High-sensitivity oxidative stress near-infrared fluorescent probe and application thereof

Abstract

The invention provides a high-sensitivity oxidative stress near-infrared fluorescent probe for in-vivo imaging and application thereof, the near-infrared fluorescent probe is a compound shown as a formula I, R1 and R2 are defined in the description, and the absorption and emission wavelength of the fluorescent compound obtained after the near-infrared fluorescent probe is activated by active oxygen is near 890 nm and even larger than 890 nm. The near-infrared fluorescent probe disclosed by the invention shows rapid and relatively high reactivity to active oxygen, and the generated fluorescent compound is high in fluorescence intensity and good in stability and can be used for monitoring the active oxygen level in a living body. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of organic dyes, and particularly relates to a highly sensitive near-infrared fluorescence probe for oxidative stress for in vivo imaging and its uses. Background Art

[0002] Compared with visible light, near-infrared light (700 - 1700 nm) has advantages such as stronger tissue penetration ability and less autofluorescence interference, making it an ideal choice for in vivo imaging. With the rapid development of molecular biology and optical technology, the penetration depth of near-infrared dyes in biological tissues can reach several centimeters, which is difficult to achieve by traditional fluorescence probes. Among them, for near-infrared fluorescence probes, based on near-infrared dyes, scientists can, through ingenious molecular engineering, make a single probe simultaneously possess targeting, imaging, and therapeutic functions, greatly improving the detection efficiency and providing new possibilities for precision medicine.

[0003] Reactive oxygen species (ROS) play a dual role in living organisms, and its dynamic balance affects the normal physiological functions of organisms. On the one hand, as an important molecule in cell signal transduction, ROS participates in and regulates processes such as cell proliferation, differentiation, and apoptosis; on the other hand, excessive ROS can lead to oxidative stress, triggering adverse processes such as DNA damage, protein denaturation, and lipid peroxidation, which are closely related to the occurrence and development of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases. Therefore, real-time and accurate detection of the dynamic changes of intracellular ROS is of great significance for understanding its biological functions and related disease mechanisms. So developing corresponding near-infrared fluorescence probes that specifically respond to ROS and produce changes in fluorescence signals (such as fluorescence intensity, wavelength, or lifetime) to capture signal changes deep in biological tissues, such probe molecular tools are indispensable for disease diagnosis, drug development, and life process research.

[0004] Existing ROS fluorescence probes have developed vigorously in the visible light region; but in the longer-wavelength near-infrared region, they still face problems such as insufficient photo / chemical stability (cyanine dyes are vulnerable to nucleophilic attack or photo-degradation), low brightness (the molar extinction coefficient of D-A-D dyes is low), and poor biocompatibility. Therefore, it is necessary to develop near-infrared ROS probes with excellent photophysical properties. Summary of the Invention

[0005] Dye EC5 has excellent photophysical properties, showing high photo-stability and chemical stability. Based on this fluorescent skeleton, the present invention designs a highly sensitive near-infrared fluorescence probe for oxidative stress for in vivo imaging.

[0006] Specifically, in the first aspect of the present invention, a compound of formula I that can be used as a near-infrared fluorescence probe for oxidative stress is provided:

[0007]

[0008] In formula I,

[0009] R 1 and R 2 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C1-C5 alkoxy group, a substituted or unsubstituted C1-C5 alkylthio group, and a halogen; the C1-C5 alkyl group, C1-C5 alkoxy group, and C1-C5 alkylthio group are optionally substituted with 1, 2, 3, or 4 halogen substituents;

[0010] R 1 and R 2 are not simultaneously a hydrogen atom.

[0011] In one or more embodiments, in formula I, R 1 and R 2 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a substituted or unsubstituted C1-C3 alkoxy group, and a halogen; the C1-C3 alkyl group and C1-C3 alkoxy group are optionally substituted with 1, 2, 3, or 4 halogen substituents.

[0012] In one or more embodiments, the compound of formula I is:

[0013]

[0014] Another aspect of the present invention provides a compound of formula II that can be used as an intermediate for preparing a compound of formula I or as a fluorescent dye:

[0015]

[0016] In formula II,

[0017] R 3 and R 4 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C1-C5 alkoxy group, a substituted or unsubstituted C1-C5 alkylthio group, and a halogen; the C1-C5 alkyl group, C1-C5 alkoxy group, and C1-C5 alkylthio group are optionally substituted with 1, 2, 3, or 4 halogen substituents;

[0018] R 3 and R 4 are not simultaneously a hydrogen atom;

[0019] 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.

[0020] In one or more embodiments, in Formula II, R 3 and R 4 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a substituted or unsubstituted C1-C3 alkoxy group, and a halogen; the C1-C3 alkyl group and the C1-C3 alkoxy group are optionally substituted with 1, 2, 3, or 4 halogen substituents.

[0021] In one or more embodiments, the compound of Formula II is or

[0022]

[0023] The present invention also provides a method for preparing the compound of Formula I according to any one of the embodiments herein, wherein the compound of Formula I is obtained by a reduction reaction from the compound of Formula II according to any one of the embodiments herein.

[0024] The present invention also provides a near-infrared fluorescence probe composition, which contains the compound of Formula I according to any one of the embodiments herein and a solvent; preferably, the solvent is a buffer solution, more preferably a HEPES buffer solution or a PBS buffer solution.

[0025] The present invention also provides the use of the compound of Formula I according to any one of the embodiments herein in the preparation of a reagent for near-infrared fluorescence imaging.

[0026] In one or more embodiments, the near-infrared fluorescence imaging is near-infrared fluorescence imaging of reactive oxygen species.

[0027] In one or more embodiments, the near-infrared fluorescence imaging is in vivo imaging.

