A near-infrared fluorescent probe and its preparation method and application

Abstract

The invention discloses a near-infrared fluorescent probe and its preparation method and application. The present invention is refluxed at 110 DEG C for 12h by prepared 1,3-dimethoxy-5-nitrosoisobenzene compound 5 and excess 48wt% hydrobromic acid aqueous solution, and the reactant is extracted, washed, dried, purified to obtain 5-nitro-1,3-benzenediol compound 6, potassium carbonate and compound 6 are added to DMF and stirred at room temperature for 0.5h, prepared compound 4 is added to the mixed system, under nitrogen light protection, refluxed at 110 DEG C for 6h, the solvent is removed under reduced pressure, and the purified mixture obtains a near-infrared fluorescent probe. The probe has the advantages of near-infrared absorption, good water solubility, high stability, etc., and can effectively detect the activity of nitroreductase.

Description

Technical Field

[0001] The present invention belongs to the field of bioluminescence imaging, and in particular relates to a near-infrared fluorescent probe and a preparation method and application thereof. Background Art

[0002] Nitroreductase (NTR) can be expressed in large quantities in Escherichia coli and cells under hypoxic conditions. In the presence of reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH), nitroreductase reduces aromatic nitro compounds to aromatic amino compounds. Nitroreductases (NTR) are a class of proteins involved in the reduction of nitro-containing compounds. Nitroreductases (NTR) play important roles in medicine and biology, such as in probes for detecting hypoxia in tumor cells and gene- and virus-directed prodrugs. Therefore, monitoring nitroreductase activity is of great scientific importance.

[0003] The fluorescence method for detecting nitroreductase activity has the advantages of high sensitivity, high resolution, and simplicity. Of course, the fluorescence method also has disadvantages. For example, some biological tissues will emit fluorescence when excited by visible light, thereby interfering with the results and imaging of fluorescence detection. In addition, fluorescent probes generally have disadvantages such as short wavelength, poor stability, and unsuitability for in vivo imaging. Summary of the Invention

[0004] The primary purpose of the present invention is to provide a near-infrared fluorescent probe with an excitation wavelength of 696nm and an emission wavelength of 723nm. Due to the sulfonic acid group it carries, the probe has the characteristics of weak biological tissue penetration ability, good water solubility and high stability.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned near-infrared fluorescent probe.

[0006] Another object of the present invention is to provide an application of the above-mentioned near-infrared fluorescent probe. As a highly water-soluble near-infrared fluorescent probe, the probe can be used for nitroreductase activity detection and further applied to in vivo detection.

[0007] The present invention is achieved by providing a near-infrared fluorescent probe, the chemical structure of which is shown in the following formula (C):

[0008]

[0009] In formula (C), R1 is selected from any one of an H group, a carboxyl group, and a sulfo group; R2 is an alkyl chain having 1 to 5 carbon atoms; R3 is selected from any one of an H group, a hydroxyl group, an amino group, a carboxyl group, a sulfo group, an alkynyl group, and an azide group; and X is selected from any one of Cl, Br, I, a periodate group, and a trifluoroacetate group.

[0010] Preferably, in formula (C):

[0011] R1 is a sulfo group, R2 is -(CH2)4-, and R3 is a sulfo group;

[0012] Or R1 is a sulfo group, R2 is -(CH2)4-, and R3 is a hydrogen group;

[0013] Or R1 is sulfo, R2 is -(CH2)4-, and R3 is carboxyl

[0014] Or R1 is sulfo, R2 is -(CH2)4-, and R3 is amino

[0015] Or R1 is sulfo, R2 is -(CH2)4-, R3 is azido,

[0016] Or R1 is a hydrogen group, R2 is -(CH2)4-, R3 is a sulfo group,

[0017] or R1 is a hydrogen group, R2 is -(CH2)4-, R3 is a hydrogen group,

[0018] Or R1 is a hydrogen group, R2 is -(CH2)4-, R3 is a carboxyl group,

[0019] Or R1 is a hydrogen group, R2 is -(CH2)4-, R3 is an amino group,

[0020] Or R1 is a hydrogen group, R2 is -(CH2)4-, R3 is an azido group,

[0021] Or R1 is carboxyl, R2 is -(CH2)4-, R3 is sulfo,

[0022] Or R1 is a carboxyl group, R2 is -(CH2)4-, and R3 is a hydrogen group,

[0023] Or R1 is a carboxyl group, R2 is -(CH2)4-, R3 is a carboxyl group,

[0024] Or R1 is a carboxyl group, R2 is -(CH2)4-, and R3 is an amino group,

[0025] Or R1 is a carboxyl group, R2 is -(CH2)4-, and R3 is an azide group.

