A near-infrared small molecule dye and its preparation method and application

Abstract

The present invention belongs to the field of biomaterial technology, specifically a near-infrared small molecule dye and its preparation method and application. The near-infrared small molecule dye of the present invention has a structure shown in general formula A, and its absorption and emission wavelengths are in the near-infrared region of 700-1700nm, with a small molecular weight, and has the characteristics of good water solubility, excellent photostability, good cell permeability, simple synthesis and easy structural modification. It can be widely used in biomedical fields such as cell labeling and imaging, fluorescent surgical navigation, multispectral imaging, and photoacoustic imaging; some structures in the small molecule dye can be used as activatable small molecule optical probes for photoacoustic detection of peroxynitrite ions;

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, in particular to the field of biomedical imaging detection, and specifically relates to a near-infrared small molecule dye and a preparation method and application thereof. Background Art

[0002] Near-infrared dyes are a class of compounds that have the ability to absorb and emit photons in the near-infrared spectral range, and have broad application potential in medical diagnosis, bioimaging, materials science and industry. Near-infrared absorbing dyes can be used for photodynamic or photothermal treatment of various skin diseases, tumors and other diseases, and can also be used as photoacoustic contrast agents for disease diagnosis; near-infrared emitting dyes can be used as fluorescent contrast agents for surgical navigation to image the edges of tissue structures or disease lesions and assist surgeons in surgical resection. In recent years, photons with wavelengths in the range of 700 to 1700 nanometers have been found to have better biological tissue penetration performance, and therefore the demand for near-infrared dyes that absorb and emit in this band in the biomedical field is increasing.

[0003] At present, the near-infrared dyes in the field are mainly developed around traditional mother bodies such as cyanine, fluoroboryl pyrrole, squaryl cyanine, porphyrin, phthalocyanine, coumarin and rhodamine. In order to extend the wavelength of the mother body to the near-infrared region, the current general strategy requires expanding the molecular structure by means of extending conjugation and fusing heteroatoms, which will lead to a decrease in the water solubility, biocompatibility and photostability of the dye. At the same time, larger and more complex structures will also make synthesis difficult and difficult to meet the needs of large-scale preparation.

[0004] Therefore, it is necessary to find new near-infrared dye precursors and explore suitable synthesis methods to develop near-infrared dyes with good solubility and photostability, small molecular structure, and long absorption and emission wavelengths. This will help promote the application of near-infrared photon technology in medicine, life sciences and other fields, and provide new solutions for fields such as diagnosis and treatment. Summary of the invention

[0005] The purpose of the present invention is to provide a class of functional dye molecules with small molecular weight, good photostability and absorption and emission peak wavelengths in the near-infrared region, as well as a preparation method and application thereof.

[0006] The near-infrared small molecule dye provided by the present invention is a new dye matrix based on a diaminofluorene cationic structure, and is a functional dye molecule with a small molecular weight and absorption and emission peak wavelengths in the near-infrared region obtained by regulating the substituents on the matrix structure.

[0007] The present invention first proposes a new strategy to construct a near-infrared dye matrix and its functionalized molecular system. Specifically:

[0008] First, the conjugated structure of the traditional dye system is constructed based on the Hückel aromaticity rule, but the present invention abandons this common practice and constructs a new anti-aromatic chromophore core based on the fluorene cation skeleton;

[0009] Secondly, by introducing amine substituents with different electron-donating abilities on the fluorene cationic skeleton and introducing a large sterically hindered aromatic ring structure at position 9 of the fluorene ring, a series of diaminofluorene cationic dyes with different absorption and emission wavelengths and excellent chemical stability were synthesized;

[0010] Finally, by performing functional modification on the dye molecule, a dye molecule with fluorescent labeling or photoacoustic sensing function is obtained.

[0011] The anti-aromatic diaminofluorene cation series dyes obtained by the present invention have ideal near-infrared absorption and emission properties in water and organic solvents, show excellent photostability and chemical stability, have a molecular weight smaller than other series of dye molecules with similar properties, are easy to chemically modify, and show excellent biocompatibility. Accordingly, the present invention demonstrates the anti-bleaching performance of this type of dye in long-term cell staining fluorescence imaging, as well as the function of a fluorescent contrast agent for liver and gallbladder metabolism and kidney metabolism clearance pathways, and also obtains a new type of activatable small molecule photoacoustic probe through functionalization, and demonstrates the photoacoustic detection effect of peroxynitrite cations.

[0012] The near-infrared small molecule dye provided by the present invention has a general structural formula as shown below, or a salt thereof, or an enantiomer, diastereomer, tautomer or solvate thereof:

[0013]

[0014] Where:

[0015] X - For anions, including ClO 4 - PF 6 - , BF 4 - , Cl - Br - ,I - , CH 3 COO - CF 3 COO - CF 3 SO 3 - and CH 3 SO 3 - ;

[0016] R 1, R 2 , R 3 , R 4 Each is independently selected from H, Cl-C6 alkyl, substituted aryl and substituted acyl;

[0017] R 5 , R 6 Each is independently selected from H, C1-C6 alkyl, C1-C6 perfluoroalkyl and C1-C6 alkoxy;

[0018] R 7 Independently selected from H, C1-C6 alkyl, and any one of the groups represented by formula I-IV:

[0019]

[0020] The curve marks the substitution position;

[0021] The near-infrared small molecule dye has a maximum absorption wavelength between 800-1150 nm, a maximum emission wavelength between 900-1250 nm, and a molecular weight between 299-603 Da.

[0022] In some embodiments, as shown in Formula A, wherein R 1 and R 2 Can form an optionally substituted n-membered nitrogen-containing heterocyclic ring with the nitrogen to which they are connected, wherein n is an integer of 4-7, and the n-membered nitrogen-containing heterocyclic ring includes azocyclobutane, pyrrolidinyl, piperidinyl, cycloheximino or indolinyl;

[0023] The R 3 and R 4 Can form an optionally substituted n-membered nitrogen-containing heterocyclic ring with the nitrogen to which they are connected, wherein n is an integer of 4-7, and the n-membered nitrogen-containing heterocyclic ring includes azocyclobutane, pyrrolidinyl, piperidinyl, cycloheximino or indolinyl;

[0024] Among them, preferably, the n-membered nitrogen-containing heterocyclic ring contains a ring oxygen atom, and more preferably, the n-membered nitrogen-containing heterocyclic ring is a morpholinyl group.

[0025] In some embodiments, the typical compound structure of Formula A is shown in Formula B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12:

[0026]

[0027] In formula B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 or B12, R 8 , R 9 , R 10 , R 11 , R12 , R 13 , R 14 , R 15 , R 16 , R 17 and R 18 Each is independently selected from H, optionally substituted C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, carboxyl, halogen and sulfonic acid.