[0028] The near-infrared fluorescence probe of the present invention can be used for fluorescence imaging in biomedicine, especially EC5-H2 and EC5-H3, which can show high reactivity to reactive oxygen species and also have a high fluorescence multiple, and can be used to monitor the level of reactive oxygen species in the living body of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 are the ultraviolet absorption spectra and fluorescence emission spectra of the dyes EC5-Me and EC5-2OMe in different solvents. Among them, Figure 1 (A) is the normalized ultraviolet absorption graph of the dye EC5-Me; Figure 1 (B) is the normalized fluorescence emission spectrum of the dye EC5-Me; Figure 1 (C) is the normalized ultraviolet absorption graph of the dye EC5-2OMe; Figure 1 (D) is the normalized fluorescence emission spectrum of the dye EC5-2OMe.

[0030] Figure 2 It is the titration fluorescence spectrum of dye EC5-H3 with reactive oxygen species in HEPES buffer. Among them, Figure 2 (A) is the ultraviolet absorption diagram of the mixture of dye EC5-H3 and ·OH with different concentrations, Figure 2 (B) is the fluorescence emission spectrum in the short wavelength range after the mixture of dye EC5-H3 and ·OH with different concentrations, Figure 2 (C) is the fluorescence emission spectrum in the long wavelength range after the mixture of dye EC5-H3 and ·OH with different concentrations, Figure 2 (D) is the fluorescence kinetic spectrum of the mixture of dye EC5-H3 and equimolar ·OH at 890 nm; Figure 2 (E) is the ultraviolet absorption diagram of the mixture of dye EC5-H3 and different concentrations of ONOO - mixture, Figure 2 (F) is the fluorescence emission spectrum in the short wavelength range after the mixture of dye EC5-H3 and different concentrations of ONOO - mixture, Figure 2 (G) is the fluorescence emission spectrum in the long wavelength range after the mixture of dye EC5-H3 and different concentrations of ONOO - mixture, Figure 2 (H) is the fluorescence kinetic spectrum of the mixture of dye EC5-H3 and equimolar ONOO - mixture at 890 nm; Figure 2 (I) is the ultraviolet absorption diagram of the mixture of dye EC5-H3 and different concentrations of ClO - mixture, Figure 2 (J) is the fluorescence emission spectrum in the short wavelength range after the mixture of dye EC5-H3 and different concentrations of ClO - mixture, Figure 2 (K) is the fluorescence emission spectrum in the long wavelength range after the mixture of dye EC5-H3 and different concentrations of ClO - mixture, Figure 2 (L) is the fluorescence kinetic spectrum of the mixture of dye EC5-H3 and equimolar ClO - mixture at 890 nm.

[0031] Figure 3 It is the fluorescence imaging of HeLa cells using EC5-H3. Among them, Figure 3 (A) is the HeLa cells treated only with dye EC5-H3, Figure 3 (B) is the HeLa cells treated with dye EC5-H3 and SIN-1, Figure 3 (C) is the HeLa cells treated with 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron(III) chloride (FeTPPS) in advance and then treated with EC5-H3 and SIN-1,Figure 3 (D) is Figure 3 (A), Figure 3 (B) and Figure 3 (C)'s relative fluorescence intensity; Figure 3 (E) is HeLa cells treated only with the dye EC5-H3, Figure 3 (F) is HeLa cells treated with the dye EC5-H3 and [lipopolysaccharide (LPS) + interferon-γ (IFN-γ)], Figure 3 (G) is HeLa cells pretreated with uric acid (UA) and then treated with EC5-H3 and [LPS + IFN-γ], Figure 3 (H) is Figure 3 (E), Figure 3 (F) and Figure 3 (G)'s relative fluorescence intensity; Figure 3 (I) is HeLa cells treated only with the dye EC5-H3, Figure 3 (J) is HeLa cells treated with the dye EC5-H3 and N-acetyl-p-aminophenol (APAP), Figure 3 (K) is HeLa cells pretreated with N-acetylcysteine (NAC) and then treated with EC5-H3 and APAP, Figure 3 (H) is Figure 3 (E), Figure 3 (F) and Figure 3 (G)'s relative fluorescence intensity.

[0032] Figure 4 is NIR-II fluorescence imaging of mice using EC5-H3. Among them, Figure 4 (A) is a graph showing the change of NIR-II fluorescence imaging of mice without APAP treatment, 150 mg / kg APAP treatment, 300 mg / kg APAP treatment, 500 mg / kg APAP treatment, and pretreatment with NAC followed by addition of APAP over time; Figure 4 (B) is the relationship between the intensity and time of NIR-II fluorescence imaging of mice without APAP treatment, 150 mg / kg APAP treatment, 300 mg / kg APAP treatment, 500 mg / kg APAP treatment, and pretreatment with NAC followed by addition of APAP; Figure 4 (C) is NIR-II fluorescence imaging of different organs of mice treated with APAP; Figure 4 (D) is a bar graph of the fluorescence intensity of different organs of mice treated with APAP.

[0033] Figure 5 is fluorescence imaging of different liver tissue cells using EC5-H3. Among them, Figure 5(A) is the co-localization map of the liver tissue of mice treated with the dye EC5-H3 + APAP after staining with hepatocyte nuclear factor 4-α (HNF4-α). Figure 5 (B) is Figure 5 the enlarged local view of (A); Figure 5 (C) is the co-localization map of the liver tissue of mice treated with the dye EC5-H3 + APAP after staining with F4 / 80. Figure 5 (D) is Figure 5 the enlarged local view of (C). Detailed implementation mode

[0034] To enable those skilled in the art to understand the features and effects of the present invention, the following is 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.

[0035] 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.

[0036] In this article, "comprising", "including", "containing" and similar expressions cover the meanings of "consisting essentially of" and "consisting of". For example, when it is disclosed herein that "A comprises B and C", it should be considered that "A consists essentially of B and C" and "A consists of B and C" have been disclosed herein.

[0037] In this article, 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 simplicity 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 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.