[0026] The present invention further discloses a method for preparing the above-mentioned near-infrared fluorescent probe, which comprises the following steps:

[0027] (1) Dissolve the hydrazine benzene compound in glacial acetic acid, add sodium acetate and 3-methyl-2-butanone, reflux at 130°C for 5 hours, and precipitate compound 1 from the reflux solution with methyl tert-butyl ether;

[0028] (2) a mixture of compound 1 and a halogenated hydrocarbon is refluxed in toluene or o-dichlorobenzene at a temperature of 110-120° C., cooled to room temperature after the reaction is completed, and the cooled reaction solution is dropwise added with ether for precipitation, and the ether is filtered off to obtain compound 2;

[0029] (3) The mixed system of DCM and DMF was cooled to 0°C, phosphorus oxychloride was added dropwise to the mixed system, and the mixture was stirred in an ice bath for 0.5 h. Cyclohexanone was slowly added to the system, and the mixture was refluxed and stirred at 80°C for 6 h. After the reaction was completed, the reaction solution was cooled to room temperature, and ice water was added dropwise and allowed to stand overnight. The mixture was filtered and washed with cold water to obtain compound 3;

[0030] (4) Under a nitrogen atmosphere, compound 2, compound 3, and sodium acetate were slowly added to an acetic anhydride solution, and the reaction system was heated to 80-110° C. and refluxed with stirring for 3 h. After the reaction was completed, the system was cooled to room temperature, and then ether was added dropwise for precipitation to obtain compound 4;

[0031] (5) Potassium carbonate and 5-nitro-1,3-diphenol were added to DMF and stirred at room temperature for 0.5 h. Compound 4 was added to the mixture, and the mixture was refluxed at 110° C. and stirred for 6 h under nitrogen protection. The solvent was removed under reduced pressure, and the mixture was purified by HPLC to obtain the probe.

[0032] Preferably, in step (2), the hydrocarbon group of the halogenated hydrocarbon is selected from any one of an alkyl chain with 3 to 5 carbon atoms, an alkynyl chain with 3 to 5 carbon atoms, an amino chain with 3 to 5 carbon atoms, a sulfonic acid chain with 4 carbon atoms, and a carboxylic acid chain with 4 carbon atoms;

[0033] The halogen of the halogenated hydrocarbon is selected from any one of Cl, Br and I.

[0034] Preferably, the halogenated hydrocarbon is selected from the group consisting of chloropropane, chlorobutane, chloropentane, bromopropane, bromobutane, bromopentane, iodopropane, iodobutane, iodopentane, 4-chloro-1-butyne, 5-chloro-1-pentyne, 6-chloro-1-hexyne, 4-bromo-1-butyne, 5-bromo-1-pentyne, 6-bromo-1-hexyne, 4-iodo-1-butyne, 5-iodo-1-pentyne, 6-iodo-1-hexyne, 2-chloroethylamine, 3-chloropropylamine, 4-chloro-1-butyne, 5-bromo-1-pentyne, 6-iodo ...4-iodo-1-butyne, 5-iodo-1-pentyne, 6-iodo-1-hexyne, 4-iodo-1-butyne, 5-iodo-1-pentyne, 6-iodo-1-hexyne, 4-iodo-1-butyne, 5-iodo-1-pentyne, 6-iodo-1-hexyne, 4-iodo-1-butyne, 5-iodo-1-pentyne, 6-iodo-1-hexyne, 4-iodo-1-butyne, 5- Any one of chlorobutylamine, 2-bromoethylamine, 3-bromopropylamine, 4-bromobutylamine, 2-iodoethylamine, 3-iodopropylamine, 4-iodobutylamine, 2-chloroethanesulfonic acid, 2-bromoethanesulfonic acid, 2-iodoethanesulfonic acid, 1,3-propane sultone, 1,4-butane sultone, 3-chloropropionic acid, 4-chlorobutyric acid, 5-chlorovaleric acid, 3-bromopropionic acid, 4-bromobutyric acid, 5-bromovaleric acid, 3-iodopropionic acid, 4-iodobutyric acid and 5-iodovaleric acid.

[0035] Preferably, in step (5), the preparation process of 5-nitro-1,3-benzenediol is:

[0036] (5-1) 3,5-Dimethoxyaniline and potassium carbonate were added to an acetonitrile solution, and a 50 wt% H2O2 aqueous solution was added to the reaction system and stirred at room temperature for 10 minutes. The reaction product was extracted, washed, dried, desolvated, and purified to obtain 1,3-dimethoxy-5-nitrobenzene, which is compound 5;

[0037] (5-2) Compound 5 was added to an excess of 48 wt% hydrobromic acid aqueous solution, and the mixture was refluxed at 110° C. with stirring for 12 h. The reaction product was extracted, washed, dried, desolventized, and purified to obtain 5-nitro-1,3-benzenediol.