[0028] in,

[0029] In the formula B5, R 8 and R 9 Each is independently H or C1-C6 alkyl;

[0030] In the formula B10 and the formula B11, R 10 , R 11 , R 12 , R 13 and R 14 Each is independently selected from H, optionally substituted C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, carboxyl, halogen and sulfonic acid;

[0031] In the formula B12, R 15 , R 16 , R 17 and R 18 Each is independently selected from H, optionally substituted C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, carboxyl, halogen and sulfonic acid;

[0032] On the other hand, the present invention synthesizes a near-infrared small molecule dye based on the parent structure of general formula A, and its structure is shown in the following formulas C1 and C2:

[0033]

[0034] In formula C1 and C2, R 19 Independently selected from any one of the groups shown in formula V-VIII:

[0035]

[0036] The near-infrared small molecule dye can be used as an activatable small molecule optical probe, and the activatable small molecule optical probe will produce a change in light signal before and after activation.

[0037] In other embodiments, the structures of the near-infrared small molecule dyes provided by the present invention are shown in the following formulas D1-D18:

[0038]

[0039] In some specific embodiments, the near-infrared small molecule dye provided by the present invention has a maximum absorption wavelength between 800-1150 nm and a maximum emission wavelength between 900-1250 nm. In other specific embodiments, the near-infrared small molecule dye provided by the present invention has a molecular weight between 299-603 Da.

[0040] Another object of the present invention is to provide a general and convenient preparation method of the compound of formula A, comprising the following steps:

[0041] Step 1: The compound represented by formula (1) and an amino compound are subjected to a Buchwald-Hartwig carbon-nitrogen coupling reaction to obtain a compound represented by formula (2);

[0042] Step 2: The compound represented by formula (2) is subjected to a nucleophilic addition reaction under the action of a phenyllithium reagent or a phenyl Grignard reagent, and then subjected to acid catalysis to obtain a compound represented by general formula A;

[0043]

[0044] In the step 1, the compound represented by formula (1) and the amino compound are subjected to a Buchwald-Hartwig carbon-nitrogen coupling reaction to obtain a compound represented by formula (2), comprising:

[0045] In an organic solvent environment, using a palladium catalyst, a phosphine ligand and a base as catalysts, under the protection of an inert gas, heating and stirring the compound represented by formula (1) and an organic amine compound to obtain a compound represented by formula (2);

[0046] Wherein, the organic amine compound is selected from one or more of the formulas IX-XX:

[0047]

[0048] in,

[0049] R 8 and R 9 Each is independently H or C1-C6 alkyl;

[0050] R 10 , R 11 , R 12 , R 13 and R 14 Each is independently selected from H, optionally substituted C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, carboxyl, halogen and sulfonic acid;

[0051] R 15 , R 16 , R 17 and R 18Each is independently selected from H, optionally substituted C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, carboxyl, halogen and sulfonic acid;

[0052] The organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ether, and 1,4-dioxane;

[0053] The palladium catalyst is selected from one or more of tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetonepalladium), and palladium acetate;

[0054] The base is selected from one or more of potassium tert-butoxide, sodium tert-butoxide, potassium carbonate, cesium carbonate, and potassium phosphate;

[0055] The phosphine ligand is selected from one or more of tri-tert-butylphosphine, triphenylphosphine and tri-n-butylphosphine.

[0056] In the step 2, the compound represented by formula (2) is subjected to a nucleophilic addition reaction under the action of a phenyllithium reagent or a phenyl Grignard reagent, and then acid-catalyzed to obtain a compound represented by general formula A, comprising:

[0057] The compound represented by formula (2) is reacted under the action of a phenyllithium reagent or a phenyl Grignard reagent, with ether as a reaction solvent, through a nucleophilic addition reaction, catalyzed by an acid catalyst, and reacted at room temperature to obtain a compound represented by general formula A;

[0058] Wherein, the phenyllithium reagent is as shown in Formula XXI:

[0059]

[0060] R 5 , R 6 Each is independently selected from H, C1-C6 alkyl, C1-C6 perfluoroalkyl and C1-C6 alkoxy;

[0061] R 7 Independently selected from H, C1-C6 alkyl, and any one of the groups represented by formula I-IV:

[0062]

[0063] The curve marks the substitution position;

[0064] The phenyl Grignard reagent is a Grignard reagent corresponding to the phenyllithium reagent;

[0065] The ether solvent is tetrahydrofuran or diethyl ether;

[0066] The acid catalyst is perchloric acid or trifluoroacetic acid.

[0067] In addition, the present invention also proposes an application of near-infrared multi-spectral fluorescence imaging based on general formula A.

[0068] Wherein, the near-infrared multi-spectral fluorescence imaging includes near-infrared in vivo multi-spectral fluorescence imaging.

[0069] Specifically, the near-infrared in vivo multi-spectral fluorescence imaging also includes the application of multi-channel fluorescence imaging of small animal in vivo organs, and more specifically, includes the application of at least two of Formulas D1-D18 in multi-channel fluorescence imaging of small animal in vivo organs.

[0070] Furthermore, the present invention also proposes a method for photoacoustic detection of peroxynitrite (ONOO) based on small molecule probes C1 and C2. - ) applications in ions.

[0071] The functional dye molecules provided by the present invention have absorption and emission wavelengths in the near-infrared region of 700-1700nm, small molecular weight, good water solubility, excellent photostability, good cell permeability, simple synthesis, easy structural modification, etc., and can be widely used in biomedical fields such as cell labeling and imaging, fluorescent surgical navigation, multispectral imaging, photoacoustic imaging, etc.; some structures in the small molecule dye can be used as activatable small molecule optical probes for photoacoustic detection of peroxynitrite ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Absorption and emission spectra of antiaromatic aminofluorene cationic dyes 1a-14a (see Examples 1-15 for details) in dichloromethane.

[0073] Figure 2 The activatable small molecule probe 18a (see Example 19 for details) is used to activate peroxynitrite (ONOO - ) The changes in the absorption and emission spectra when ions respond.

[0074] Figure 3 Endothelial (EC) cells were stained with dye 3a (see Examples 3 and 20) and commercial near-infrared dye Cy7-Cl as a control, and then excited with 808 nm laser. 2×10 4 mW / mm 2 Comparison of cell fluorescence imaging images and image signal-to-noise ratio collected before and after 1000 frames of continuous shooting at high power density and 100ms exposure time.

[0075] Figure 4 These are the results of near-infrared multispectral fluorescence imaging of multiple organs in mice using dyes 2a and 15a (see Examples 2, 16, and 21 for details).