[0038] In this article, unless otherwise specified, the percentage refers to the mass percentage and the ratio refers to the mass ratio.

[0039] In this article, when describing the embodiments or examples, it should be understood that it is not used to limit the present invention to these embodiments or examples. On the contrary, all alternatives, improvements and equivalents of the methods and materials described in the present invention can be covered within the scope defined by the claims.

[0040] In this document, for the sake of brevity in description, not all possible combinations of all the technical features in each implementation or example are described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation or example can be combined arbitrarily, and all possible combinations should be considered to be within the scope described in this specification.

[0041] In certain chemical groups defined herein, the total number of carbon atoms present in the group is represented by a simplified symbol in front. For example, C1-C4 alkyl refers to an alkyl group as defined below having a total of 1 to 4 carbon atoms. The total number of carbon atoms in the simplified symbol does not include the carbon that may be present in the substituents of the group.

[0042] As used herein, "halo" or "halogen" refers to the elements of Group 17 with atomic numbers from 9 to 85. "Halogen" or "halo atom" refers to F, Cl, Br, and I. "Halo-substituted" means substituted by an atom selected from F, Cl, Br, and I. "Halide anion" refers to an anion formed by a halogen atom gaining one electron, including Cl — 、Br — 、I — 、F — 。

[0043] As used herein, "amino" refers to -NH 2 。

[0044] As used herein, "sulfate" refers to SO 4 2- 。

[0045] As used herein, "nitrate" refers to NO 3 - 。

[0046] As used herein, "carbonate" refers to CO 3 - 。

[0047] As used herein, "bicarbonate" refers to HCO 3 - 。

[0048] As used herein, "acetate" refers to CH 3 COO - 。

[0049] As used herein, "oxalate" refers to C 2 O 4 2- 。

[0050] As used herein, "trifluoroacetate" refers to CF 3 COO - 。

[0051] As used herein, "alkyl" refers to a straight-chain or branched-chain monovalent saturated hydrocarbon group having a specified number of carbon atoms, and specific alkyl groups are those having 1 to 5 carbon atoms ("C1-C5 alkyl"), preferably containing 1-3 carbon atoms (C1-C3 alkyl). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and n-pentyl.

[0052] As used herein, "aryl" 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, aryl contains 6 to 14 ring carbon atoms, preferably C6-C10 aryl. 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, azulyl, biphenyl, biphenylene, and fluorenyl.

[0053] As used herein, "alkoxy" refers to alkyl-O-, and preferred alkoxy groups are C1-C5 alkoxy groups, such as 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.

[0054] As used herein, "alkylthio" refers to a group in which the hydrogen in mercapto is replaced by the alkyl group described above, i.e., R'S-, where R' is an alkyl group. In some embodiments, the alkylthio group may be a mercapto group substituted by a C1-C5 alkyl group (C1-C5 alkylthio), a mercapto group substituted by a C1-C4 alkyl group (C1-C4 alkylthio), a mercapto group substituted by a C1-C3 alkyl group (C1-C3 alkylthio), and a mercapto group substituted by a C1-C2 alkyl group (C1-C2 alkylthio).

[0055] As used herein, as a group or as part of another group, the term "heteroaryl" or "heteroaromatic ring" refers to a conjugated ring system group or moiety having carbon atoms (e.g., 1 to 11 carbon atoms, 1 to 10 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms) and heteroatoms selected from nitrogen, oxygen, and sulfur (e.g., 1 to 6 heteroatoms, such as 1, 2, 3, 4, or 5 heteroatoms) within the ring. In some embodiments, the heteroaryl may contain 5 to 12 ring atoms, 5 to 10 ring carbon atoms, 5 to 8 ring atoms, 5 to 7 ring atoms, or 5 to 6 ring atoms. Unless otherwise specifically indicated herein, the heteroaryl may be a monocyclic, bicyclic, tricyclic, or higher ring system, and may also be fused to an aryl or heterocyclic group as defined above. Examples of heteroaryl fused to an aryl include, but are not limited to, benzopyridyl, benzopyrazolyl, benzimidazolyl, benzopyrrolyl, etc. The nitrogen, carbon, or sulfur atoms in the heteroaryl may be optionally oxidized; the nitrogen atoms may be optionally quaternized. For the purposes of the present disclosure, in some embodiments, the heteroaryl is a stable 5- to 12-membered aromatic group containing 1 to 5 (e.g., 1, 2, 3, 4, or 5) heteroatoms selected from nitrogen, oxygen, and sulfur, and in some embodiments, is a stable 5- to 10-membered aromatic group containing 1 to 4 (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur or a 5- to 6-membered aromatic group containing 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically provided herein, the heteroaryl may be optionally substituted.

[0056] Non-limiting examples of heteroaryl include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, benzothienyl, oxatriazolyl, cinnolinyl, quinazolinyl, indolizidinyl, phenanthrolinyl, isoxazolyl, phenoxazinyl, phenothiazinyl, 4,5,6,7-tetrahydrobenzo[b]thienyl, naphthopyridyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[4,3-a]pyrazinyl, [1,2,4]triazolo[4,3-c]pyrimidinyl, [1,2,4]triazolo[4,3-a]pyridyl, imidazo[1,2-a]pyridyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, etc.

[0057] As used herein, "optionally" or "optional" means that the subsequent described event or condition may or may not occur, and this description includes both the occurrence and non-occurrence of the event or condition. For example, "optionally substituted aryl" means that the aryl is substituted or unsubstituted, and this description includes both the substituted aryl and the unsubstituted aryl. The "optionally" substituents described in the claims and the specification of the present invention are selected from alkyl, alkoxy, alkylthio, halogen, and amino.