[0038] Preferably, in steps (5-1) and (5-2), the extraction is to extract the mixture three times with ethyl acetate, the washing is to wash with saturated brine, the drying is to dry with anhydrous Na2SO4, and the solvent removal is to evaporate the solvent under vacuum; and the purification is to purify the residue of the mixture by column chromatography.

[0039] The present invention further discloses the application of the near-infrared fluorescent probe in detecting nitroreductase activity.

[0040] The present invention overcomes the shortcomings of the prior art and provides a near-infrared fluorescent probe and its preparation method and application. The preparation process of the near-infrared fluorescent probe of the present invention is as follows:

[0041] (1) Preparation of Compound 1

[0042]

[0043] The hydrazinobenzene compound was dissolved in glacial acetic acid, and sodium acetate and 3-methyl-2-butanone were added. The mixture was refluxed at 130°C for 5 hours. Methyl tert-butyl ether precipitated as a red solid, which was compound 1.

[0044] (2) Preparation of Compound 2

[0045]

[0046] Compound 1 is reacted with a halogenated hydrocarbon (wherein the hydrocarbon group is an alkyl chain having 3 to 5 carbon atoms, or an alkynyl chain having 3 to 5 carbon atoms, or an amino chain having 3 to 5 carbon atoms, or a sulfonic acid chain having 4 carbon atoms, or a carboxylic acid chain having 4 carbon atoms, and the halogen is one of Cl, Br, and I) under reflux in toluene or o-dichlorobenzene at 110°C. After completion of the reaction, the reaction mixture is cooled to room temperature, and the cooled reaction solution is added dropwise to diethyl ether for precipitation. The diethyl ether is then filtered off to obtain compound 2.

[0047] (3) Preparation of Compound 3

[0048]

[0049] The mixed system of DCM and DMF was cooled to 0°C, phosphorus oxychloride was added dropwise to the mixed system at 0°C, and the mixture was stirred for 0.5 h in an ice bath. Then, cyclohexanone was slowly added to the system, and the mixture was refluxed and stirred at 80°C for 6 h. After the reaction was completed, the reaction solution was cooled to room temperature, added dropwise to ice water, and allowed to stand overnight. After filtration, it was washed with cold water to obtain a yellow solid compound 3.

[0050] (4) Preparation of Compound 4

[0051]

[0052] Under a nitrogen atmosphere, compound 2, compound 3 and sodium acetate were slowly added to the acetic anhydride solution, and the reaction system was heated to 80°C and refluxed with stirring for 3 hours. After the reaction was completed, it was cooled to room temperature and then added dropwise to diethyl ether to precipitate a green solid, which was compound 4.

[0053] (5) Preparation of Compound 5

[0054]

[0055] 3,5-Dimethoxyaniline and potassium carbonate were added to an acetonitrile solution. A 50 wt% aqueous solution of H₂O₂ was added to the reaction system and stirred at room temperature for 10 minutes. After completion of the reaction, the mixture was extracted three times with ethyl acetate and washed with saturated brine. The mixture was dried over anhydrous Na₂SO₄. The solvent was evaporated under vacuum. The mixture was purified by column chromatography to obtain 1,3-dimethoxy-5-nitrobenzene, Compound 5.

[0056] (6) Preparation of Compound 6

[0057]

[0058] Compound 5 was added to an excess of 48 wt% aqueous hydrobromic acid and stirred under reflux at 110°C for 12 h. After the reaction, the mixture was extracted three times with ethyl acetate and washed with saturated brine. The mixture was dried over anhydrous Na2SO4. The solvent was evaporated under vacuum. The mixture was purified by column chromatography using petroleum ether / ethyl acetate. 5-nitro-1,3-benzenediol was obtained as compound 6.

[0059] (7) Preparation of Compound C

[0060]

[0061] Potassium carbonate and compound 6 were added to DMF and stirred at room temperature for 0.5 h. Compound 4 was added to the mixed system, and the mixture was refluxed and stirred at 110° C. for 6 h under nitrogen protection and light protection. The solvent was removed under reduced pressure, and the mixture was purified by HPLC to obtain compound C, wherein, in formula (C), R1 is selected from any one of H group, carboxyl group, and sulfo group; R2 is an alkyl chain with 1 to 5 carbon atoms; R3 is selected from any one of H group, hydroxyl group, amino group, carboxyl group, sulfo group, alkynyl group, and azido group; and X is selected from any one of Cl, Br, I, periodate group, and trifluoroacetate group.

[0062] The obtained compound C is the near-infrared fluorescent probe of the present invention, which has the advantages of near-infrared absorption, good water solubility, high stability, etc., and can effectively detect the activity of nitroreductase.