[0076] Figure 5The activatable small molecule probe 18a (see Examples 19 and 22 for details) was used to detect the peroxynitrite (ONOO) in a mouse brain injury model. - ) ions’ photoacoustic imaging detection results. DETAILED DESCRIPTION

[0077] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described with the following specific embodiments, but the present invention is by no means limited to these examples. The following description is only a preferred embodiment of the present invention, which is only used to explain the present invention and cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that any modification, substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0078] Embodiment 1:

[0079] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 1a, the compound structure and specific synthesis route are as follows:

[0080]

[0081] The specific steps are as follows:

[0082] (1) Synthesis of intermediate 1

[0083] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), tert-butyl carbamate, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, diluted with dichloromethane after cooling, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 1. 1 H NMR (400 MHz, CDCl 3 )δ7.72(d,J=1.5Hz,2H),7.53(d,J=8.1Hz,2H),7.11(dd,J=8.1,1.9Hz,2H),6.82(s,2H),1.54(s,18H); 13 CNMR (101MHz, CDCl 3 )δ152.24(C),145.62(C),144.42(C),129.77(C),125.23(C),117.75(C),110.31(C),28.42(CH3);

[0084] (2) Synthesis of dye 1a

[0085] The method is anhydrous and oxygen-free operation. 2,6-dimethylbromobenzene is dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium in n-hexane solution is added dropwise, and stirring is continued for 30 minutes. An anhydrous tetrahydrofuran solution of intermediate 1 is added, and the mixture is warmed to room temperature and stirred for 30 minutes. Then, excess water is added to quench the mixture, and the mixture is extracted with dichloromethane, washed with saturated brine three times, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain an oily substance. A mixed solvent of trifluoroacetic acid and dichloromethane (volume ratio of 1:1) is added, and the mixture is stirred at room temperature for 3 hours. The solvent is removed by rotary evaporation, and the mixture is separated by column chromatography (dichloromethane: methanol = 50:1) to obtain dye 1a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 1 is 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ8.68(s,4H),7.33–7.18(m,3H),6.95(s,2H),6.59(d,J=8.4Hz,2H),6.18(d,J=8.4Hz,2H),2.16(s,6H); 13 C NMR (101 MHz, CDCl 3 )δ172.07(C),160.87(C),147.57(C),135.41(C),134.64(C),131.10(C),129.86 (C),128.87(C),128.37(C),114.44(C),114.15(C),20.31(CH3); HRMS(ESI)calcd for C 21 H 19 N 2 [M] + 299.1543, found 299.1694.

[0086] Embodiment 2:

[0087] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 2a, the compound structure and specific synthesis route are as follows:

[0088]

[0089] The specific steps are as follows:

[0090] (1) Synthesis of intermediate 2

[0091] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), tert-butyl methylcarbamate, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, diluted with dichloromethane after cooling, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 2. 1 H NMR (400 MHz, CDCl 3 )δ7.60(d,J=8.0Hz,2H),7.47(d,J=1.7Hz,2H),7.15(dd,J=8.0,1.9Hz,2H),3.32(s,6H),1.50(s,18H); 13 C NMR (101 MHz, CDCl 3 )δ154.23(C),149.79(C),144.66(C),131.24(C),124.97(C),124.64(C),117.18(C),81.42(C),37.20(CH3),28.46(CH3);

[0092] (2) Synthesis of dye 2a

[0093] The method is anhydrous and oxygen-free operation. 2,6-dimethylbromobenzene is dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium in n-hexane solution is added dropwise, and stirring is continued for 30 minutes. An anhydrous tetrahydrofuran solution of intermediate 2 is added, and the mixture is warmed to room temperature and stirred for 30 minutes. Then, excess water is added to quench the mixture, and the mixture is extracted with dichloromethane, washed with saturated brine three times, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain an oily substance. A mixed solvent of trifluoroacetic acid and dichloromethane (volume ratio of 1:1) is added, and the mixture is stirred at room temperature for 3 hours. The solvent is removed by rotary evaporation, and the mixture is separated by column chromatography (dichloromethane: methanol = 50:1) to obtain dye 2a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 2 is 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ9.11(s,2H),7.26–7.21(m,1H),7.19(d,J=1.2Hz,2H),7.11(d,J=7.6Hz,2H) ,6.61(d,J=8.7Hz,2H),6.06(dd,J=8.7,1.3Hz,2H),3.09(s,6H),2.17(s,6H); 13 C NMR (101 MHz, CDCl 3)δ171.59(C),159.38(C),146.59(C),135.65(C),133.93(C),131.10(C),129.95(C),12 9.32(C),127.88(C),114.50(C),109.90(C),30.98(CH3),20.29(CH3); HRMS(ESI)calcd for C 23 H 23 N 2 [M] + 327.1856, found 327.1960.

[0094] Embodiment 3:

[0095] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 3a, the compound structure and specific synthesis route are as follows:

[0096]

[0097] The specific steps are as follows:

[0098] (1) Synthesis of intermediate 3

[0099] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), a tetrahydrofuran solution of dimethylamine, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, cooled and diluted with dichloromethane, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 3. 1 H NMR (400 MHz, CDCl 3 )δ7.48(d,J=8.4Hz,2H),6.75(d,J=2.2Hz,2H),6.43(dd,J=8.4,2.3Hz,2H),3.09(s,12H); 13 C NMR (101 MHz, CDCl 3 )δ191.76(C),154.59(C),146.19(C),125.34(C),124.47(C),110.38(C),103.19(C),40.72(CH3);

[0100] (2) Synthesis of dye 3a

[0101] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 3 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 3a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 3 was 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.67(d,J=2.1Hz,2H),7.24(d,J=7.5Hz,1H),7.13(d,J=7.6Hz,2H),6.5 8(d,J=8.9Hz,2H),6.08(dd,J=8.9,2.1Hz,2H),3.44(s,12H),2.22(s,6H); 13 C NMR (101 MHz, CDCl 3 )δ158.36(C),147.72(C),135.39(C),132.95(C),129.20(C),127.71(C),113.62(C),111.21(C),42.17(CH3),20.09(CH3); HRMS(ESI)calcd for C 25 H 27 N 2 [M] + 355.2169, found 355.2269.