[0058] 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 "suitable substituents". Herein, 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 substituents. For example, when the substituent of ethyl is 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 allowed valence of the substituting atom 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 suitable substituents 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 designated as "unsubstituted", chemical moieties mentioned herein should be understood to include substituted variants. For example, mention of an "aryl" group or moiety implicitly includes both unsubstituted aryl groups and substituted variants.

[0059] Throughout this application, the above-mentioned "suitable substituents" should be understood to include, but are not limited to, alkyl, halogen, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino, aryl, heteroaryl, etc. described herein; these groups as substituents, including alkyl, alkoxy, aryl, and heteroaryl themselves, are also optionally substituted, e.g., they can also be optionally substituted by one or more groups selected from alkyl, halogen, alkoxy, amino, monoalkylamino, dialkylamino, aryl, and heteroaryl.

[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 asymmetric centers, the (Z) and (E) isomers of double bonds, etc. Therefore, the 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] This invention also provides a fluorescent dye or a dye composition, which contains the compound of formula I of this invention and an optional solvent. Solvents applicable to this invention include, but are not limited to, buffers, preferably HEPES buffer or PBS buffer.

[0062] This invention provides the use of the compounds of this invention in the preparation of reagents for use in cell fluorescence imaging or animal fluorescence imaging. The cells can be cells, such as live Hela cells. The animals can be mammals, such as mice or humans.

[0063] The following further elaborates this invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate this invention and not to limit the scope of this 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 stated, percentages and parts are calculated by weight.

[0064] General synthetic method for EC5 series compounds

[0065]

[0066] (1) Synthesis of compound 2: Dissolve 10 g (1 eq) of 2-bromo-4-fluorobenzaldehyde, 10.2 g (1.5 eq) of potassium carbonate, and 10.8 g (3.0 eq) of diethylamine in 100 mL of N,N-dimethylformamide, react at 60 °C for 24 h. After the reaction is completed, rotary evaporate to remove the reaction solvent, dissolve the crude product in 200 mL of hot ethanol, filter while hot, and obtain 11.9 g of compound 2 (orange-yellow solid), with a yield of 94%. 1 H NMR(400MHz,CDCl 3 )δ10.05(s,1H),7.78(d,J=8.4Hz,1H),6.77(d,J=2.5Hz,1H),6.61(dd,J=8.4Hz,2.5Hz,1H),3.41(q,J=7.1Hz,4H),1.22(t,J=7.1Hz,6H).HRMS(EI)calcd.for C 11 H 14 BrNO,[M] +: 255.0259, found: 255.0258.

[0067] (2) Synthesis of Compound 3: 10 g (1 eq) of Compound 2, 4.9 g (2.0 eq) of ethylene glycol, 11.6 g (2.0 eq) of triethyl orthoformate, and 0.1 eq of catalytic amount of p-toluenesulfonic acid were dissolved in 100 mL of toluene. The mixture was refluxed at 110 °C for 2 h, neutralized with saturated sodium bicarbonate solution, extracted with dichloromethane, and then the organic solvent was removed by rotary evaporation to obtain 8.6 g of Compound 3 (brown oily liquid) with a yield of 73%. 1 H NMR (400 MHz, CDCl 3 ) δ 7.38 (d, J = 8.8 Hz, 1H), 6.79 (d, J = 2.2 Hz, 1H), 6.59 (dd, J = 8.8 Hz, 2.2 Hz, 1H), 6.01 (s, 1H), 4.16 - 4.10 (m, 2H), 4.07 - 4.01 (m, 2H), 3.33 (q, J = 7.0 Hz, 4H), 1.14 (t, J = 7.0 Hz, 6H). HRMS (EI) calcd. for C 13 H 18 BrNO 2 , [M] + : 299.0521, found: 299.0522.

[0068] (3) Synthesis of Compound 4: 21 g (1 eq) of Compound 3 was dissolved in 200 mL of anhydrous tetrahydrofuran. The reaction temperature was controlled at -78 °C. Under the protection of inert gas, 33.6 mL of n-butyllithium was slowly added, and the reaction mixture was stirred at -78 °C for 20 min to prepare the active lithium reagent. Then, 10.8 mL (2.0 eq) of DMF was added to the reaction system, and the temperature was slowly raised to room temperature. After reacting for 2 h, saturated ammonium chloride aqueous solution was added, and the mixture was extracted with ethyl acetate. After concentration, it was purified by silica gel column chromatography (PE:EA:TEA = 100:1:0.5, v / v) to obtain 12.4 g of Compound 4 (yellow oily liquid) with a yield of 71%. 1 H NMR (400 MHz, CDCl 3 ) δ 10.39 (s, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.18 (d, J = 2.9 Hz, 1H), 6.83 (dd, J = 8.6 Hz, 2.9 Hz, 1H), 6.22 (s, 1H), 4.17 - 4.11 (m, 2H), 4.10 - 4.04 (m, 2H), 3.40 (q, J = 7.1 Hz, 4H), 1.17 (t, J = 7.1 Hz, 6H). HRMS (EI) calcd. for C 14 H 19 NO3 ,[M] + : 249.1365, found: 249.1364.

[0069] (4) Synthesis of Compound 5: Dissolve 12 g (2.0 eq) of Compound 4 and 3.7 g (1.0 eq) of 1,4 - cyclohexanedione monoethylene ketal in 20 mL of ethanol. Add 2 mL of 30% sodium hydroxide solution thereto and react at room temperature for 4 h. A solid precipitates during the reaction. Filter and wash the filter cake with 30 mL of ice - cold ethanol to obtain 13.5 g of Compound 5 (yellow solid), with a yield of 91%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.13 (s, 2H), 7.40 (d, J = 8.7 Hz, 2H), 6.63 (dd, J = 8.7, 1.9 Hz, 2H), 6.43 (d, J = 1.9 Hz, 2H), 5.78 (s, 2H), 4.17 - 4.09 (m, 4H), 4.00 - 3.92 (m, 4H), 3.85 (s, 4H), 3.35 (q, J = 7.0 Hz, 8H), 2.92 (s, 4H), 1.15 (t, J = 7.0 Hz, 12H). HRMS (EI) calcd. for C 36 H 46 N 2 O 7 ,[M] + : 618.3305, found: 618.3306.

