Near-infrared small-molecule fluorescent dye with pentadecanedioic conjugated chain as well as preparation method and application of near-infrared small-molecule fluorescent dye

Abstract

The invention relates to a near-infrared small-molecule fluorescent dye with a pentadecamethine conjugated chain and a preparation method and application thereof, the near-infrared small-molecule fluorescent dye has a structure as shown in a general formula A, and is a novel dye matrix based on two benzoindole structures connected by a pentadecamethine chain, the functional dye molecule with the absorption and emission peak wavelength in the near-infrared region is obtained through regulation and control of substituent groups on a parent structure, and different substituent groups can be introduced to a fifteen-membered methine chain and an indole N atom. The absorption and emission wavelength is in a near-infrared region of 1100-1300nm, and the material has the characteristics of high brightness, excellent light stability, low cytotoxicity, simplicity and convenience in synthesis, easiness in structure modification and the like. After water-soluble modification, the polymer can be widely applied to the biomedical fields of tumor targeting imaging, living body angiography and the like. General formula A # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomaterial technology, in particular to the field of biomedical imaging detection, and specifically to a near-infrared small molecule fluorescent dye with a pentamethine conjugated chain, and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging has good applications in pathology and physiology due to its fast response, high spatiotemporal resolution, and real-time imaging, such as early diagnosis and treatment of diseases and research on their mechanisms. Compared with other clinical imaging methods, such as positron emission tomography (PET), computed tomography (CT), and single-photon emission computed tomography (SPECT), fluorescence imaging has the advantage of not emitting harmful radiation. Organic small molecule fluorescent probes, due to their low toxicity and rapid metabolism, have good safety and biocompatibility among many fluorescent probes such as carbon nanotubes, rare earth nanoparticles, and quantum dots, and have applications in angiography, lymph node angiography, and targeted tumor imaging.

[0003] However, the main bottleneck of fluorescence imaging lies in the shallow tissue penetration depth and the low signal-to-noise ratio caused by the high absorption, scattering and autofluorescence of biological tissues. In recent years, near-infrared II imaging (NIR-II, 1000-1700nm), also known as shortwave infrared imaging (SWIR), has great prospects because it avoids the autofluorescence region of organisms and has less light scattering, and has higher resolution and tissue penetration depth compared with traditional visible light and near-infrared I imaging. In addition, the near-infrared II region is also divided into near-infrared IIa (1300-1400nm) and near-infrared IIb (1500-1700nm) regions. Although the tissue absorption coefficient of the near-infrared II region is slightly higher than that of the near-infrared I region, the scattered light and autofluorescence are significantly reduced, making the near-infrared II region have higher resolution and penetration depth. Although the near-infrared II region fluorescent probes have the advantages of long wavelength and high contrast, the types of conjugated chains of fluorescent probe molecules are few, the molar absorption coefficient and fluorescence quantum yield are still low, and the modifiability on the conjugated chain is limited.

[0004] In terms of fluorescent dye performance, as the polymethine chain lengthens, the absorption and fluorescence wavelength of cyanine dyes are significantly improved, which improves important performance parameters such as the penetration depth and signal-to-background ratio of dye molecules in biological imaging. However, there is currently a lack of methods for synthesizing long polymethine chains.

[0005] However, in terms of synthesis, as the polymethine chain becomes longer, the synthesis of cyanine dyes becomes more difficult and the stability of the dyes is also poor.

[0006] Therefore, designing and synthesizing near-infrared region II organic small molecule fluorescent probes with high molar absorption coefficient, high brightness, excellent photostability, low cytotoxicity, simple synthesis, novel structure and easy modification is an important goal in the field of fluorescent probe synthesis. Summary of the invention

[0007] The purpose of the present invention is to provide a near-infrared small molecule fluorescent dye with a 15-membered methine conjugated chain and its preparation method and application in order to solve the above problems, and the structure is easy to modify. The near-infrared small molecule dye provided by the present invention is a new dye matrix based on two benzindole structures connected by a 15-membered methine chain. The functional dye molecule with the absorption and emission peak wavelength in the near-infrared region obtained by regulating the substituents on the matrix structure can introduce different substituents on the 15-membered methine chain and the indole N atom. In the process of lengthening the chain length of the polymethine chain, the present application improves the important performance parameters of the fluorescent dye such as penetration depth and signal-to-background ratio. In the process of lengthening the chain length of the polymethine chain, the present application is simple to synthesize and ensures the stability of the dye.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The first object of the present invention is to provide a near-infrared small molecule fluorescent dye having a pentamethine conjugated chain, wherein the small molecule fluorescent dye comprises one or more of the structure shown in general formula A and the solvate of the structure shown in general formula A, wherein the general formula A is as follows:

[0010]

[0011] Where:

[0012] R1 and R2 are each independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring, chlorine atom;

[0013] R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne, C1-C6 sulfonic acid substituted alkane;

[0014] n is independently selected from 1 or 2;

[0015] X is independently selected from a bromine atom, tetraphenylboric acid, and tetrakis(pentafluorophenyl)boric acid.

[0016] Furthermore, the near-infrared small molecule dye has a maximum absorption wavelength between 1100-1300 nm, a maximum emission wavelength between 1150-1300 nm, and a molecular weight between 700-1300 Da.

[0017] Furthermore, the brightness of the near-infrared small molecule dye reaches 4.8-115.1M -1 cm -1 , the molar extinction coefficient reaches 4.8×10 4 -2.6×10 5 M -1 cm -1 .

[0018] Furthermore, the structure of the small molecule fluorescent dye is the structure shown in general formula A, or is an isomer of the structure shown in general formula A or a solvate of the structure shown in general formula A.

[0019] Further preferably, the structure of the small molecule fluorescent dye is the structure shown in general formula A.

[0020] Furthermore, the solvate of the structure represented by the general formula A is a solvate formed by the general formula A and a solvent such as dichloromethane, methanol, and dimethyl sulfoxide.