[0102] Embodiment 4:

[0103] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 4a, the compound structure and specific synthesis route are as follows:

[0104]

[0105] The specific steps are as follows:

[0106] (1) Synthesis of intermediate 3

[0107] The synthesis of intermediate 3 is as described in Example 3;

[0108] (2) Synthesis of dye 4a

[0109] In anhydrous and oxygen-free operation, 2,6-dimethoxybromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 3 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 4a. The molar ratio of 2,6-dimethoxybromobenzene, n-butyl lithium, and intermediate 3 was 1:1:0.5. 1 HNMR (400MHz, DMSO) δ7.49(t,J=8.4Hz,1H),7.44(d,J=2.1Hz,2H),6.84(d,J=8.5Hz ,2H),6.78(d,J=9.0Hz,2H),6.33(dd,J=9.0,2.1Hz,2H),3.73(s,6H),3.34(s,12H); 13 C NMR(101MHz,DMSO)δ158.53(C),157.76(C),147.30(C),133.99(C),133.58(C),129. 92(C),112.23(C),112.14(C),105.49(C),56.67(CH3),42.06(CH3); HRMS(ESI)calcd forC 25 H 27 N 2 O 2 [M] + 387.2067, found 387.2234.

[0110] Embodiment 5:

[0111] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 5a, the compound structure and specific synthesis route are as follows:

[0112]

[0113] The specific steps are as follows:

[0114] (1) Synthesis of intermediate 4

[0115] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), diethylamine, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, diluted with dichloromethane after cooling, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 4. 1 H NMR (400 MHz, CDCl3 )δ7.48(d,J=8.4Hz,2H),6.72(d,J=2.3Hz,2H),6.43(dd,J=8.5,2.3Hz,2H),3.47(q,J=7.1Hz,8H),1.24(t,J=7.1Hz,12H); 13 C NMR (101 MHz, CDCl 3 )δ191.38(C),152.27(C),146.46(C),125.63(C),123.92(C),109.90(C),102.55(C),44.98(CH2),13.01(CH3);

[0116] (2) Synthesis of dye 5a

[0117] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 4 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 5a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 4 was 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.65(s,2H),7.27–7.22(m,1H),7.13(d,J=7.7Hz,2H),6.58(d,J=8.9Hz,2 H),6.09(s,2H),3.75(d,J=6.4Hz,8H),2.23(s,6H),1.37(t,J=7.1Hz,12H); 13 CNMR (101MHz, CDCl 3 )δ156.73(C),148.18(C),135.66(C),133.13(C),131.20(C),129.32(C),127.88( C),113.40(C),110.92(C),47.19(CH2),20.33(CH3),13.97(CH3); HRMS(ESI)calcd for C 29 H 35 N 2 [M] + 411.2795, found 411.2924.

[0118] Embodiment 6:

[0119] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 6a, the compound structure and specific synthesis route are as follows:

[0120]

[0121] The specific steps are as follows:

[0122] (1) Synthesis of Intermediate 5

[0123] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), azetidine, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, diluted with dichloromethane after cooling, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 5. 1 H NMR (400 MHz, CDCl 3 )δ7.45(d,J=8.0Hz,2H),6.39(s,2H),6.11(d,J=8.1Hz,2H),4.01(t,J=7.1Hz,9H),2.42(p,J=7.0Hz,5H); 13 C NMR (101 MHz, CDCl 3 )δ155.43(C),145.78(C),125.21(C),124.89(C),109.04(C),102.06(C),51.81(CH2),16.67(CH2);

[0124] (2) Synthesis of dye 6a

[0125] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 5 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 6a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 5 was 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.34(s,2H),7.23(d,J=7.4Hz,1H),7.12(d,J=7.6Hz,2H),6.55(d,J=8.6Hz,2 H),5.78(d,J=8.6Hz,2H),4.49(t,J=7.6Hz,8H),2.60–2.47(m,4H),2.19(s,6H);13 C NMR(101MHz,DMSO)δ156.66(C),146.28(C),135.50(C),132.48(C),129.81(C),128.91(C), 128.42(C),111.43(C),110.71(C),53.46(CH2),20.25(CH3),16.22(CH2); HRMS(ESI)calcd for C 27 H 27 N 2 [M] + 379.2169, found 379.2245.

[0126] Embodiment 7:

[0127] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 7a, the compound structure and specific synthesis route are as follows:

[0128]

[0129] The specific steps are as follows:

[0130] (1) Synthesis of intermediate 6

[0131] Under anhydrous and oxygen-free conditions, 3,6-dibromofluorenone (compound 1), 3,3-difluoroazetidine, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, cooled and diluted with dichloromethane, the insoluble matter was removed by filtration, and the solvent was removed by rotary evaporation to obtain a crude product, which was washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 6. 1 H NMR (400 MHz, CDCl 3 )δ7.50(d,J=8.1Hz,2H),6.46(d,J=1.9Hz,2H),6.23(dd,J=8.1,2.0Hz,2H),4.35(t,J=11.6Hz,8H); 13 C NMR (101 MHz, CDCl 3 )δ191.34(C),153.52(C),145.73(C),126.51(C),125.63(C),115.68(C),111.08(C),103.63(C),63.23(CH2);

[0132] (2) Synthesis of dye 7a

[0133] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 6 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 7a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 6 was 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.36(d,J=2.1Hz,2H),7.26(t,J=7.6Hz,1H),7.13(d,J=7.6Hz,2H),6.63( d,J=8.6Hz,2H),5.82(dd,J=8.6,2.1Hz,2H),4.87–4.68(m,8H),2.20(s,6H); 13 CNMR (101MHz, CDCl 3 )δ178.35(C),157.17(C),148.14(C),135.34(C),134.53(C),131.39(C),130.43(C),129.95( C),128.14(C),114.48(C),113.34(C),110.91(C),64.40(CH2),20.29(CH3); HRMS(ESI)calcd for C 27 H 23 F 4 N 2 [M] + 451.1792, found 451.2016.

[0134] Embodiment 8:

[0135] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 8a, the compound structure and specific synthesis route are as follows:

[0136]

[0137] The specific steps are as follows:

[0138] (1) Synthesis of intermediate 7

[0139] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), tetrahydropyrrole, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, diluted with dichloromethane after cooling, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 7. 1 H NMR (400 MHz, CDCl 3 )δ7.48(d,J=8.3Hz,2H),6.63(d,J=2.1Hz,2H),6.30(dd,J=8.3,2.2Hz,2H),3.48–3.36(m,8H),2.12–1.98(m,8H); 13 CNMR (101MHz, CDCl 3 )δ191.67(C),151.93(C),146.10(C),125.26(C),123.93(C),110.14(C),103.21(C),48.02(CH2),25.54(CH2);

[0140] (2) Synthesis of dye 8a

[0141] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 7 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 8a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 7 was 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.56(d,J=1.8Hz,2H),7.24(dd,J=8.1,7.1Hz,1H),7.12(d,J=7.7Hz,2H),6.55(d,J=8 .8Hz,2H),5.97(dd,J=8.8,1.7Hz,2H),3.81(s,8H),2.21(s,6H),2.09(t,J=6.6Hz,8H); 13 C NMR (101 MHz, CDCl 3)δ172.57(C),155.62(C),147.49(C),135.42(C),132.66(C),131.06(C),129.52(C),129.06( C),127.65(C),114.47(C),111.73(C),50.19(CH3),25.14(CH2),20.04(CH2); HRMS(ESI)calcd for C 29 H 31 N 2 [M] + 407.2482, found407.2552.