[0070] (5) Synthesis of Compound 6: Dissolve 2 g (1.0 eq) of Compound 5 and 1.5 eq of 2 - phenoxyphenyl lithium in 80 mL of anhydrous tetrahydrofuran and react at 0 °C for 0.5 h. After the reaction is completed, add saturated ammonium chloride aqueous solution, extract with ethyl acetate, and concentrate to obtain the crude product. Without separation, redissolve it directly in 100 mL of dichloromethane, add 1 mL of methanesulfonic acid, and react at room temperature for 1 h. After the reaction is completed, add saturated sodium bicarbonate solution for neutralization, extract with dichloromethane, and concentrate the organic solvent, then purify by silica gel column chromatography (PE:DCM = 20:1, v / v) to obtain 1.3 g of Compound 6 (orange - red solid), with a yield of 67%. 1 H NMR (400 MHz, CDCl 3)δ8.85(s,2H),7.84(d,J=9.2Hz,2H),7.27-7.25(m,2H),7.16-7.12(m,4H),7.03(dd,J=9.2Hz,2.4Hz,2H),6.77-6.70(m,4H),6.52(d,J=2.4Hz,2H),3.38(q,J=7.1Hz,8H),1.14(t,J=7.1Hz,12H).HRMS(ESI)calcd.for C 42 H 38 N 2 O 2 ,[M+H] + :603.3007,found:603.3009.

[0071] (6)Synthesis of EC5 series compounds

[0072] 6.1 Preparation of organolithium reagent

[0073] In formula IV, R 1 and R 2 are as described above.

[0074] Dissolve 5eq, 4.15 mmol of the compound of formula IV ( In formula V, R 1 and R 2 are as described above, and X is a halogen) in 60 mL of anhydrous tetrahydrofuran. Control the reaction temperature at -78 °C. Under the protection of inert gas, slowly transfer 1.66 mL of n-butyllithium, and keep stirring at -78 °C for 20 min to prepare the active lithium reagent.

[0075] 6.2 Preparation of EC5 series compounds

[0076] Dissolve 1eq, 0.83 mmol of compound 6 ( ) in 60 mL of anhydrous tetrahydrofuran to obtain an organic phase. Transfer the lithium reagent to the above organic phase under ice bath conditions. After reacting for 1 h, add 10 mL of saturated ammonium chloride aqueous solution, extract with 90 mL of dichloromethane, and remove the solvent by vacuum evaporation to obtain a crude product. Redissolve the obtained crude product in 100 mL of dichloromethane, add 0.5 mL of methanesulfonic acid, react for 3 h, then add 30 mL of saturated sodium bicarbonate solution, and extract with 90 mL of dichloromethane. Finally, purify by silica gel column chromatography (CH 2 Cl 2 :MeOH = 40:1, v / v) to obtain EC5 series compounds.

[0077] Example 1: Synthesis of compound EC5-Me

[0078]

[0079] Dissolve 0.71 g of compound 1-bromo-2-methylbenzene in 60 mL of anhydrous tetrahydrofuran. Control the reaction temperature at -78 °C. Under the protection of an inert gas, slowly transfer 1.66 mL of n-butyllithium, and maintain the reaction temperature at -78 °C and stir for 20 min to prepare an active lithium reagent. Dissolve 0.5 g of compound 6( ) in 40 mL of anhydrous tetrahydrofuran to obtain an organic phase. Transfer the lithium reagent to the above organic phase under an ice bath condition. After reacting for 40 min, add 10 mL of saturated ammonium chloride aqueous solution, extract with 90 mL of dichloromethane, and remove the solvent by vacuum evaporation to obtain a crude product. Redissolve the obtained crude product in 60 mL of dichloromethane, add 0.5 mL of methanesulfonic acid, react for 3 h, then add 30 mL of saturated sodium bicarbonate solution, and extract with 90 mL of dichloromethane. Finally, purify by silica gel column chromatography (CH 2 Cl 2 :MeOH = 40:1, v / v) to obtain 0.4 g of a purple-red solid, which is compound EC5-Me , with a yield of 65%. 1 H NMR (400 MHz, CDCl 3 ) δ 7.66 - 7.62 (m, 1H), 7.57 - 7.53 (m, 6H), 7.42 (d, J = 7.2 Hz, 1H), 7.35 - 7.33 (m, 2H), 7.30 - 7.28 (m, 2H), 7.08 - 7.05 (m, 4H), 6.94 (q, J = 8.6 Hz, 2H), 6.80 (d, J = 7.6 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.54 (s, 2H), 3.57 (q, J = 7.2 Hz, 8H), 2.27 (s, 3H), 1.26 (t, J = 7.2 Hz, 12H); 13C NMR (101 MHz, CDCl3) δ 153.03, 148.78, 148.82, 148.50, 141.13, 139.02, 135.39, 130.91, 130.11, 130.04, 130.01, 129.81, 129.12, 129.02, 128.61, 126.24, 126.19, 125.78, 124.22, 124.17, 117.33, 117.20, 106.82, 47.42, 45.90, 19.89, 13.12. HRMS (ESI) calcd. for C 49 H 45 N 2 O + , [M] + : 677.3526, found: 677.3531.