[0063] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0064] (1) The preparation method of the present invention is simple, easy to separate and purify, and has a high yield;

[0065] (2) The near-infrared fluorescent probe of the present invention has the advantages of near-infrared absorption, good water solubility, and high stability;

[0066] (3) The emission and absorption wavelengths of the near-infrared fluorescent probe molecules of the present invention are located in the near-infrared light region, which can effectively reduce the interference of background fluorescence;

[0067] (4) The near-infrared fluorescent probe molecules described in the present invention can effectively detect the activity of nitroreductase, and can be used as probes for detecting the hypoxia of tumor cells, as well as gene- and virus-directed prodrugs, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 is the nuclear magnetic resonance spectrum of the near-infrared fluorescent probe 1 in Example 1 of the present invention;

[0069] Figure 2 is the LCMS spectrum of the near-infrared fluorescent probe 1 in Example 1 of the present invention;

[0070] Figure 3 is the ultraviolet absorption spectrum of the near-infrared fluorescent probe 1 in Example 1 of the present invention;

[0071] Figure 4 This is the fluorescence emission spectrum of the near-infrared fluorescent probe 1 in the embodiment of the present invention;

[0072] Figure 5 This is the result of detecting the activity of nitroreductase in Escherichia coli in the application examples of the present invention. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0074] Example 1

[0075] The synthetic route of the near-infrared fluorescent probe molecule C1 is:

[0076]

[0077] (1) Synthesis of Compound 1

[0078] 4-Hydrazinobenzenesulfonic acid (2.5 g, 13.3 mmol) was dissolved in 15 mL of glacial acetic acid, and sodium acetate (2.18 g, 26.6 mmol) and 3-methyl-2-butanone (4.2 mL, 39.4 mmol) were added. The mixture was refluxed at 130°C for 5 h. After reflux, the mixture was cooled to room temperature and slowly added dropwise to 200 mL of methyl tert-butyl ether. A red precipitate was formed, which was filtered and washed with acetone to obtain a red solid, Compound 1.

[0079] (2) Synthesis of Compound 2

[0080] Compound 1 (1.22 g, 6.0 mmol) and 1,4-butane sultone (3.21 mL, 4.30 g, 35 mmol) were suspended in o-dichlorobenzene and refluxed at 120°C for 8 h. After the reaction was completed, the mixture was cooled to room temperature. The cooled reaction solution was dropwise added into methyl tert-butyl ether for precipitation. The mixture was filtered and washed with acetone to obtain compound 2.

[0081] (3) Synthesis of Compound 3

[0082] DMF (10 mL, 136.5 mmol) and dichloromethane (10 mL) were stirred evenly and cooled to 0°C. A mixed solution of phosphorus oxychloride (8.75 mL, 57.5 mmol) and 1.25 mL of dichloromethane was added dropwise at 0°C. After ice bathing for 30 min, cyclohexanone (2.64 mL, 25 mmol) was slowly added dropwise to the above solution. The mixture was refluxed at 80°C for 6 h. After the reaction, the reaction solution was diluted with DCM and poured into ice water and allowed to stand overnight. The mixture was filtered and washed with water to obtain a yellow solid, which was compound 3.

[0083] (4) Synthesis of Compound 4

[0084] Compound 3 (0.5 mmol, 86 mg) and sodium acetate (0.5 mmol, 41 mg) were suspended in a mixture of 10 mL of methanol and 10 mL of acetic anhydride, and then compound 2 (1 mmol, 376 mg) was slowly added to the above solution. The reaction system was heated to 80°C and refluxed with stirring for 3 h. After the reaction was completed, it was cooled to room temperature and then tert-butyl methyl was added dropwise to produce a green precipitate. The tert-butyl methyl ether was removed by centrifugation to obtain a green solid, which was compound 4.

[0085] (5) Synthesis of Compound 5

[0086] 3,5-Dimethoxyaniline (10 mmol, 1.53 g) and potassium carbonate (10 mmol, 1.37 g) were added to 10 mL of acetonitrile solution. A 50 wt% aqueous solution of H2O2 (30.0 mmol) was slowly added to the reaction system under ice-bath conditions and stirred for 10 minutes. After completion of the reaction, the mixture was extracted three times with ethyl acetate and washed with saturated brine. The mixture was dried over anhydrous Na2SO4. The solvent was evaporated under vacuum. The mixture was purified by column chromatography to obtain 1,3-dimethoxy-5-nitrobenzene, which is Compound 5.