[0142] Embodiment 9:

[0143] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 9a, the compound structure and specific synthesis route are as follows:

[0144]

[0145] The specific steps are as follows:

[0146] (1) Synthesis of Intermediate 8

[0147] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), piperidine, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, cooled and diluted with dichloromethane, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 8. 1 H NMR (400 MHz, CDCl 3 )δ7.49(d,J=8.4Hz,2H),6.97(s,2H),6.65(dd,J=8.4,1.8Hz,2H),3.45–3.33(m,8H),1.75–1.61(m,12H); 13 C NMR (101 MHz, CDCl 3 )δ191.44(C),155.81(C),146.03(C),125.67(C),125.39(C),113.16(C),106.06(C),49.32(CH2),25.64(CH2),24.49(CH2);

[0148] (2) Synthesis of dye 9a

[0149] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 8 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 9a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 8 was 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.83(s,2H),7.28–7.22(m,1H),7.13(d,J=7.6Hz,2H),6.62(d,J=8.9Hz,2H),6.30(s,2H),3.86(s,8H),2.21(s,6H),1.89–1.73(m,12H); 13 C NMR (101 MHz, CDCl 3 )δ

[0150] 157.23(C),148.06(C),135.71(C),133.09(C),131.27(C),129.61(C),129.31(C),127.89(C) ,113.74(C),111.38(C),50.53(CH2),27.01(CH2),24.50(CH2),20.34(CH3); HRMS(ESI)calcd for C 31 H 35 N 2 [M] + 435.2795, found 435.3144.

[0151] Embodiment 10:

[0152] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 10a, the compound structure and specific synthesis route are as follows:

[0153]

[0154] The specific steps are as follows:

[0155] (1) Synthesis of Intermediate 9

[0156] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), hexamethyleneimine, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, diluted with dichloromethane after cooling, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 9. 1 H NMR (400 MHz, CDCl 3 )δ7.47(d,J=8.4Hz,2H),6.75(d,J=2.2Hz,2H),6.45(dd,J=8.5,2.3Hz,2H),3.64–3.50(m,8H),1.89–1.77(m,8H),1.62–1.52(m,8H); 13 C NMR (101 MHz, CDCl 3 )δ153.38(C),146.35(C),125.52(C),123.97(C),109.74(C),102.43(C),49.90(CH2),27.73(CH2),27.02(CH2);

[0157] (2) Synthesis of Dye 10a

[0158] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 9 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 10a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 9 was 1:1:0.5. 1 HNMR(400MHz,DMSO)δ7.63(d,J=2.2Hz,2H),7.32(dd,J=8.3,6.9Hz,1H),7.23(d,J=7.7Hz,2H),6.63(d, J=9.0Hz,2H),6.43(dd,J=9.1,2.2Hz,2H),3.87(t,J=5.5Hz,8H),2.17(s,6H),1.80(s,9H),1.55(s,8H); 13C NMR(101MHz,DMSO)δ170.44(C),157.22(C),147.57(C),135.51(C),133.26 (C),131.25(C),129.88(C),129.15(C),128.42(C),113.09(C),112.47(C),

[0159] 52.24(CH3),27.74(CH2),26.21(CH2),20.37(CH2); HRMS(ESI)calcd for C 33 H 39 N 2 [M] + 463.3108, found 463.3435.

[0160] Embodiment 11:

[0161] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 11a, the compound structure and specific synthesis route are as follows:

[0162]

[0163] The specific steps are as follows:

[0164] (1) Synthesis of Intermediate 9

[0165] The synthesis of intermediate 9 is described in Example 10;

[0166] (2) Synthesis of Dye 11a

[0167] In anhydrous and oxygen-free operation, 2,6-dimethoxybromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 9 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 11a. The molar ratio of 2,6-dimethoxybromobenzene, n-butyl lithium, and intermediate 9 was 1:1:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.43(d,J=2.2Hz,2H),7.39(t,J=8.4Hz,1H),6.72(d,J=9.0Hz,2H),6.65(d,J=8.5Hz,2H),6. 10(dd,J=9.0,2.2Hz,2H),3.91–3.72(m,8H),3.77(s,6H),1.95–1.84(m,8H),1.60–1.55(m,8H); 13C NMR (101 MHz, CDCl 3 )δ166.42(C),158.50(C),157.17(C),148.24(C),134.13(C),132.63(C),130.34(C),112.11(C),110.58(C),109.75(C),104.53(C),

[0168] 56.24(CH3),52.21(CH2),27.90(CH2),26.61(CH2); HRMS(ESI)calcd forC 33 H 39 N 2 O 2 [M] +

[0169] 495.3006, found 495.3227.

[0170] Embodiment 12:

[0171] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 12a, the compound structure and specific synthesis route are as follows:

[0172]

[0173] The specific steps are as follows:

[0174] (1) Synthesis of Intermediate 10

[0175] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), N-methylaniline, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, cooled and diluted with dichloromethane, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 10. 1 H NMR (400 MHz, CDCl 3 )δ7.46(d,J=8.3Hz,2H),7.43–7.37(m,4H),7.25–7.18(m,6H),6.83(d,J=2.1Hz,2H),6.57(dd,J=8.3,2.1Hz,2H),3.40(s,6H); 13 C NMR (101 MHz, CDCl 3)δ191.47(C),154.00(C),147.78(C),145.82(C),130.02(C),126.36(C) ,125.87(C),125.45(C),125.26(C),114.40(C),106.80(C),40.96(CH3);

[0176] (2) Synthesis of Dye 12a

[0177] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 10 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 12a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 10 was 1:1:0.5. 1 HNMR (400MHz, CDCl 3 )δ7.60(d,J=2.2Hz,2H),7.50(dd,J=10.5,4.8Hz,4H),7.43–7.36(m,2H),7.26–7.20(m,4H),7.09 (d,J=7.7Hz,2H),6.98(s,1H),6.42(d,J=8.9Hz,2H),5.88–5.85(m,2H),3.83(s,6H),2.22(s,6H); 13 C NMR (101 MHz, CDCl 3 )δ175.57(C),158.99(C),148.63(C),144.83(C),135.45(C),133.12(C),131.07(C),130.47(C),129.62(C),12 8.76(C),128.23(C),127.97(C),126.73(C),114.20(C),114.10(C),43.23(CH3),20.35(CH3); HRMS(ESI)calcd for C 35 H 31 N 2 [M] + 479.2482, found 479.2677.