[0080] Example 2: Synthesis of Compound EC5-2OMe

[0081]

[0082] Dissolve 0.72 g of compound 1-bromo-2-(methoxymethyl)benzene in 40 mL of anhydrous tetrahydrofuran. Control the reaction temperature at -78 °C. Under the protection of an inert gas, slowly transfer 1.33 mL of n-butyllithium, and maintain the reaction temperature at -78 °C and stir for 20 min to prepare an active lithium reagent. Dissolve 0.4 g of compound 6 ( ) in 60 mL of anhydrous tetrahydrofuran to obtain an organic phase. Transfer the lithium reagent to the above organic phase under an ice bath condition. After reacting for 40 min, add 10 mL of saturated ammonium chloride aqueous solution, extract with 90 mL of dichloromethane, and remove the solvent by vacuum evaporation to obtain a crude product. Redissolve the obtained crude product in 60 mL of dichloromethane, add 0.5 mL of methanesulfonic acid, react for 3 h, then add 30 mL of saturated sodium bicarbonate solution, and extract with 90 mL of dichloromethane. Finally, purify by silica gel column chromatography (CH 2 Cl 2 :MeOH = 40:1, v / v) to obtain 0.2 g of a purple-red solid, which is compound EC5-2OMe ( ), with a yield of 65%. 1 H NMR (400 MHz, CDCl 3 ) δ 7.76 - 7.64 (m, 3H), 7.61 (d, J = 9.4 Hz, 2H), 7.32 - 7.27 (m, 3H), 7.25 - 7.24 (m, 1H), 7.03 - 6.97 (m, 6H), 6.91 (dd, J = 8.3, 5.7 Hz, 4H), 6.51 (s, 2H), 3.78 (s, 6H), 3.53 (q, J = 7.0 Hz, 8H), 1.22 (t, J = 7.0 Hz, 12H); 13C NMR (151 MHz, CDCl3) δ 163.90, 158.01, 152.72, 148.93, 148.64, 148.56, 141.30, 139.14, 139.12, 135.34, 135.32, 132.21, 130.62, 130.23, 130.11, 128.51, 128.40, 127.20, 125.71, 125.69, 124.00, 117.02, 116.71, 106.68, 104.32, 56.31, 47.73, 45.69, 13.02. HRMS (ESI) calcd. for C 50 H 47 N 2 O 3+ ,[M] + : 723.3582, found: 723.3585.

[0083] Example 3: Synthesis of Compound EC5-H2

[0084]

[0085] Dissolve 50 mg of compound EC5-Me in 30 mL of ethanol, add 11 mg of sodium borohydride thereto, react at room temperature for 0.5 h, then rotary evaporate under reduced pressure to remove the excess solvent, and then purify by silica gel column chromatography (PE:EA = 60:1, v / v) to obtain 19 mg of a yellowish-green solid, which is compound EC5-H2( ), and the yield is 40%. 1 H NMR (400 MHz, CDCl3) δ 7.45 - 7.38 (m, 3H), 7.34 (d, J = 9.0 Hz, 2H), 7.20 - 7.17 (m, 4H), 7.15 (dd, J = 4.2, 1.5 Hz, 1H), 7.01 (s, 1H), 6.93 - 6.83 (m, 4H), 6.78 (s, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 2.2 Hz, 1H), 6.34 (dd, J = 8.4, 2.5 Hz, 1H), 6.12 (d, J = 2.5 Hz, 1H), 5.87 (s, 1H), 3.61 (d, J = 23.0 Hz, 1H), 3.43 (d, J = 23.0 Hz, 1H), 3.31 (q, J = 7.4 Hz, 4H), 3.18 (q, J = 7.4 Hz, 4H), 2.28 (s, 3H), 1.06 (d, J = 7.4 Hz, 7H), 1.02 (d, J = 7.4 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ 146.60, 145.81, 145.62, 144.03, 136.84, 134.04, 131.63, 131.12, 131.11, 130.70, 129.80, 129.71, 128.92, 127.93, 127.54, 126.89, 125.04, 123.61, 123.52, 123.22, 116.13, 115.84, 111.42, 111.11, 105.32, 47.03, 44.42, 44.31, 29.80, 19.61, 12.72, 12.61. HRMS (ESI) calcd. for C 49 H 46 N 2 O, [M+H] +:679.3685, found:679.3691.

[0086] Example 4: Synthesis of Compound EC5-H3

[0087]

[0088] Dissolve 100 mg of Compound EC5-2OMe in 30 mL of ethanol. Add 25 mg of sodium borohydride thereto. After reacting at room temperature for 0.5 h, rotary evaporate under reduced pressure to remove the excess solvent, and then purify by silica gel column chromatography (PE:EA = 50:1, v / v) to obtain 88 mg of a yellowish-green solid, which is Compound EC5-H3( ), with a yield of 93%. 1 H NMR(400 MHz, CDCl3)δ7.50 - 7.42(m, 3H), 7.37(d, J = 9.1 Hz, 1H), 7.18(d, J = 4.0 Hz, 4H), 6.97(s, 1H), 6.94 - 6.88(m, 3H), 6.87(dd, J = 9.1, 2.6 Hz, 1H), 6.78(d, J = 8.3 Hz, 2H), 6.74(d, J = 8.3 Hz, 1H), 6.48(d, J = 2.5 Hz, 1H), 6.36(dd, J = 8.4, 2.5 Hz, 1H), 6.45(d, J = 2.5 Hz, 1H), 5.82(s, 1H), 3.78(s, 6H), 3.60(s, 2H), 3.30(q, J = 7.2 Hz, 4H), 3.22(q, J = 7.2 Hz, 4H), 1.09(t, J = 7.2 Hz, 6H), 1.04(t, J = 7.2 Hz, 6H); 13C NMR(151 MHz, CDCl3)δ158.30, 148.61, 146.42, 146.13, 145.54, 144.44, 134.43, 134.29, 131.56, 130.45, 130.52, 129.21, 129.00, 128.94, 128.73, 128.32, 127.81, 127.34, 127.28, 125.23, 123.42, 122.21, 122.01, 116.53, 115.84, 115.78, 111.54, 111.43, 105.82, 104.31, 56.00, 47.21, 44.42, 44.40, 32.33, 12.74, 12.45. HRMS(ESI) calcd. for C 50 H 48 N 2 O 3 , [M + H] +: 725.3738, found: 725.3677.