[0021] Further preferably, the structure of the small molecule fluorescent dye is any one of the following formulas B1-B14:

[0022]

[0023]

[0024] The second object of the present invention is to provide a method for preparing the above-mentioned near-infrared small molecule dye, comprising the following steps:

[0025] Step 1: a carbon-nitrogen coupling reaction of a compound represented by formula (1) with a halide or sultone compound to obtain a compound represented by formula (2);

[0026] Step 2: The compound represented by formula (2) and the aldehyde compound undergo a dehydration condensation reaction to obtain a compound represented by formula (3);

[0027] Step 3: The compound represented by formula (3) is subjected to Suzuki coupling reaction to obtain a compound represented by formula (4);

[0028] Step 4: The compound represented by formula (3) or formula (4) is subjected to Knoevenagel condensation reaction with cyclopentanone to obtain a compound represented by formula (5);

[0029] Step 5, the compound represented by formula (5) is subjected to nucleophilic addition reaction under the action of lithium reagent or Grignard reagent to obtain the compound represented by general formula A after the nucleophilic addition reaction or the compound represented by general formula A is obtained by reacting with the corresponding anion salt after the nucleophilic addition reaction;

[0030] Among them, formula (1) is Formula (2) is Formula (3) is Formula (4) is Formula (5) is

[0031] Furthermore, the preparation method comprises the following process:

[0032]

[0033] Furthermore, in step 1, the compound represented by formula (1) is subjected to a carbon-nitrogen coupling reaction with a halide or a sultone compound to obtain a compound represented by formula (2), comprising:

[0034] In a first organic solvent environment, heating and stirring the compound represented by formula (1) and a halide or the compound represented by formula (1) and a sultone compound to obtain a compound represented by formula (2);

[0035] The halide is an alkyl or terminal alkyne halide;

[0036] The halogenated compound or sultone compound has a structure as shown in any of the following formulas C1, C2, and C3:

[0037]

[0038] In formula C1, C2, C3:

[0039] R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne;

[0040] wherein C1 and C2 are halogenated compounds, and C3 is a sultone compound;

[0041] The first organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ether, 1,4-dioxane, and glacial acetic acid.

[0042] Furthermore, in step 1, the conditions for heating and stirring are as follows: the heating temperature is 100-120° C., the stirring rate is 500-1000 rpm, and the heating and stirring time is 24-72 h.

[0043] Furthermore, the molar ratio of the compound represented by formula (1) to the halide or the compound represented by formula (1) to the sultone compound is 1:1.2-1:10.

[0044] Furthermore, in step 2, the compound represented by formula (2) and the aldehyde compound undergo a dehydration condensation reaction to obtain a compound represented by formula (3), comprising:

[0045] In a second organic solvent environment, the compound represented by formula (2) and the aldehyde compound are stirred to obtain a compound represented by formula (3);

[0046] The aldehyde compound has a structure as shown in any of the following formulas D1 and D2:

[0047]

[0048] The second organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, acetic acid, tetrahydrofuran, ether, 1,4-dioxane, ethanol, and methanol.

[0049] Furthermore, in step 2, the stirring conditions are as follows: the stirring rate is 500-1000 rpm, and the stirring time is 9-24 h.

[0050] Furthermore, the molar ratio of the compound represented by formula (2) to the aldehyde compound is 1:1-1:3.

[0051] Furthermore, in step 3, the compound represented by formula (3) is subjected to a Suzuki coupling reaction to obtain a compound represented by formula (4), comprising:

[0052] In a third organic solvent environment, under the protection of an inert gas, the compound represented by formula (3) and the boric acid compound are heated and stirred with a palladium catalyst and a base as a catalyst to obtain a compound represented by formula (4) through a Suzuki coupling reaction;

[0053] The boric acid compound has a structure as shown in the following formula E:

[0054] R1-B(OH)2

[0055] E

[0056] In formula E:

[0057] R1 is independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring;

[0058] The palladium catalyst is selected from one or more of tetrakistriphenylphosphine palladium, tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetonepalladium), and palladium acetate;

[0059] The base is selected from one or more of potassium tert-butoxide, sodium tert-butoxide, potassium carbonate, cesium carbonate, and potassium phosphate;

[0060] The third organic solvent is selected from one or more of benzene, toluene, xylene, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and diethyl ether.

[0061] Furthermore, in step three, under the protection of inert gas, the conditions for heating and stirring are as follows: heating temperature is 80-120° C., stirring rate is 500-1000 rpm, and heating and stirring time is 12-48 h.

[0062] Furthermore, the molar ratio of the compound represented by formula (3) to the boric acid compound is 1:1-1:3.

[0063] Furthermore, in step 4, the compound represented by formula (3) or formula (4) is subjected to Knoevenagel condensation reaction with cyclopentanone to obtain a compound represented by formula (5), comprising:

[0064] In a fourth organic solvent environment, the compound represented by formula (3) or formula (4) and cyclopentanone are heated and stirred with a base, wherein the base serves as a catalyst, and a Knoevenagel condensation reaction is performed to obtain a compound represented by formula (5);

[0065] The base is selected from one or more of sodium hydroxide, sodium tert-butoxide, sodium ethoxide, and potassium hydroxide;

[0066] The fourth organic solvent is selected from one or more of tetrahydrofuran, diethyl ether, 1,4-dioxane, ethanol, and methanol.

[0067] Furthermore, in step 4, the conditions for heating and stirring are as follows: the heating temperature is 80-120° C., the stirring rate is 500-1000 rpm, and the heating and stirring time is 12-72 h.

[0068] Furthermore, the molar ratio of the compound represented by formula (3) to cyclopentanone is 1:0.5-1:1.