[0178] Embodiment 13:

[0179] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 13a, the compound structure and specific synthesis route are as follows:

[0180]

[0181] The specific steps are as follows:

[0182] (1) Synthesis of intermediate 11

[0183] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), diphenylamine, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, cooled and diluted with dichloromethane, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 11. 1 H NMR (400 MHz, CDCl 3 )δ7.48(d,J=8.2Hz,2H),7.33–7.27(m,8H),7.16–7.07(m,12H),7.01(d,J=1.9Hz,2H),6.77(dd,J=8.2,2.0Hz,2H); 13 CNMR (101MHz, CDCl 3 )δ191.01(C),153.53(C),146.82(C),145.34(C),129.70(C),128.60(C),125.71(C),125.27(C),124.52(C),121.40(C),113.45(C);

[0184] (2) Synthesis of dye 13a

[0185] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 11 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 13a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 11 was 1:1:0.5. 1 HNMR (400MHz, CDCl 3 )δ7.45(t,J=7.7Hz,8H),7.34(t,J=7.4Hz,4H),7.26–7.22(m,9H),7.13(d,J=7.6Hz,2H) ,6.75(d,J=2.0Hz,2H),6.63(d,J=8.8Hz,2H),6.22(dd,J=8.8,1.8Hz,2H),2.28(s,6H);13 C NMR (101 MHz, CDCl 3 )δ175.99(C),158.30(C),147.34(C),143.64(C),135.63(C),134.39(C),133.64(C),130.47(C),1 30.08(C),128.70(C),128.17(C),127.62(C),118.48(C),116.80(C),20.60(CH3); HRMS(ESI)calcd for C 45 H 35 N 2 [M] + 603.2795, found 603.3048.

[0186] Embodiment 14:

[0187] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 14a, the compound structure and specific synthesis route are as follows:

[0188]

[0189] The specific steps are as follows:

[0190] (1) Synthesis of intermediate 12

[0191] In anhydrous and oxygen-free operation, 3,6-dibromofluorenone (compound 1), indoline, tri(dibenzylideneacetone)dipalladium, tri-tert-butylphosphine and sodium tert-butoxide were added in a molar ratio of 1:4:0.025:0.075:6, dissolved in toluene, reacted at 60°C for 12 hours, cooled and diluted with dichloromethane, filtered to remove insoluble matter, and then rotary evaporated to remove the solvent to obtain a crude product, washed with ethyl acetate, filtered, and the solid was collected to obtain intermediate 12. 1 H NMR (400 MHz, CDCl 3 )δ7.63(d,J=8.2Hz,2H),7.34(d,J=2.0Hz,2H),7.31(d,J=8.0Hz,2H),7.24(d,J=7.3Hz,2H),7.17(t,J=7. 7Hz,2H),7.04(dd,J=8.2,2.0Hz,2H),6.87(t,J=7.4Hz,2H),4.10(t,J=8.3Hz,4H),3.19(t,J=8.3Hz,4H); 13 C NMR (101 MHz, CDCl 3)δ149.14(C),145.68(C),145.35(C),132.37(C),127.57(C),127.42(C),125.57(C), 125.54(C),120.73(C),115.52(C),110.33(C),107.83(C),52.46(CH2),28.28(CH2);

[0192] (2) Synthesis of dye 14a

[0193] In anhydrous and oxygen-free operation, 2,6-dimethylbromobenzene was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then n-butyl lithium n-hexane solution was added dropwise, and stirring was continued for 30 minutes. Anhydrous tetrahydrofuran solution of intermediate 12 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 3a. The molar ratio of 2,6-dimethylbromobenzene, n-butyl lithium, and intermediate 12 was 1:1:0.5. 1 HNMR (400MHz, CDCl 3 )δ7.87(s,2H),7.43(d,J=8.1Hz,2H),7.35–7.26(m,5H),7.19–7.09(m,4H),6.91(d,J=8. 9Hz, 2H), 6.71 (d, J = 8.8Hz, 2H), 4.67 (t, J = 7.5Hz, 4H), 3.30 (t, J = 7.4Hz, 4H), 2.27 (s, 6H); 13 C NMR (101 MHz, CDCl 3 )δ172.23(C),153.17(C),147.80(C),142.61(C),136.93(C),135.75(C),132.87(C),132.50(C),129.66(C),128.29(C),12 8.07(C),126.50(C),125.93(C),117.30(C),116.73(C),114.39(C),54.74(CH3),28.63(CH2),20.49(CH2); HRMS(ESI)calcd for C 37 H 31 N 2 [M] + 503.2482, found 503.2802.

[0194] Embodiment 15:

[0195] The dyes 1a-14a of the general formula A prepared in the above Examples 1-14 were prepared into 20 mM DMSO solutions as mother solutions, and then diluted into 20 μM dichloromethane solutions, and their UV-visible absorption spectra and fluorescence emission spectra were scanned and plotted, as shown in the attached figure. Figure 1 The relevant photophysical properties are shown in Table 1. It can be seen that the near-infrared absorption peak and emission peak of the general formula A series dyes are between 800-1200nm.

[0196] Table 1 Photophysical properties of dyes 1a-14a of formula A series

[0197]

[0198]

[0199] Embodiment 16:

[0200] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 15a, the compound structure and specific synthesis route are as follows:

[0201]

[0202] The specific steps are as follows:

[0203] (1) Synthesis of intermediate 13

[0204] Under anhydrous and oxygen-free conditions, 2,6-dimethyl-1,4-dibromobenzene (Compound 2) was dissolved in tetrahydrofuran solvent, stirred at -78°C for 5 minutes, and then a solution of n-butyl lithium in n-hexane was added dropwise, and stirring was continued for 60 minutes. Di-tert-butyl dicarbonate was dissolved in anhydrous tetrahydrofuran, added dropwise to the above reaction system, and the mixture was warmed to room temperature and stirred for 12 hours. Water was added to quench the reaction, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The intermediate 13 was obtained by column chromatography separation (pure petroleum ether). The molar ratio of compound 2, n-butyl lithium, and di-tert-butyl dicarbonate was 1:1:1;

[0205] (2) Synthesis of Intermediate 3

[0206] The synthesis of intermediate 3 is as described in Example 3;