[0087] (6) Synthesis of Compound 6

[0088] Compound 5 (1 mmol, 183 mg) was added to an excess of 48 wt% aqueous hydrobromic acid (8 mL, 70 mmol) and stirred under reflux at 110°C for 12 h. After the reaction, the mixture was extracted three times with ethyl acetate and washed with saturated brine. Drying was performed over anhydrous Na2SO4, and the solvent was evaporated under vacuum. The mixture was purified by column chromatography using petroleum ether / ethyl acetate to obtain 5-nitro-1,3-benzenediol, which is compound 6.

[0089] (7) Synthesis of Compound C1

[0090] Compound 6 (0.425 mmol, 66 mg) and potassium carbonate (0.425 mmol, 56 mg) were added to 5 mL of DMF and stirred at room temperature under nitrogen for 0.5 h. Compound 4 (0.17 mmol, 150 mg) was added to the mixture and stirred under reflux at 50°C for 6 h under nitrogen and light protection. After the reaction, the mixture was purified by HPLC to obtain compound C1, which was characterized. Figure 1 、 2 As shown, the chemical structure of the chemical C1 is:

[0091]

[0092] Example 2

[0093] The synthetic route of the near-infrared fluorescent probe molecule C2 is:

[0094]

[0095] (1) Synthesis of Compound 1

[0096] 4-Hydrazinobenzenesulfonic acid (2.5 g, 13.3 mmol) was dissolved in 15 mL of glacial acetic acid, and sodium acetate (2.18 g, 26.6 mmol) and 3-methyl-2-butanone (4.2 mL, 39.4 mmol) were added. The mixture was refluxed at 130°C for 5 h. After reflux, the mixture was cooled to room temperature and slowly added dropwise to 200 mL of methyl tert-butyl ether. A red precipitate was formed, which was filtered and washed with acetone to obtain a red solid, Compound 1.

[0097] (2) Synthesis of Compound 2

[0098] Compound 1 (1.22 g, 6.0 mmol) and 5-bromovaleric acid (6.33 g, 35 mmol) were suspended in o-dichlorobenzene and refluxed at 120°C for 8 h. After the reaction was completed, the mixture was cooled to room temperature. The cooled reaction solution was dropwise added into methyl tert-butyl ether for precipitation. The mixture was filtered and washed with acetone to obtain compound 2.

[0099] (3) Synthesis of Compound 3

[0100] DMF (10 mL, 136.5 mmol) and dichloromethane (10 mL) were stirred evenly and cooled to 0°C. A mixed solution of phosphorus oxychloride (8.75 mL, 57.5 mmol) and 1.25 mL of dichloromethane was added dropwise at 0°C. After ice bathing for 30 min, cyclohexanone (2.64 mL, 25 mmol) was slowly added dropwise to the above solution. The mixture was refluxed at 80°C for 6 h. After the reaction, the reaction solution was diluted with DCM and poured into ice water and allowed to stand overnight. The mixture was filtered and washed with water to obtain a yellow solid, which was compound 3.

[0101] (4) Synthesis of Compound 4

[0102] Compound 3 (0.5 mmol, 86 mg) and sodium acetate (0.5 mmol, 41 mg) were suspended in a mixture of 10 mL of methanol and 10 mL of acetic anhydride, and then compound 2 (1 mmol, 376 mg) was slowly added to the above solution. The reaction system was heated to 80°C and refluxed with stirring for 3 h. After the reaction was completed, it was cooled to room temperature and then tert-butyl methyl was added dropwise to produce a green precipitate. The tert-butyl methyl ether was removed by centrifugation to obtain a green solid, which was compound 4.

[0103] (5) Synthesis of Compound 5

[0104] 3,5-Dimethoxyaniline (10 mmol, 1.53 g) and potassium carbonate (10 mmol, 1.37 g) were added to 10 mL of acetonitrile solution. A 50 wt% aqueous solution of H2O2 (30.0 mmol) was slowly added to the reaction system under ice-cooling conditions and stirred for 10 minutes. After completion of the reaction, the mixture was extracted three times with ethyl acetate and washed with saturated brine. The mixture was dried over anhydrous Na2SO4. The solvent was evaporated under vacuum. The mixture was purified by column chromatography to obtain 1,3-dimethoxy-5-nitrobenzene, Compound 5.

[0105] (6) Synthesis of Compound 6

[0106] Compound 5 (1 mmol, 183 mg) was added to an excess of 48 wt% aqueous hydrobromic acid solution (8 mL, 70 mmol) and refluxed at 110°C with stirring for 12 h. After the reaction, the mixture was extracted three times with ethyl acetate, washed with saturated brine, and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum, and the mixture was purified by column chromatography using petroleum ether / ethyl acetate to obtain 5-nitro-1,3-diphenol, which is compound 6.