[0069] Furthermore, in step 5, the compound represented by formula (5) is subjected to a nucleophilic addition reaction under the action of a lithium reagent or a Grignard reagent, and the compound represented by general formula A is obtained after the nucleophilic addition reaction, or part of the compound obtained after the nucleophilic addition reaction is reacted with a corresponding anion salt to obtain a compound represented by general formula A, including:

[0070] In a fifth organic solvent environment, under the protection of an inert gas, reacting the compound represented by formula (5) with a lithium reagent or reacting the compound represented by formula (5) with a Grignard reagent at low temperature, undergoing a nucleophilic addition reaction, and obtaining a compound represented by general formula A after the nucleophilic addition reaction, or exchanging anions with a corresponding anion salt of a part of the compound obtained after the nucleophilic addition reaction, to obtain a compound represented by general formula A;

[0071] The lithium reagent or Grignard reagent has a structure as shown in any of the following formulas F1 and F2:

[0072]

[0073] In formula F1 and F2:

[0074] R2 are each independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring;

[0075] The anion salt is selected from one or more of sodium tetraphenylborate and potassium tetrakis(pentafluorobenzene)borate;

[0076] The fifth organic solvent is selected from one or more of super dry tetrahydrofuran, diethyl ether, and 1,4-dioxane.

[0077] Furthermore, in step 5, under the protection of inert gas, the conditions for the low-temperature reaction are as follows: the low-temperature is -87°C, the stirring rate is 500-1000rpm, and the stirring time is 0.5-3h.

[0078] Furthermore, the molar ratio of the compound represented by formula (5) to the lithium reagent or the molar ratio of the compound represented by formula (5) to the Grignard reagent is 1:1-1:20.

[0079] Furthermore, the molar ratio of the compound represented by formula (5) to the anion salt is 1:1-1:20.

[0080] The near-infrared small molecule dye provided by the present invention is a new dye matrix based on two benzindole structures connected by a 15-membered methine chain. The absorption and emission peak wavelengths of the functional dye molecules obtained by regulating the substituents on the matrix structure are in the near-infrared region. Different substituents can be introduced on the 15-membered methine chain and the indole N atom. Its maximum absorption wavelength is between 1100-1300nm, the maximum emission wavelength is between 1150-1300nm; the molecular weight is between 700-1300Da, and the brightness is high at 4.8-115.1M -1 cm -1 , with a molar extinction coefficient as high as 4.8×10 4 -2.6×10 5 M -1 cm -1 , excellent photostability, low cytotoxicity, simple synthesis and easy structure modification.

[0081] The third object of the present invention is to provide an application of a near-infrared small molecule fluorescent dye having a pentamethine conjugated chain. The near-infrared small molecule dye provided by the present invention can be used for tumor targeted imaging, in vivo vascular imaging, fluorescence imaging, etc.

[0082] Compared with the prior art, the beneficial effects of the present invention are embodied in the following aspects:

[0083] The present invention provides a near-infrared small molecule fluorescent dye with a pentamethine conjugated chain and a preparation method thereof, which has a longer absorption and emission wavelength, can perform NIR-IIb imaging, and has a better signal-to-background ratio (SNR) and imaging effect compared to traditional visible and NIR-I imaging. It makes up for the shortcomings of NIR-II imaging, such as fewer molecular structures and shorter wavelengths. Its maximum absorption wavelength is between 1100-1300nm, and its maximum emission wavelength is between 1150-1300nm. It has the characteristics of high brightness, high molar extinction coefficient, excellent photostability, low cytotoxicity, simple synthesis, and easy structural modification, and can be used for tumor targeted imaging, in vivo vascular imaging, NIR-II fluorescence imaging, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The normalized absorption spectra and photoluminescence (PL) spectra of the small molecule fluorescent dyes B1-B10 of the present invention in dichloromethane are shown.

[0085] Figure 2 The small molecule fluorescent dye B3 and B7 nanoparticles and ICG of the present invention were respectively prepared into PBS solutions with a concentration of 0.15mM, and the fluorescence imaging test photos under different long-pass filters LP1300 and LP1500 were obtained using a chemiluminescence fluorescence imaging analysis system, where (a) LP1300, (b) LP1500.

[0086] Figure 3 The small molecule fluorescent dye B3 of the present invention was prepared into a PBS solution with a concentration of 0-150 μM nanoparticles. 4T1 cells, 8000 cells / well, were incubated with the B3 material for 12 hours. Schematic diagram of the survival rate of 4T1 cells.

[0087] Figure 4 A schematic diagram of NIR-IIb imaging of hind limb blood vessels of Balb / c-nude mice using the small molecule fluorescent dye B3 nanoparticles of the present invention formulated into a PBS solution with a concentration of 0.15 mM.

[0088] Figure 5 Schematic diagram of mouse NIR-II vascular imaging using the small molecule fluorescent dye B3 of the present invention.

[0089] Figure 6 Schematic diagram of mouse NIR-II tumor imaging using the small molecule fluorescent dye B3 of the present invention. DETAILED DESCRIPTION

[0090] The present invention is described in detail below in conjunction with specific embodiments, but is by no means intended to limit the present invention. Features such as preparation methods, materials, structures or composition ratios not explicitly described in this technical solution are considered to be common technical features disclosed in the prior art.

[0091] The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several modifications and improvements may be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0092] In this embodiment, the hydrogen nuclear magnetic resonance spectrum (1H NMR) of the compound is measured by Bruker AVANCE III HD 400 or Bruker AVANCE III HD 500; all reagents used are commercially available reagents.

[0093] Example 1

[0094] This example provides a preparation of a fluorescent dye B1, and the compound structure and specific synthesis route are as follows:

[0095]

[0096] (1) Synthesis of Compound B1-(2)

[0097] 2,3,3-trimethyl-4,5-benzo-3H-indole (1.0 g, 4.8 mmol), acetonitrile (36 mL), and 1-iodopropane (702 μL, 7.2 mmol) were added to a 100 mL round-bottom flask, and the reaction was heated for 2 days at 110° C. and 800 rpm. After cooling to room temperature, the mixture was concentrated by rotary evaporation, and the remaining oily solution was washed with ether to obtain the product B1-(2), a dark brown solid of 1.34 g, with a yield of 97%. H-NMR (400MHz, CDCl3, 25℃) δ = 8.08 (2H, t, J = 8Hz, ar.CH) and 8.04(1H,d,J=8Hz,ar.CH)part.overlapped,7.92(1H,d,J=8Hz,ar.CH),7.83(1H,d,J=8Hz,ar.CH),7.66(1H,t,J=8Hz,ar.CH),4.8 0(2H,t,J=8Hz,-NCH2-),3.20(3H,s,-CH3),2.11-2.02(2H,m,-NCH2CH2CH2),1.87(6H,s,-CH3),1.11(3H,t,J=8Hz,-NCH2CH2CH3); 13C-NMR (100MHz, CDCl3, 25℃) δ = 195.3, 189.7, 150.1, 138.5, 138.0, 136.9, 13 3.5,132.1,131.2,130.0,127.3,55.8,55.2,22.6,21.6,16.8,11.2; ESI-MS m / z calculated for C 18 H 22 N + 252.17; obs.:252.2

