeLa cells (cell concentration 1.0x10 6 / mL) to make the final concentrations 0 μM, 1 μM, 2 μM, 4 μM, 10 μM, 15 μM, 20 μM, and 30 μM, respectively. The CCK-8 method was used to determine the cell viability, and the test results are as Figure 5 shown. As can be seen from Figure 5 , the compound EdC3n shows low toxicity to HeLa cells.

Claims

1. Compound of formula A: In formula A, R1, R2, R3 and R4 are each independently selected from substituted or unsubstituted C1-C6 alkyl groups, and said C1-C6 alkyl groups are optionally substituted by 1, 2, 3 or 4 substituents selected from amino, hydroxyl and halogen; R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted C1-C6 amide group, a substituted or unsubstituted C1-C6 ester group and a substituted or unsubstituted C6-C14 aryl group; the C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 amide group, C1-C6 ester group and C6-C14 aryl group are optionally substituted by 1, 2, 3 or 4 substituents selected from amino, hydroxyl and halogen; Y - Selected from one or more of sulfate radical 1 / 2, chloride ion, fluoride ion, bromide ion, nitrate ion, carbonate radical 1 / 2, bicarbonate radical, acetate ion, oxalate radical 1 / 2 and trifluoroacetate ion.

2. The compound of formula A according to claim 1, wherein R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted C1-C6 amide group, a substituted or unsubstituted C1-C6 ester group and a substituted or unsubstituted C6-C14 aryl group; the C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 amide group, C1-C6 ester group and C6-C14 aryl group are optionally substituted by 1, 2, 3 or 4 substituents selected from amino and hydroxyl.

3. The compound of formula A according to claim 1, characterized in that, R1, R2, R3 and R4 are the same, and R1, R2, R3 and R4 are selected from C1-C6 alkyl groups.

4. The compound of formula A according to claim 1, characterized in that, R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C1-C6 amide group, a C1-C6 ester group and a C6-C14 aryl group.

5. The compound of formula A according to claim 1, wherein R1, R2, R3 and R4 are the same, and R1, R2, R3 and R4 are selected from C1-C2 alkyl groups; R5, R6, R7, R8 and R9 are each independently selected from: a hydrogen atom, a carboxyl group, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 amide group, a C1-C4 ester group and a phenyl group.

6. The compound of formula A according to claim 1, wherein R1, R2, R3 and R4 are the same, and R1, R2, R3 and R4 are selected from C1-C2 alkyl groups; R5 is a hydrogen atom; R9 is a carboxyl group; R6, R7 and R8 are each independently selected from: a hydrogen atom, a carboxyl group, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 amide group, a C1-C4 ester group and a phenyl group.

7. The compound of formula A according to claim 1, wherein R1, R2, R3 and R4 are the same, and R1, R2, R3 and R4 are selected from C1-C2 alkyl groups, preferably, R1, R2, R3 and R4 are methyl groups; R5 and R9 are the same, and R5 and R9 are selected from C1-C4 alkyl groups and a hydrogen atom; R6, R7 and R8 are each independently selected from: a hydrogen atom, a carboxyl group, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C1-C4 amide group, a C1-C4 ester group and a phenyl group.

8. The compound of formula A according to claim 1, characterized in that, The compound of formula A is 9. A fluorescent probe composition, characterized in that, The fluorescent probe composition contains the compound of formula A according to any one of claims 1-8 and a solvent; preferably, the solvent is a buffer solution, more preferably HEPES buffer solution or PBS buffer solution.

10. Use of the compound of formula A according to any one of claims 1-8 in the preparation of a reagent for biological fluorescence imaging; preferably, the biological fluorescence imaging is fluorescence imaging of a physiological or pathological process related to viscosity change, and / or the biological fluorescence imaging is directional imaging of mitochondria.

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