[0107] (7) Synthesis of Compound C2

[0108] Compound 6 (0.425 mmol, 66 mg) and potassium carbonate (0.425 mmol, 56 mg) were added to 5 mL of DMF and stirred at room temperature under nitrogen for 0.5 h. Compound 4 (0.17 mmol, 150 mg) was added to the mixture, and the mixture was refluxed at 50°C under nitrogen and light protection for 6 h. After the reaction, the mixture was purified by HPLC to obtain compound C2.

[0109]

[0110] C2 NMR data: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 11.42 (s, 1H), 8.51 (d, J = 15.32Hz, 2H), 7.95 (s, 1H), 7.77 (s, 2H), 7.59 (s, 1H), 7.38 (s, 1H), 7.2 4(s,1H),7.11(s,1H),6.71(d,J=15.26Hz,2H),4.48(s,2H),2.74(s,4H),2.58(s,2H),1.92(s,2H),1.83(s,4H),1.74(s,6H),. 13C NMR (100MHz, DMSO-d6): δ (ppm) 178.46, 173.24, 159.36, 156.04, 149.52, 149.44, 147.14, 147.12, 145.22, 142.24, 140.62, 126.42, 125 .34,124.64,122.62,111.12,106.94,106.12,105.62,104.54,54.74,52.42,33.72,30.84,29.92,27.42,26.64,24.74,24.74,22.92.

[0111] Example 3

[0112] The synthetic route of the near-infrared fluorescent probe molecule C3 is:

[0113]

[0114] (1) Synthesis of Compound 1

[0115] 4-Hydrazinobenzenesulfonic acid (2.5 g, 13.3 mmol) was dissolved in 15 mL of glacial acetic acid, and sodium acetate (2.18 g, 26.6 mmol) and 3-methyl-2-butanone (4.2 mL, 39.4 mmol) were added. The mixture was refluxed at 130°C for 5 h. After reflux, the mixture was cooled to room temperature and slowly added dropwise to 200 mL of methyl tert-butyl ether. A red precipitate was formed, which was filtered and washed with acetone to obtain a red solid, Compound 1.

[0116] (2) Synthesis of Compound 2

[0117] Compound 1 (1.22 g, 6.0 mmol) and 6-iodo-1-hexyne (7.28 g, 35 mmol) were suspended in o-dichlorobenzene and refluxed at 120°C for 8 h. After the reaction, the mixture was cooled to room temperature. The cooled reaction solution was dropwise added into methyl tert-butyl ether for precipitation. The mixture was filtered and washed with acetone to obtain compound 2.

[0118] (3) Synthesis of Compound 3

[0119] DMF (10 mL, 136.5 mmol) and dichloromethane (10 mL) were stirred evenly and cooled to 0°C. A mixed solution of phosphorus oxychloride (8.75 mL, 57.5 mmol) and 1.25 mL of dichloromethane was added dropwise at 0°C. After ice bathing for 30 min, cyclohexanone (2.64 mL, 25 mmol) was slowly added dropwise to the above solution. The mixture was refluxed at 80°C for 6 h. After the reaction, the reaction solution was diluted with DCM and poured into ice water and allowed to stand overnight. The mixture was filtered and washed with water to obtain a yellow solid, which was compound 3.

[0120] (4) Synthesis of Compound 4

[0121] Compound 3 (0.5 mmol, 86 mg) and sodium acetate (0.5 mmol, 41 mg) were suspended in a mixture of 10 mL of methanol and 10 mL of acetic anhydride, and then compound 2 (1 mmol, 376 mg) was slowly added to the above solution. The reaction system was heated to 80°C and refluxed with stirring for 3 h. After the reaction was completed, it was cooled to room temperature and then tert-butyl methyl was added dropwise to produce a green precipitate. The tert-butyl methyl ether was removed by centrifugation to obtain a green solid, which was compound 4.

[0122] (5) Synthesis of Compound 5

[0123] 3,5-Dimethoxyaniline (10 mmol, 1.53 g) and potassium carbonate (10 mmol, 1.37 g) were added to 10 mL of acetonitrile solution. A 50 wt% aqueous solution of H₂O₂ (30.0 mmol) was slowly added to the reaction system under ice-cooling and stirred for 10 minutes. After completion of the reaction, the mixture was extracted three times with ethyl acetate, washed with saturated brine, and dried over anhydrous Na₂SO₄. The solvent was evaporated under vacuum, and the mixture was purified by column chromatography to obtain 1,3-dimethoxy-5-nitrobenzene, Compound 5.