[0098] (2) Synthesis of Compound B1-(3)

[0099] A mixture of compound B1-(2) (5 mmol, 1.9 g) and 2-chloro-1-formyl-3-(hydroxymethylene)cyclohexene (6 mmol, 1.0 g) was dissolved in a mixture of toluene (37 mL) and acetic acid (13 mL), stirred at room temperature for 12 h at a stirring rate of 800 rpm, and concentrated by rotary evaporation. The obtained compound was purified by silica gel column chromatography (PE:EA=9:1) to obtain compound B1-(3), a red solid 1.0 mmol, 427 mg, with an isolated yield of 21.1%.

[0100] 1 H NMR(400MHz,Chloroform-d)δ10.30(s,1H),8.07(d,J=8.6Hz,1H),8.00(d,J=12.6Hz, 1H),7.85(d,J=8.2Hz,1H),7.80(d,J=8.7Hz,1H),7.56–7.46(m,1H),7.36–7.29(m,1H ),7.13(d,J=8.7Hz,1H),5.57(d,J=12.8Hz,1H),3.79(t,J=6.0Hz,2H),2.61(t,J=5.9 Hz,2H),2.52(t,J=6.0Hz,2H),2.01(s,6H),1.89–1.78(m,4H),1.06(t,J=7.2Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ190.8,164.3,148.8,141.6,131.3,130.1,129.9,129.6,129.5,128.9,128.3 ,127.0,122.8,122.7,121.7,109.4,92.5,48.6,44.2,27.5,26.7,24.6,21.0,20.1,11.7.HRMS:calculated for C 26 H28 ClNO[M+H] + 406.1932, found 406.1942.

[0101] (3) Synthesis of Compound B1-(4)

[0102] A mixture of B1-(3) (1mmol, 410mg), phenylboronic acid (2mmol, 250mg), K2CO3 (2.2mmol, 300mg), and Pd(PPh3)4 (tetrakistriphenylphosphine palladium) (0.06mmol, 60mg) was added to a Shrek bottle under argon protection. Dry DMF (0.1mmol, 8.5mL) and deionized water (0.08mmol, 1.5mL) were added to the bottle and stirred at 90°C for 24h at a stirring rate of 800rpm. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated by rotary evaporation. Compound B1-(4) was purified by silica gel column chromatography (PE:EA=9:1) to obtain a red solid 0.87mmol, 389mg, with an isolated yield of 87.0%.

[0103] 1 H NMR(400MHz,Chloroform-d)δ9.26(s,1H),7.85(d,J=8.6Hz,1H),7.83(d,J=8.1Hz,1H),7 .76(d,J=8.7Hz,1H),7.47–7.37(m,4H),7.25–7.19(m,3H),7.01(d,J=8.7Hz,1H),6.57(d, J=12.9Hz,1H),5.55(d,J=12.9Hz,1H),3.69(t,J=7.2Hz,2H),2.59(t,J=5.8Hz,2H),2.52( t,J=6.1Hz,2H),1.92–1.83(m,2H),1.82–1.70(m,2H),1.43(s,6H),1.00(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ193.1,162.7,158.5,141.7,137.6,133.9,131.1,130.3,129.9,129.8,129.4,129.1,128.8 ,128.6,127.9,127.5,126.7,122.4,121.6,109.3,92.5,47.8,44.1,27.0,25.4,22.5,21.5,20.0,11.7.HRMS:calculated for C 32 H33 NO[M+H] + 448.2635, found 448.2638.

[0104] (4) Synthesis of Compound B1-(5)

[0105] A mixture of B1-(4) (0.5mmol, 225mg), cyclopentanone (0.25mmol, 23μL) and NaOH (2mmol, 80mg) was added to a round-bottomed bottle. 0.2mmol, 4mL of anhydrous ethanol (EtOH) was added to the bottle, stirred at 80°C for 48h, with a stirring rate of 800rpm. After cooling to room temperature, the mixture was filtered and washed with ethanol to obtain compound B1-(5), a blue solid of 0.19mmol, 183mg, with an isolated yield of 77%. 1 H NMR(400MHz,Chloroform-d)δ7.85(d,J=8.4Hz,2H),7.75(d,J=8.0Hz,2H),7.68(d,J=8.7Hz,2H ),7.43–7.35(m,8H),7.22–7.18(m,2H),7.14–7.08(m,4H),7.02–6.96(m,4H),6.23(d,J=12.8Hz ,2H),5.48(d,J=12.8Hz,2H),3.65(t,J=7.0Hz,4H),3.05–2.97(m,4H),2.78(t,J=5.9Hz,4H),2 .58(t,J=5.6Hz,4H),1.98–1.90(m,4H),1.80–1.72(m,4H),1.43(s,12H),1.00(t,J=7.4Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ159.5,150.0,142.1,140.1,135.0,134.4,131.1,130.5,130.1,129.7,129.5,129.2,129.1,1 29.0,128.7,128.0,127.0,126.5,122.0,121.7,109.1,92.8,47.4,44.0,28.7,26.9,16.7,25.0,22.5,20.0,11.7.MALDI-FT ICR MS:calculated forC 69 H 70 N2O[M] + 942.5488, found 942.5444.