[0207] (3) Synthesis of dye 15a

[0208] The intermediate 13 was dissolved in tetrahydrofuran solvent in anhydrous and oxygen-free conditions, stirred at -78°C for 5 minutes, and then a n-pentane solution of tert-butyl lithium was added dropwise, and stirring continued for 30 minutes. An anhydrous tetrahydrofuran solution of intermediate 3 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and the mixture was stirred for 10 minutes. The precipitate was precipitated, and the solid was filtered and collected. The solid was dissolved in a mixed solvent of trifluoroacetic acid and dichloromethane (volume ratio of 1:1), stirred at room temperature for 3 hours, and the solvent was removed by rotary evaporation. The dye 15a was obtained by column chromatography separation (dichloromethane: methanol = 50:1). The molar ratio of intermediate 13, tert-butyl lithium, and intermediate 3 was 1:2:0.5. 1 H NMR (400MHz, DMSO) δ7.79(s,2H),7.62(d,J=1.7Hz,2H),6.65(d,J=9.0Hz,2H),6.37(dd,J=9.0,1.7Hz,2H),3.38(s,12H),2.21(s,6H). 13 C NMR(101MHz,DMSO)δ169.03(C),167.70(C),158.07(C),146.97(C),136.31(C),135.88(C),132.79(C) ,131.96(C),129.14(C),128.78(C),113.62(C),113.04(C),42.31(CH3),20.20(CH3).HRMS(ESI)calcd for C 26 H 27 N 2 O 2 [M] + 399.2067, found 399.2165.

[0209] Embodiment 17:

[0210] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 16a, the compound structure and specific synthesis route are as follows:

[0211]

[0212] The specific steps are as follows:

[0213] (1) Synthesis of intermediate 13

[0214] The synthesis of intermediate 13 is described in Example 16;

[0215] (2) Synthesis of Intermediate 2

[0216] The synthesis of intermediate 2 is as follows:

[0217] (3) Synthesis of dye 16a

[0218] The intermediate 13 was dissolved in tetrahydrofuran solvent in anhydrous and oxygen-free operation, stirred at -78°C for 5 minutes, and then a n-pentane solution of tert-butyl lithium was added dropwise, and stirring was continued for 30 minutes. An anhydrous tetrahydrofuran solution of intermediate 2 was added, and the mixture was warmed to room temperature and stirred for 30 minutes, and then an excess of water was added to quench, extracted with dichloromethane, and washed with saturated brine three times, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain an oily substance, a mixed solvent of trifluoroacetic acid and dichloromethane (volume ratio of 1:1) was added, and stirred at room temperature for 3 hours, toluene was added and the solvent was repeatedly removed by rotary evaporation, and the precipitate was precipitated with ether and petroleum ether and washed to obtain dye 16a. The molar ratio of intermediate 13, tert-butyl lithium, and intermediate 2 was 1:2:0.5. 1 H NMR (400MHz, DMSO) δ9.29 (s, 2H), 7.78 (s, 2H), 7.20 (s, 2H), 6.65 (d, J = 7.8Hz, 2H), 6.23 (d, J = 8.5Hz, 2H), 3.07 (s, 6H), 2.22 (s, 6H); 13 C NMR(101MHz,DMSO)δ169.18(C),167.68(C),159.42(C),147.03(C),136.32(C),135.82(C) ,133.45(C),131.95(C),129.09(C),128.77(C),31.17(CH3),20.20(CH3); HRMS(ESI)calcd for C 24 H 23 N 2 O 2 [M] + 371.1754, found 371.1864.

[0219] Embodiment 18:

[0220] Preparation of antiaromatic diaminofluorene cationic fluorescent dye 17a, the compound structure and specific synthesis route are as follows:

[0221]

[0222] The specific steps are as follows:

[0223] (1) Synthesis of intermediate 14

[0224] 2-Bromo-5-iodo-1,3-xylene (Compound 3) was dissolved in diisopropylamine, and di(triphenylphosphine)palladium dichloride, cuprous iodide and trimethylsilylene were added, and stirred at room temperature for 4 hours. The reaction solution was diluted with ethyl acetate and washed with water three times, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The intermediate 14 was separated by column chromatography (pure petroleum ether). The molar ratio of compound 3, di(triphenylphosphine)palladium dichloride, cuprous iodide and trimethylsilylene was 1:0.03:0.06:1.2;

[0225] (1) Synthesis of intermediate 15

[0226] The intermediate 14 was dissolved in methanol and potassium carbonate was added, and stirred at room temperature for 12 hours. The reaction solution was diluted with water, extracted with ether three times, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the intermediate 15. The molar ratio of the intermediate 14 to methanol was 1:2;

[0227] (2) Synthesis of Intermediate 3

[0228] The synthesis of intermediate 3 is as described in Example 3;

[0229] (3) Synthesis of dye 17a

[0230] The intermediate 15 was dissolved in tetrahydrofuran solvent in anhydrous and oxygen-free operation, stirred at -78°C for 5 minutes, and then n-butyl lithium in n-hexane solution was added dropwise, and stirring was continued for 30 minutes. The anhydrous tetrahydrofuran solution of intermediate 3 was added, and the mixture was warmed to room temperature and stirred for 30 minutes. An excess of 10% perchloric acid solution was added, and stirred for 10 minutes. The precipitate was precipitated, filtered, and the solid was collected to obtain dye 17a. The molar ratio of intermediate 15, n-butyl lithium, and intermediate 3 was 1:2:0.5. 1 H NMR (400 MHz, CDCl 3 )δ7.64(s,2H),7.27(s,2H),6.54(d,J=8.9Hz,2H),6.08(d,J=8.9Hz,2H),3.44(s,12H),3.11(s,1H),2.19(s,6H); 13 C NMR (101 MHz, CDCl 3 )δ172.04(C),158.60(C),147.82(C),136.04(C),132.92(C),131.93(C),131.46(C),129.59(C),1 23.17(C),113.94(C),111.47(C),83.23(C),78.26(CH),42.40(CH3),20.12(CH3); HRMS(ESI)calcd for C 27 H 27N 2 [M] + 379.2169, found 379.2277.