[0124] (6) Synthesis of Compound 6

[0125] Compound 5 (1 mmol, 183 mg) was added to an excess of 48 wt% aqueous hydrobromic acid (8 mL, 70 mmol) and stirred at reflux at 110°C for 12 h. After the reaction, the mixture was extracted three times with ethyl acetate and washed with saturated brine. The mixture was dried over anhydrous Na2SO4. The solvent was evaporated under vacuum. The mixture was purified by column chromatography using petroleum ether / ethyl acetate. 5-nitro-1,3-benzenediol was obtained as compound 6.

[0126] (7) Synthesis of Compound C3

[0127] Compound 6 (0.425 mmol, 66 mg) and potassium carbonate (0.425 mmol, 56 mg) were added to 5 mL of DMF and stirred at room temperature under nitrogen for 0.5 h. Compound 4 (0.17 mmol, 150 mg) was added to the mixture, and the mixture was refluxed at 50°C under nitrogen and light protection for 6 h. After the reaction, the mixture was purified by HPLC to obtain compound C3.

[0128]

[0129] C2 NMR data: 1H NMR (400MHz, DMSO-d6): δ (ppm) 11.44 (s, 1H), 8.71 (d, J = 15.42Hz, 2H), 7.85 (s, 1H), 7.72 (s, 2H), 7.62 (s, 1H), 7.34 (s, 1H), 7.18 (s, 1 H),7.12(s,1H),6.51(d,J=15.12Hz,2H),4.28(s,2H),2.76(s,4H),2.54(s,2H),1.96(s,2H),1.89(s,1H),1.83(s,4H),1.64(s,6H). 13 C NMR (100MHz, DMSO-d6): δ (ppm) 176.46, 172.44, 160.32, 157.14, 150.42, 148.84, 147.14, 147.12, 144.62, 142.44, 141.42, 126.82, 125.54 ,123.44,122.22,111.32,106.64,106.02,105.32,104.64,104.24,6 8.74,51.32,32.82,30.64,29.72,27.62,25.84,24.44,24.44,22.12.

[0130] Examples 4 to 10

[0131] The embodiment of the present invention is basically the same as embodiment 1, except for the selection in step (2) and the resulting product, as shown in Table 1 below:

[0132] Table 1

[0133]

[0134]

[0135] Effect Example Photophysical Property Test

[0136] Compound C1 synthesized in Example 1 was prepared into a mother solution with a concentration of 1 mM using ultrapure water. 20 μL of the mother solution was taken out and diluted 100 times with 1800 μL of ultrapure water. The solution was then poured into a quartz cuvette and placed in a UV absorption spectrometer for testing. The maximum absorption peak of compound 7 in water was 696 nm. Figure 3 Then take out 20 μL of the above mother solution and add it to 1800 μL 1× PBS buffer solution to dilute it 100 times, then pour it into a quartz cuvette and put it into the fluorescence spectrometer for testing, as shown. Figure 4 As shown, compound 7 was found to have no fluorescence.

[0137] Application Example Detection of Nitroreductase Activity in Escherichia coli

[0138] 0.5 mL of overnight cultured E. coli was taken out and diluted 20-fold with 9.5 mL of fresh LB medium and cultured for another 2 h. The E. coli was separated by centrifugation and then resuspended in 50 mM Tris-HCl (pH 7.5) for lysis. Protein and nitroreductase were separated by centrifugation. Compound 7 was added to the lysate at a final concentration of 25 μM and NADH at a final concentration of 50 μM. The mixture was protected from light and incubated at 37°C for 1.5 h. After the reaction, the mixture was diluted twice and poured into a quartz cuvette. The maximum emission wavelength was measured at 723 nm by a fluorescence spectrometer. Figure 5 shown.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe, characterized in that The chemical structure of the probe is shown in the following formula (C): In formula (C), R1 is selected from any one of a carboxyl group and a sulfonic acid group; R2 is an alkyl chain having 1 to 5 carbon atoms; R3 is selected from any one of a hydroxyl group, an amino group, a carboxyl group, a sulfonic acid group, an alkynyl group, and an azide group; and X is selected from any one of Cl, Br, I, periodate, and trifluoroacetate.

2. The near-infrared fluorescent probe molecule according to claim 1, wherein In formula (C): R1 is a sulfonic acid group, R2 is -(CH2)4-, and R3 is a sulfonic acid group; Or R1 is a sulfonic acid group, R2 is -(CH2)4-, and R3 is a carboxyl group Or R1 is a sulfonic acid group, R2 is -(CH2)4-, and R3 is an amino group Or R1 is a sulfonic acid group, R2 is -(CH2)4-, and R3 is an azide group, Or R1 is a carboxyl group, R2 is -(CH2)4-, and R3 is a sulfonic acid group, Or R1 is a carboxyl group, R2 is -(CH2)4-, R3 is a carboxyl group, Or R1 is a carboxyl group, R2 is -(CH2)4-, and R3 is an amino group; or R1 is a carboxyl group, R2 is -(CH2)4-, and R3 is an azide group.