[0106] (5) Synthesis of Compound B1

[0107] Add B1-(5) (0.02mmol, 20mg) to a Shrek bottle and protect with argon. Add ultra-dry THF (ultra-dry tetrahydrofuran) (2mL) to the bottle and cool to 0°C in an ice-water bath. Then drop phenylmagnesium bromide 1M (0.04mmol, 40μL) into the solution. Stir in a low-temperature reactor for 0.5h at a stirring rate of 800rpm, and add 2mL of methanol to quench the reaction. The remaining liquid is purified by alumina (300-400 mesh) chromatography (DCM:MeOH=25:1) to obtain compound B1, a black solid 0.004mmol, 4.2mg, with an isolated yield of 19.3%.

[0108] 1 H NMR(400MHz,Chloroform-d)δ8.00–7.79(m,6H),7.52–7.49(m,2H),7.41–7.28(m,11H),7.05–6.87(m,11H),6.61(d,J=7.1Hz,2H),6.17(d,J =14.2Hz,1H),4.42(t,J=6.5Hz,3H),3.00–2.65(m,9H),2.08–1.98(m,8H),1.91–1.82(m,4H),1.41(s,12H),1.03(t,J=7.3Hz,6H).MALDI-FT ICR MS:calculated for C 75 H 75 BrN2[M-Br] + 1003.5925,found1003.5925,[M-2H-Br] + 1001.5768, found 1001.5726.

[0109] Example 2

[0110] This example provides a preparation of a fluorescent dye B3, and the compound structure and specific synthesis route are as follows:

[0111]

[0112] (1) Compound B1-(2) is the same as in the synthesis of B1.

[0113] (2) Synthesis of Compound B3-(3)

[0114] A mixture of B1-(2) (10 mmol, 3.8 g) and 2-chloro-1-formyl-3-(hydroxymethylene)cyclopentene (15 mmol, 2.4 g) was dissolved in a mixture of toluene (347 mmol, 37 mL) and acetic acid 209 (mmol, 13 mL), stirred at room temperature for 12 h at a stirring rate of 800 rpm, and concentrated by rotary evaporation. The residual organic matter was purified by silica gel column chromatography (PE:EA=9:1) to obtain compound B1-(3), a red solid 1.6 mmol 636.2 mg, with an isolated yield of 16.3%.

[0115] 1 H NMR(400MHz,Chloroform-d)δ10.01(s,1H),8.02(d,J=8.5Hz,1H),7.81(d,J=8.2Hz,1H),7.76(d,J=8.7Hz,1H),7.50–7.39(m,2H),7.31–7.26(m,1 H),7.08(d,J=8.7Hz,1H),5.31(d,J=12.9Hz,1H),3.75(t,J=6.2Hz,2H), 2.80–2.73(m,4H),1.94(s,6H),1.84–1.77(m,2H),1.02(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ186.7,162.5,149.4,141.8,135.6,133.9,130.0,129.8,129.6,129.2, 129.0,126.9,125.0,122.7,121.7,109.4,93.4,48.4,27.6,26.2,24.9,20.0,11.7.HRMS:calculated forC 25 H 26 ClNO[M+H] + 392.1776, found 392.1776.

[0116] (3) Synthesis of Compound B3-(4)

[0117] A mixture of B1-(3) (0.5mmol, 200mg), 4-methoxyphenylboronic acid (1mmol, 152mg), K2CO3 (1mmol, 138mg) and Pd(PPh3)4 (0.2mmol, 231mg) was added to a Shrek bottle and protected by argon. Dry DMF (0.1mmol, 8.5mL) and deionized water 0.08 (mmol, 1.5mL) were added to the bottle and stirred at 90°C for 24h at a stirring rate of 800rpm. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate. The composite organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. Compound B3-(4) was purified by silica gel flash chromatography (PE:EA=6:1) to obtain a red solid 0.39mmol, 179mg, with an isolated yield of 77.4%.

[0118] 1 H NMR(400MHz,Chloroform-d)δ9.57(s,1H),7.92(d,J=8.6Hz,1H),7.77(d,J=8.2Hz,1H) ,7.71(d,J=8.7Hz,1H),7.44–7.37(m,1H),7.31(d,J=8.6Hz,2H),7.25–7.20(m,1H),7. 06–7.00(m,3H),6.94(d,J=12.8Hz,1H),5.38(d,J=12.8Hz,1H),3.92(s,3H),3.70(t,J =7.2Hz,2H),2.89–2.80(m,4H),1.83–1.74(m,2H),1.71(s,6H),1.01(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ189.6,162.4,160.9,160.0,142.0,140.5,140.0,131.0,129.8,129.7,129.4,129.0,128,7,126 .7,126.2,125.4,122.3,121.6,116.1,114.8,113.8,109.3,94.2,55.4,47.8,44.0,27.7,27.2,26.5,20.0,11.7.MALDI-FTICR MS: calculated for C 32 H 33 NO2[M] + 463.2511, found 463.2495.

[0119] (4) Synthesis of Compound B3-(5)

[0120] A mixture of B3-(4) (0.1 mmol, 46 mg), cyclopentanone (0.05 mmol, 4.5 μL) and NaOH (0.2 mmol, 8 mg) was added to a round-bottomed bottle. Ethanol (1 mL) was added to the bottle and stirred at 80°C for 48 h at a stirring rate of 800 rpm. After cooling to room temperature, the reaction mixture was filtered and washed with ethanol to obtain compound B3-(5) as a blue solid (0.04 mmol, 41 mg) with an isolated yield of 85%.

[0121] 1 H NMR(400MHz,Chloroform-d)δ7.90(d,J=8.5Hz,2H),7.74(d,J=8.1Hz,2H),7.67(d ,J=8.7Hz,2H),7.41–7.35(m,2H),7.22–7.15(m,6H),7.05–6.87(m,8H),6.68(d,J= 12.6Hz,2H),5.36(d,J=11.8Hz,2H),3.94–3.85(m,6H),3.70–3.62(m,4H),3.17–2 .99(m,4H),2.90–2.78(m,4H),1.82–1.73(m,16H),1.00(t,J=7.4Hz,6H).MALDI-FT ICR MS: calculated for C 69 H 70 N2O3[M] + 974.5386, found 974.5343.