[0089] Test Example 1

[0090] Dissolve EC5-Me in chloroform (CH 3 Cl), acetonitrile (CH 3 CN), dimethyl sulfoxide (DMSO), and HEPES / DMF buffer (10 mM, pH = 7.4, HEPES:DMF = 1:1, v / v) respectively to prepare a solution with a concentration of 5 μM. Use a Shimadzu UV-2600i instrument to detect its corresponding absorption spectrum and a PTI-QM4 steady-state fluorescence spectrometer from Horiba Instruments (Shanghai) Co., Ltd. to test the fluorescence emission spectrum. Dissolve EC5-2OMe in chloroform (CH 3 Cl), acetonitrile (CH 3 CN), dimethyl sulfoxide (DMSO), and HEPES / DMF buffer (10 mM, pH = 7.4, HEPES:DMF = 1:1, v / v) respectively to prepare a solution with a concentration of 5 μM. Use a Shimadzu UV-2600i instrument to detect its corresponding absorption spectrum and fluorescence emission spectrum. The results are as Figure 1 shown; and calculate their molar extinction coefficients, fluorescence quantum yields, fluorescence brightness, and fluorescence lifetimes respectively. The results are shown in Table 1.

[0091] Among them, the fluorescence quantum yield is the relative fluorescence quantum yield. Φ reference sample is the absolute fluorescence quantum yield of EC5-Me in chloroform, which is 0.19. Refer to the reference (DOI: 10.1016 / j.cclet.2023.108154). The specific calculation formula is as follows:

[0092]

[0093] 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.

[0094] From Figure 1 it can be seen that the ultraviolet absorption wavelengths of compounds EC5-Me and EC5-2OMe are around 800 nm to around 900 nm; in chloroform, the maximum emission wavelength of fluorescence emission of compounds EC5-Me and EC5-2OMe is around 875 nm, and with the change of the dye dissolution solvent, the corresponding maximum emission wavelength of fluorescence emission will have a red shift within 40 nm.

[0095] Table 1: Photophysical Properties of Dyes EC5-Me and EC5-2OMe

[0096]

[0097] As can be seen from Table 1, both the EC5-Me and EC5-2OMe dyes of the present invention have high molar extinction coefficients and quantum yields, and at the same time have good fluorescence brightness, and can be used as fluorescence probes for bioimaging.

[0098] Test Example 2

[0099] Dissolve EC5-H3 in HEPES buffer solution (10 mM, pH = 7.4, containing 50% DMF) to prepare a solution with a concentration of 5 μM. Use the Shimadzu UV-2600i instrument and set the excitation light wavelength to 840 nm to measure the ultraviolet absorption spectrum and fluorescence emission spectrum after mixing EC5-H3 with different concentrations of ROS (·OH, ONOO - or ClO - ). The spectral data results are as Figure 2 A-C, Figure 2 E-G, Figure 2 I-K shown; use the PTI-QM4 steady-state fluorescence spectrometer of Horiba Instruments (Shanghai) Co., Ltd. and set the excitation light wavelength to 840 nm to measure the change in fluorescence intensity over time at the fluorescence emission wavelength of 890 nm of EC5-H3 under the condition of adding an equivalent amount of ROS (·OH, ·OONO or ClO·). The data are as Figure 2 D, Figure 2 H, Figure 2 L shown.

[0100] As can be seen from Figure 2 , ROS (·OH, ONOO - and ClO - ) can all react with EC5-H3 to obtain EC5-2OMe, thereby generating a fluorescence signal at a long wavelength (around 890 nm), and as the concentration of reactive oxygen increases, the intensity of the generated fluorescence signal also increases. At the same time, the fluorescence emission intensity at its short wavelength (around 530 nm) becomes weaker and weaker, and the signal interference at 890 nm can be ignored; at the same time, as can be seen from Figure 2 , EC5-H3 has a relatively fast response to the above three kinds of ROS, especially to ·OH, and almost immediately generates a corresponding fluorescence signal when added.

[0101] Test Example 3

[0102] Blank control group: Prepare a 10 μM PBS buffer solution of EC5-H3, take 500 μL and incubate it with Hela cells for about 1 hour. Perform confocal micro-fluorescence imaging on the incubated cells (Leica, DMi8 inverted microscope instrument), and the shooting parameters are λ ex = 808 nm, λ em= 850 - 890 nm. The results are as shown in Figure 3 A, Figure 3 E, Figure 3 as shown in I.

[0103] Experimental group:

[0104] SIN-1 stimulation group: First, add 500 μM of SIN-1 and incubate for 2 hours. Then, prepare EC5-H3 into a 10 μM PBS buffer solution, take 500 μL and co-incubate with Hela cells for about 1 hour; perform confocal microscopy fluorescence imaging on the incubated cells, and the imaging parameters are λ ex = 808 nm, λ em = 850 - 890 nm. The results are as shown in Figure 3 B.

[0105] LPS / IFN-γ stimulation group: First, add 1 μg / ml of LPS and 50 ng / mL of IFN-γ and incubate for 4 hours. Prepare EC5-H3 into a 10 μM PBS buffer solution and co-incubate with Hela cells for about 1 hour; perform confocal microscopy fluorescence imaging on the incubated cells, and the imaging parameters are λ ex = 808 nm, λ em = 850 - 890 nm. The results are as shown in Figure 3 F.

[0106] APAP stimulation group: First, add 1 mM of APAP and incubate for 2 hours. Then, prepare EC5-H3 into a 10 μM PBS buffer solution, take 500 μL and co-incubate with Hela cells for about 1 hour;, perform confocal microscopy fluorescence imaging on the incubated cells, and the imaging parameters are λ ex = 808 nm, λ em = 850 - 890 nm. The results are as shown in Figure 3 K.