[0231] Embodiment 19:

[0232] Preparation of activatable small molecule probe 18a and ONOO - Response study, compound structure and specific synthesis route are as follows:

[0233]

[0234] The specific steps are as follows:

[0235] Anhydrous operation, compound 4 was dissolved in dry THF, stirred under ice bath and triethylamine and triphosgene were added, and stirring was continued for 4 hours. The reaction solution was filtered with diatomaceous earth, and the solvent was removed by rotary evaporation to obtain an oily chloroformate intermediate. Dye 2a was dissolved in dichloromethane, N, N-diisopropylethylamine (DIPEA) was added under ice bath and stirred for 5 minutes. The chloroformate intermediate was dissolved in dichloromethane and added dropwise to the solution of dye 2a, and stirring was continued for 30 minutes. After the reaction was completed, the reaction solution was diluted with dichloromethane and washed with 0.1M hydrochloric acid, dried over anhydrous sodium sulfate, most of the solvent was removed by rotary evaporation, and a large amount of ether was added to precipitate the crude product. The precipitate was separated by preparative high performance liquid chromatography (methanol / water, adding 0.1% trifluoroacetic acid) to obtain dye 18a. The molar ratio of compound 4, triethylamine, triphosgene, dye 2a, and DIPEA was 1.5:2.25:0.75:1:4. 1 H NMR (400 MHz, CDCl 3 )δ8.03–7.75(m,2H),7.47–7.29(m,3H),7.21–7.06(m,2H),6.91(dd,J=48.3,8.6Hz,2H),6.80–6.57 (m,3H),6.42(d,J=9.0Hz,1H),5.24(s,2H),3.47(s,3H),3.29(s,3H),2.17(s,6H).HRMS(ESI)calcd for C 31 H 29 BN 2 O 4 [M+H] + 505.2299, found 505.2292;

[0236] Dye 18a has the ability to respond to ONOO - The phenylboronic acid group is eliminated by cascade, and has no near-infrared absorption and emission. -The resulting dye 2a has near-infrared absorption and emission, and is therefore a responsive ONOO - Probe. ONOO for dye 18a - To investigate the response, dye 18a was prepared into a 50 μM 1× PBS solution, and 5-25 μM concentrations of ONOO - The solution was added and the absorption spectrum of the dye was collected by UV-visible spectrophotometer. - The samples without ONOO - The fluorescence emission of the two samples was measured by near-infrared fluorescence spectrometer. The results are shown in the attached Figure 2 As shown, it shows that dye 18a is effective for ONOO - Has good response effect.

[0237] Embodiment 20:

[0238] Ultra-stable long-term cell staining imaging experiment of antiaromatic diaminofluorene cationic fluorescent dyes. Take dye 3a as an example.

[0239] The specific steps are as follows:

[0240] Endothelial cells were cultured in a 35 mm culture dish overnight, and the dye 3a was diluted to 1 μM in 1×PBS solution. The commercial near-infrared dye Cy7-Cl was selected as the control group and also diluted to 1 μM in 1×PBS solution. After staining for 30 minutes, each was washed three times with 1×PBS solution and imaged under a near-infrared zone II fluorescence microscope. The excitation wavelength used was 808 nm, and the fluorescence signal above 1000 nm was collected. The excitation power was 5 W (power density 2×10 4 mW / mm 2 ), exposure time 100ms, and continuously collect 1000 frames of signals, and plot the changes in the signal-to-noise ratio of the cell fluorescence signal in each frame. The results are shown in the attached figure. Figure 3 As shown in the figure, the signal-to-noise ratio of the cell fluorescence signal of the commercial near-infrared dye Cy7-Cl dropped sharply within a few seconds of 5W irradiation, and its photostability was weak. However, under the same imaging conditions, the dye 3a still maintained its good imaging signal-to-noise ratio after 1000 repeated exposures, indicating that it has high photostability.

[0241] Embodiment 21:

[0242] Dual-channel mouse organ fluorescence imaging experiment using antiaromatic diaminofluorene cationic fluorescent dyes. Dyes 2a and 15a have significantly different absorption spectra and large differences in oil-water partition coefficients (LogD), so they can show different metabolic conditions in mice, and distinguish the fluorescence signals from the two dyes through multi-spectral unmixing. Take dyes 2a and 15a as an example. The specific steps are as follows:

[0243] Dyes 2a and 15a were prepared into DMSO solutions with a concentration of 20mM as mother solutions. First, the dyes were diluted to 1×PBS solutions with a concentration of 20μM in a 2mL centrifuge tube and placed under a near-infrared camera. They were excited in sequence with 730nm, 808nm, and 940nm lasers of equal power, and fluorescence signals with wavelengths above 1000nm were collected. The collected simulated spectra were used as reference spectra for subsequent multi-channel unmixing. Then, the two dye mother solutions were diluted to 1mM with 1×PBS (5% F127 was added to 2a). A 6-8 week old Balb / c mouse was anesthetized and depilated, and 100μL of the above two dye solutions were injected through the tail vein. After metabolism for 5 minutes, the same excitation and collection conditions as the reference spectrum were used. The mouse was placed under a near-infrared camera and excited in sequence with 730nm, 808nm, and 940nm, and fluorescence signals with wavelengths above 1000nm were collected. The image results were unmixed using the reference spectrum collected in vitro as described above. The results are shown in the attached Figure 4 As shown in the figure, it can be seen that the dye successfully realizes the fluorescence imaging of multiple organs and can successfully analyze the fluorescence signals from different dyes, which has good application value in multi-color fluorescence imaging of living organisms.

[0244] Embodiment 22:

[0245] Antiaromatic diaminofluorene cation-responsive probe for overproduced ONOO during brain injury in mice - Multispectral photoacoustic detection of dye 18a with ONOO - The responsiveness can realize OFF-ON response in near-infrared photoacoustic imaging, and can further distinguish the signals of deoxygenated / oxygenated hemoglobin in vivo by unmixing multi-spectral photoacoustic signals to realize ONOO in vivo - Detection. Taking dye 18a as an example, the specific steps are as follows:

[0246] A 6-8 week old Balb / c mouse was anesthetized and a 40g metal block was dropped from a height of 20cm to hit one side of the mouse's head at a depth of 1.5mm to create a brain injury model. The intact head on the other side was used as a self-control. After 12 hours, 200μL of a 200μM dye 18a solution in 1×PBS was injected intravenously, and after 30 minutes, the mouse was placed in a small animal photoacoustic imaging system to collect photoacoustic signals at 680nm-970nm. The system's preset deoxygenated / oxygenated hemoglobin standard spectrum and the pre-collected photoacoustic spectrum of dye 2a were used as reference spectra for multi-spectral unmixing to perform spectral analysis and identify the photoacoustic signal of dye 2a generated after the response. The results are shown in the attached figure. Figure 5 As shown. It can be seen that the probe molecule successfully achieved ONOO in vivo - It has the potential to be further expanded into a new small molecule probe platform for a variety of in vivo sensing and analytical applications.

Claims

1. A near-infrared small molecule dye, characterized in that: The structure is any one of the following formulas D 6-D 14, D18:

2. Use of the near-infrared small molecule dye as described in claim 1 in near-infrared multi-spectral fluorescence imaging.

Images