[0122] (5) Synthesis of Compound B3

[0123] B3-(5) (0.02mmol, 20mg) was added to a Schlenk bottle under argon protection. Ultra-dry THF (2mL) was added to the bottle and cooled to 0°C in an ice-water bath. 1M 4-methoxyphenylmagnesium bromide (0.2mmol, 0.2mL) was then dropped into the solution. Stirred in a low-temperature reactor for 0.5h at a stirring rate of 800rpm, and 2mL of methanol was added to quench the reaction. Compound 4c was purified by basic alumina 300-400 mesh column chromatography (DCM:MeOH=25:1) to obtain a black solid (0.009mmol, 9.7mg) with an isolated yield of 41.2%.

[0124] 1H NMR(400MHz,Chloroform-d)δ7.98–7.77(m,6H),7.49–7.30(m,6H),7.15–7.08(m,4H),6.95–6.88(m,8H),6.82–6.75(m,4H),6.68–6.64(m,1H),6 .07(d,J=13.8Hz,1H),4.69–4.12(m,4H),3.75–3.60(m,9H),3.25–3.00( m,8H),2.05–1.78(m,8H),1.67(s,12H),0.97(t,J=7.2Hz,6H).MALDI-FT ICR MS:calculated for C 66 H 67 BrN2O3[M-Br] + 1065.5929,found 1065.5929,[M-2H-Br] + 1063.5772, found1063.5616.

[0125] Example 3

[0126] This embodiment provides a preparation of a fluorescent dye B13, and the compound structure and specific synthesis route are as follows:

[0127]

[0128] (1) Compound B1-(2) is the same as in the synthesis of B1.

[0129] (2) Compound B3-(3) is the same as in the synthesis of B3.

[0130] (3) Synthesis of Compound B13-(4)

[0131] A mixture of B3-(3) (0.5mmol, 197mg), phenylboronic acid (1mmol, 122mg), K2CO3, (1mmol, 138mg) and Pd(PPh3)4 (0.05mmol, 50mg) was added to a Shrek bottle under argon protection. Dry DMF (0.05mmol, 4mL) and deionized water (0.04mmol, 0.8mL) were added to the bottle and stirred at 90°C for 24h at a stirring rate of 800rpm. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated by rotary evaporation. The obtained organic matter was purified by column chromatography on silica gel (PE:EA=6:1) to obtain compound B13-(4), a red solid 0.45mmol, 197mg, with an isolated yield of 90.9%.

[0132] 1 H NMR(400MHz,Chloroform-d)δ9.55(s,1H),7.90(d,J=8.4Hz,1H),7.77(d,J=8.4Hz, 1H),7.71(d,J=8.8Hz,1H),7.53–7.44(m,3H),7.42–7.33(m,3H),7.25–7.20(m,1H) ,7.03(d,J=8.7Hz,1H),6.88(d,J=12.1Hz,1H),5.37(d,J=13.1Hz,1H),3.69(t,J=6 .7Hz,2H),2.92–2.81(m,4H),1.83–1.72(m,2H),1.68(s,6H),1.01(t,J=6.8Hz,3H). 13 CNMR(101MHz,Chloroform-d)δ189.6,163.0,161.2,141.9,140.5,140.2,133.3,129.8,129.8,129.7,129.6,129.5,128.9, 128.8,128.7,128.3,126.7,126.6,122.4,121.6,115.4,109.3,94.2,47.9,44.0,27.8,27.2,26.5,11.7.HRMS:calculated for C 31 H 31 NO[M+H] + 434.2478, found 434.2494.

[0133] (4) Synthesis of Compound B13-(5)

[0134] A mixture of B13-(4) 0.2mmol, 87mg, cyclopentanone 0.1mmol, 8.9μL, and NaOH 4mmol, 32mg was added to a round bottom bottle. 2mL of ethanol was added to the bottle and stirred at 80°C for 48h at a stirring rate of 800rpm. After cooling to room temperature, the reaction mixture was filtered and washed with ethanol to obtain compound B13-(5) as a blue solid 0.08mmol, 76mg with an isolated yield of 83%.

[0135] 1H NMR(400MHz,Chloroform-d)δ7.89(d,J=9.0Hz,2H),7.74(d,J=8.1Hz,2H),7.67(d ,J=8.7Hz,2H),7.46–7.33(m,8H),7.25–7.14(m,8H),6.99(d,J=8.8Hz,2H),6.62( d,J=12.6Hz,2H),5.35(d,J=11.4Hz,2H),3.75–3.58(m,4H),3.21–2.97(m,8H),2. 94–2.77(m,4H),1.80–1.72(m,4H),1.68(s,12H),1.00(t,J=7.4Hz,6H).MALDI-FT ICR MS: calculated for C 67 H 66 N2O[M] + 914.5175, found 914.5050.

[0136] (5) Synthesis of Compound B13

[0137] B13-(5), 0.01mmol, 9mg was added to a Schlenk bottle under argon protection. Ultra-dry THF (200mmol, 2mL) was added to the bottle and cooled to 0°C in an ice-water bath. Subsequently, 1M phenylmagnesium bromide (0.2mmol, 0.2mL) was dropped into the solution. Stirred in an ice-water bath for 0.5h at a stirring rate of 800rpm, and 2mL of methanol was added to quench the reaction. The obtained organic matter was purified by basic alumina (300-400 mesh) column chromatography (DCM:MeOH=25:1) to obtain 4mg of the compound as a black solid (0.004mmol, 4.0mg), with an isolated yield of 41.0%. The obtained solid was dissolved in acetonitrile, sodium tetraphenylborate (0.2 mmol, 68 mg) was added, and stirred at room temperature for 5 hours at a stirring rate of 800 rpm. The obtained organic matter was purified by basic alumina (300-400 mesh) column chromatography (DCM: MeOH 99:1) to obtain compound B13, 2.4 mg, a black solid (0.002 mmol, 2.4 mg), and the isolation yield was 46.3%.