3. The method for preparing the near-infrared fluorescent probe according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) A hydrazine benzene compound was dissolved in glacial acetic acid, sodium acetate and 3-methyl-2-butanone were added, and the mixture was refluxed at 130°C for 5 hours. Compound I of the following structural formula was precipitated from the reflux solution using methyl tert-butyl ether; (2) A mixture of Compound I and a halide, or Compound I and 1,3-propane sultone, or Compound I and 1,4-butane sultone is refluxed in toluene or o-dichlorobenzene at a temperature of 110-120°C. After the reaction is completed, the mixture is cooled to room temperature. The cooled reaction solution is dropwise added with ether for precipitation, and the ether is filtered off to obtain Compound II with the following structural formula: (3) The mixed system of DCM and DMF was cooled to 0°C, phosphorus oxychloride was added dropwise to the mixed system, and the mixture was stirred in an ice bath for 0.5 h. Cyclohexanone was slowly added to the system, and the mixture was refluxed and stirred at 80°C for 6 h. After the reaction was completed, the reaction solution was cooled to room temperature, and ice water was added dropwise and allowed to stand overnight. The mixture was filtered and washed with cold water to obtain compound III with the following structural formula: (4) Under a nitrogen atmosphere, the compound II, compound III and sodium acetate were slowly added to the acetic anhydride solution, and the reaction system was heated to 80-110°C and refluxed with stirring for 3 h. After the reaction was completed, the mixture was cooled to room temperature and then ether was added dropwise for precipitation to obtain compound IV with the following structural formula: (5) Potassium carbonate and 5-nitro-1,3-diphenol were added to DMF and stirred at room temperature for 0.5 h. Compound IV was added to the mixture, and the mixture was refluxed at 110°C for 6 h under nitrogen and light protection. The solvent was removed under reduced pressure and the mixture was purified by HPLC to obtain Compound C with the following structural formula: 。 4. The method according to claim 3, wherein In step (2), the hydrocarbon group of the halide is selected from any one of an alkyl chain with 3 to 5 carbon atoms, an alkynyl chain with 3 to 5 carbon atoms, an amino chain with 3 to 5 carbon atoms, a sulfonic acid chain with 4 carbon atoms, and a carboxylic acid chain with 4 carbon atoms; The halogen of the halide is selected from any one of Cl, Br and I.

5. The method according to claim 4, wherein The halide is selected from chloropropane, chlorobutane, chloropentane, bromopropane, bromobutane, bromopentane, iodopropane, iodobutane, iodopentane, 4-chloro-1-butyne, 5-chloro-1-pentyne, 6-chloro-1-hexyne, 4-bromo-1-butyne, 5-bromo-1-pentyne, 6-bromo-1-hexyne, 4-iodo-1-butyne, 5-iodo-1-pentyne, 6-iodo-1-hexyne, 2-chloroethylamine , 3-chloropropylamine, 4-chlorobutylamine, 2-bromoethylamine, 3-bromopropylamine, 4-bromobutylamine, 2-iodoethylamine, 3-iodopropylamine, 4-iodobutylamine, 2-chloroethanesulfonic acid, 2-bromoethanesulfonic acid, 2-iodoethanesulfonic acid, 3-chloropropionic acid, 4-chlorobutyric acid, 5-chlorovaleric acid, 3-bromopropionic acid, 4-bromobutyric acid, 5-bromovaleric acid, 3-iodopropionic acid, 4-iodobutyric acid and 5-iodovaleric acid.

6. The method according to claim 3, wherein In step (5), the preparation process of 5-nitro-1,3-benzenediol is as follows: (5-1) 3,5-Dimethoxyaniline and potassium carbonate were added to an acetonitrile solution, and a 50 wt % H2O2 aqueous solution was added to the reaction system and stirred at room temperature for 10 minutes. The reaction product was extracted, washed, dried, desolvated, and purified to obtain 1,3-dimethoxy-5-nitrobenzene, which is compound V. (5-2) Compound V was added to an excess of 48 wt % aqueous hydrobromic acid solution and stirred under reflux at 110° C. for 12 h. The reaction product was extracted, washed, dried, desolvated, and purified to obtain 5-nitro-1,3-diphenol.

7. The method according to claim 6, wherein In steps (5-1) and (5-2), the extraction is to extract the mixture three times with ethyl acetate, the washing is to wash with saturated brine, the drying is to dry with anhydrous Na2SO4, and the solvent removal is to evaporate the solvent under vacuum; and the purification is to purify the residue of the mixture by column chromatography.

8. Use of the near-infrared fluorescent probe according to claim 1 in detecting nitroreductase activity.

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