[0107] Control group:

[0108] SIN-1 + FeTPPS group: The difference from the SIN-1 stimulation group is only that 10 μM of FeTPPS is added in advance for treatment. The results are as shown in Figure 3 C.

[0109] LPS / IFN-γ stimulation group: The difference from the LPS / IFN-γ stimulation group is only that 200 μM of uric acid is added in advance for treatment. The results are as shown in Figure 3 G.

[0110] APAP stimulation group: The difference from the APAP stimulation group is only that 200 μM of NAC is added in advance for treatment. The results are as shown in Figure 3 K.

[0111] From Figure 3 As can be seen, EC5-H3 can react with ROS generated in cells to produce a fluorescence signal, which can be used for fluorescence imaging of the ROS process in cells.

[0112] Test Example 4

[0113] Control group: Prepare a 200 μM PBS buffer solution of EC5-H3, take 200 μL and inject it into a mouse through the tail vein. After anesthetizing the mouse, place it on a near-infrared in vivo fluorescence imaging platform, and use an excitation light with λ ex = 808 nm and a 1100 nm long-pass filter to take near-infrared fluorescence images of the mouse at different treatment times. The results are as Figure 4 shown in

[0114] Injury group: Inject 500 μL of 150 mg / kg, 300 mg / kg, and 500 mg / kg APAP into the abdominal cavity of the mouse respectively, and then inject 200 μL of a 200 μM PBS buffer solution of EC5-H3 into the mouse through the tail vein. After anesthetizing the mouse, place it on a near-infrared in vivo fluorescence imaging platform, and use an excitation light with λ ex = 808 nm and a 1100 nm long-pass filter to take near-infrared fluorescence images of the mouse at different treatment times. The results are as Figure 4 shown in

[0115] Treatment group: Inject 500 μL of 300 mg / kg NAC into the abdominal cavity of the mouse, then inject 500 μL of 300 mg / kg APAP into the abdominal cavity of the mouse, and then inject 200 μL of a 200 μM PBS buffer solution of EC5-H3 into the mouse through the tail vein. After anesthetizing the mouse, place it on a near-infrared in vivo fluorescence imaging platform, and use an excitation light with λ ex = 808 nm and a 1100 nm long-pass filter to take near-infrared fluorescence images of the mouse at different treatment times. The results are as Figure 4 shown in

[0116] From Figure 4 As can be seen, as metabolism proceeds, the fluorescence intensity at the liver of the injury group gradually increases, far higher than that of the control group and the treatment group, indicating that EC5-H3 can also be used for in vivo fluorescence imaging of the ROS process in mice.

[0117] Test Example 5

[0118] For mice treated with APAP (500 μL of 78 mM APAP solution was injected via the tail vein, with PBS buffer as the solvent, and the APAP concentration was 300 mg / kg), after injecting 200 μL of 200 μM EC5-H3 via the tail vein, the mice were sacrificed and liver tissues were isolated, and the tissues were stained with HNF4-α or F4 / 80 respectively. The results are shown in the figure.

[0119] It can be seen from Figure 5 that macrophages have better overlap with the signal in the EC5-H3 probe channel, indicating that EC5-H3 can label inflammation well.

Claims

1. Compounds of formula I: In Formula I, R1 and R2 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C1-C5 alkoxy group, a substituted or unsubstituted C1-C5 alkylthiol group and a halogen; the C1-C5 alkyl group, the C1-C5 alkoxy group and the C1-C5 alkylthiol group are optionally substituted by 1, 2, 3 or 4 halogen substituents; R1 and R2 are not hydrogen atoms at the same time.

2. The compound of formula (I) according to claim 1, characterized in that R1 and R2 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a substituted or unsubstituted C1-C3 alkoxy group and a halogen; the C1-C3 alkyl group and the C1-C3 alkoxy group are optionally substituted by 1, 2, 3 or 4 halogen substituents.

3. The compound of formula I according to claim 1, characterized in that The compound of formula I is:

4. Compound of formula II: In Formula II, R3 and R4 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C1-C5 alkoxy group, a substituted or unsubstituted C1-C5 alkylthiol group and a halogen; the C1-C5 alkyl group, the C1-C5 alkoxy group and the C1-C5 alkylthiol group are optionally substituted by 1, 2, 3 or 4 halogen substituents; R3 and R4 are not hydrogen atoms at the same time; Y - One or more selected from 1 / 2 sulfate, chloride, fluoride, bromide, nitrate, 1 / 2 carbonate, bicarbonate, acetate, 1 / 2 oxalate and trifluoroacetate.

5. The compound of formula II as claimed in claim 4, characterized in that R3 and R4 are each independently selected from: a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a substituted or unsubstituted C1-C3 alkoxy group and a halogen; the C1-C3 alkyl group and the C1-C3 alkoxy group are optionally substituted by 1, 2, 3 or 4 halogen substituents.

6. The compound of formula II as claimed in claim 4, characterized in that The compound of formula II is 7. A method for preparing a compound of formula I according to any one of claims 1 to 3, characterized in that: The compound of formula I is obtained by reducing the compound of formula II according to any one of claims 4 to 6.

8. A near-infrared fluorescent probe composition, characterized in that: The near-infrared fluorescent probe composition contains the compound of formula I according to any one of claims 1 to 3 and a solvent; preferably, the solvent is a buffer, more preferably a HEPES buffer or a PBS buffer.

9. Use of the compound of formula I according to any one of claims 1 to 3 in the preparation of an agent for near-infrared fluorescence imaging.

10. The use according to claim 9, characterized in that The near-infrared fluorescence imaging is reactive oxygen near-infrared fluorescence imaging, and / or the near-infrared fluorescence shaping is in vivo imaging.

Images