[0138] 1H NMR(400MHz,Chloroform-d)δ7.96–7.92(m,2H),7.90–7.81(m,4H),7.49–7.39(m,13H),7.08–6.99(m,10H),6.89–6.82(m,3H),5.84(d,J= 14.0Hz,1H),3.79(t,J=7.2Hz,2H),3.67(t,J=6.8Hz,2H),3.49–2.88(m,8H),1.79–1.62(m,20H),0.91(t,J=7.2Hz,6H).HRMS:calculated for C 73 H 71 BrN2[M-Br] + 975.5612, found 975.5620.

[0139] Example 4

[0140] This example provides a preparation of a fluorescent dye B7, and the compound structure and specific synthesis route are as follows:

[0141]

[0142] (1) Compound B1-(2) is the same as in the synthesis of B1.

[0143] (2) Compound B3-(3) is the same as in the synthesis of B3.

[0144] (3) Compound B13-(4) is the same as in the synthesis of B13.

[0145] (4) Compound B13-(5) is the same as in the synthesis of B13.

[0146] (5) Synthesis of Compound B7

[0147] B13-(5) (0.01mmol, 9mg) was added to a Schlenk bottle under argon protection. Ultra-dry THF (200mmol, 2mL) was added to the bottle and cooled to 0°C in an ice-water bath. Subsequently, 1M phenylmagnesium bromide (0.2mmol, 0.2mL) was dropped into the solution. Stirred in an ice-water bath for 0.5h at a stirring rate of 800rpm, and 2mL of methanol was added to quench the reaction. The obtained organic matter was purified by basic alumina (300-400 mesh) column chromatography (DCM:MeOH=25:1) to obtain compound B7, 4mg, black solid (0.004mmol, 4.0mg), with an isolated yield of 41.0%.

[0148] 1H NMR(400MHz,Chloroform-d)δ7.96–7.92(m,2H),7.90–7.81(m,4H),7.49–7.39(m,13H),7.08–6.99(m,10H),6.89–6.82(m,3H),5.84(d,J= 14.0Hz,1H),3.79(t,J=7.2Hz,2H),3.67(t,J=6.8Hz,2H),3.49–2.88(m,8H),1.79–1.62(m,20H),0.91(t,J=7.2Hz,6H).HRMS:calculated for C 73 H 71 BrN2[M-Br] + 975.5612, found 975.5620.

[0149] The preparation methods of dyes B2, B4-B12 in general formula A are basically the same as those in Example 1 under the same reaction conditions, except that the corresponding reactants used are different.

[0150] The preparation method of dyes B13-B14 of general formula A is basically the same as the reaction conditions of Example 3, except that the corresponding reactants used are different.

[0151] Test Case

[0152] The dyes B1-B10 of the general formula A prepared in the above example were prepared into a concentration of 1×10 -5 M in DCM solution, scan and plot its UV-visible-near infrared absorption spectrum and fluorescence emission spectrum, as shown in the attached Figure 1 The relevant photophysical properties are shown in Table 1. The maximum absorption wavelength (λ abs ) is between 1100-1300nm, and the maximum emission wavelength (λ em ) is between 1150-1300nm, and the molar extinction coefficient (ε) is 4.8×10 4 -2.6×10 5 M -1 cm -1 The brightness is between 4.8-115.1M -1 cm -1 The fluorescence quantum yield (Φ) is higher.

[0153] Table 1 Photophysical data of Part A dyes.

[0154]

[0155] The dye B3 and B7 nanoparticles of the general formula A prepared in the above embodiment and ICG (indocyanine green) were respectively prepared into PBS solutions with a concentration of 0.15 mM, and fluorescence imaging test photos under different long-pass filters LP1300 and LP1500 were obtained using a chemiluminescence fluorescence imaging analysis system, as shown in the attached figure. Figure 2 As shown by Figure 2 It can be seen that the brightness of B3 and B7 at LP1300 is 5.1 and 4.1 times that of ICG at the same concentration. The brightness of B3 and B7 at LP1500 is 9.6 and 5.1 times that of ICG at the same concentration.

[0156] The dye B3 of general formula A prepared in the above examples was prepared into PBS solution with a concentration of 0-150 μM nanoparticles. 4T1 cells, 8000 cells / well, were incubated with B3 material for 12 hours. The survival rate of 4T1 cells is shown in the attached figure. Figure 3 As shown by Figure 3 It can be seen that B3 nanoparticles have no obvious cytotoxicity at a concentration of 150 μM.

[0157] The dye B3 nanoparticles of the general formula A prepared in the above example were prepared into a PBS solution with a concentration of 0.15 mM, and the NIR-IIb imaging of the hind limb blood vessels of Balb / c-nude mice was performed as shown in the attached figure. Figure 4 As shown by Figure 4 It can be seen that B3 nanoparticles can be used to image the hind limb blood vessels of mice, and can clearly distinguish the femoral artery, vein and multiple calf blood vessels, with a signal-to-background ratio of up to 6.8.

[0158] The dye B3 of the general formula A prepared in the above example was used for NIR-II vascular imaging of mice. Figure 5 As shown by Figure 5 It can be seen that B3 can clearly distinguish the blood vessels in the chest, abdomen and hind limbs of mice.

[0159] The dye B3 of general formula A prepared in the above example was used for NIR-II tumor imaging of mice. Figure 6 As shown by Figure 6 It can be seen that B3 is enriched in mouse in situ breast tumors, and the tumor background signal ratio to normal tissue can reach 4.8.

[0160] The above is a description of the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned description of the embodiments for the convenience of ordinary technicians in the technical field to understand and use the invention. It is obvious that those familiar with the technology in the field can easily make various modifications to these embodiments and apply the general principles described here to other embodiments without creative work. Therefore, the present invention is not limited to the above-mentioned embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A near-infrared small molecule fluorescent dye having a pentamethine conjugated chain, characterized in that: The small molecule fluorescent dye includes one or more of the structure shown in general formula A and the solvate of the structure shown in general formula A, and the general formula A is as follows: Where: R1 and R2 are each independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring, chlorine atom; R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne, C1-C6 sulfonic acid substituted alkane; n is independently selected from 1 or 2; X is independently selected from a bromine atom, tetraphenylboric acid, and tetrakis(pentafluorophenyl)boric acid.

2. A near-infrared small molecule dye having a pentamethine conjugated chain according to claim 1, characterized in that: The near-infrared small molecule dye has a maximum absorption wavelength between 1100-1300 nm, a maximum emission wavelength between 1150-1300 nm, and a molecular weight between 700-1300 Da; The brightness of the near-infrared small molecule dye is 4.8-115.1M -1 cm -1 , the molar extinction coefficient is 4.8×10 4 -2.6×10 5 M -1 cm -1 .

3. A near-infrared small molecule dye having a pentamethine conjugated chain according to claim 1, characterized in that: The structure of the small molecule fluorescent dye is any one of the following formulas B1-B14:

4. A method for preparing a near-infrared small molecule dye having a pentamethine conjugated chain as claimed in any one of claims 1 to 3, characterized in that: The steps include: Step 1: Carbon-nitrogen coupling reaction of the compound represented by formula (1) with a halide or sultone compound. The compound represented by formula (2) is obtained; Step 2: The compound represented by formula (2) and the aldehyde compound undergo a dehydration condensation reaction to obtain a compound represented by formula (3); Step 3: The compound represented by formula (3) is subjected to Suzuki coupling reaction to obtain a compound represented by formula (4); Step 4: The compound represented by formula (3) or formula (4) is subjected to Knoevenagel condensation reaction with cyclopentanone to obtain a compound represented by formula (5); Step 5, the compound represented by formula (5) is subjected to nucleophilic addition reaction under the action of lithium reagent or Grignard reagent to obtain the compound represented by general formula A or further reacts with the corresponding anion salt to obtain the compound represented by general formula A; Among them, formula (1) is Formula (2) is Formula (3) is Formula (4) is Formula (5) is 5. The method for preparing a near-infrared small molecule dye having a pentamethine conjugated chain according to claim 4, characterized in that: In step 1, the compound represented by formula (1) is subjected to carbon-nitrogen coupling reaction with a halide or sultone compound to obtain a compound represented by formula (2), which comprises the following process: In a first organic solvent environment, heating and stirring the compound represented by formula (1) and a halide or the compound represented by formula (1) and a sultone compound to obtain a compound represented by formula (2); The halogenated compound or sultone compound has a structure as shown in any of the following formulas C1, C2, and C3. Show: In formula C1-C3: R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne; The first organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ether, 1,4-dioxane, and glacial acetic acid.

6. The method for preparing a near-infrared small molecule dye having a pentamethine conjugated chain according to claim 4, characterized in that: In step 2, the compound represented by formula (2) and the aldehyde compound undergo a dehydration condensation reaction to obtain a compound represented by formula (3), which includes the following process: In a second organic solvent environment, the compound represented by formula (2) and the aldehyde compound are stirred to obtain a compound represented by formula (3); The aldehyde compound has a structure as shown in any of the following formulas D1 and D2: The second organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, acetic acid, tetrahydrofuran, ether, 1,4-dioxane, ethanol, and methanol.

7. The method for preparing a near-infrared small molecule dye having a pentamethine conjugated chain according to claim 4, characterized in that: In step 3, the compound represented by formula (3) undergoes a Suzuki coupling reaction to obtain a compound represented by formula (4), which includes the following process: In a third organic solvent environment, under the protection of an inert gas, the compound represented by formula (3) and the boric acid compound are heated and stirred with a palladium catalyst and a base as a catalyst to obtain a compound represented by formula (4) through a Suzuki coupling reaction; The boric acid compound has a structure as shown in the following formula E: R1-B(OH)2 E Where: R1 is independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring; The palladium catalyst is selected from one or more of tetrakistriphenylphosphine palladium, tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetonepalladium), and palladium acetate; The base is selected from one or more of potassium tert-butoxide, sodium tert-butoxide, potassium carbonate, cesium carbonate, and potassium phosphate; The third organic solvent is selected from one or more of benzene, toluene, xylene, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and diethyl ether.

8. The method for preparing a near-infrared small molecule dye having a pentamethine conjugated chain according to claim 4, characterized in that: In step 4, the compound represented by formula (3) or formula (4) and cyclopentanone undergo Knoevenagel condensation reaction to obtain the compound represented by formula (5), which includes the following process: In a fourth organic solvent environment, the compound represented by formula (3) or formula (4) is heated and stirred with cyclopentanone and a base as a catalyst to obtain a compound represented by formula (5) through a Knoevenagel condensation reaction; The base is selected from one or more of sodium hydroxide, sodium tert-butoxide, sodium ethoxide, and potassium hydroxide; The fourth organic solvent is selected from one or more of tetrahydrofuran, diethyl ether, 1,4-dioxane, ethanol, and methanol.

9. The method for preparing a near-infrared small molecule dye having a pentamethine conjugated chain according to claim 4, characterized in that: In step 5, the compound represented by formula (5) undergoes a nucleophilic addition reaction under the action of a lithium reagent or a Grignard reagent to obtain a compound represented by general formula A, or further reacts with a corresponding anion salt to obtain a compound represented by general formula A, comprising the following process: In a fifth organic solvent environment, under the protection of an inert gas, reacting the compound represented by formula (5) with a lithium reagent or a Grignard reagent at low temperature, undergoing a nucleophilic addition reaction to obtain a compound represented by general formula A, or further reacting with a corresponding anion salt to exchange anions to obtain a compound represented by general formula A; The lithium reagent or Grignard reagent has a structure as shown in any of the following formulas F1 and F2: Where: R2 are each independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring; The anion salt is selected from one or more of sodium tetraphenylborate and potassium tetrakis(pentafluorobenzene)borate; The fifth organic solvent is selected from one or more of super dry tetrahydrofuran, diethyl ether, and 1,4-dioxane.

10. Use of a near-infrared small molecule dye having a 15-methine conjugated chain as claimed in any one of claims 1 to 3 or a near-infrared small molecule dye having a 15-methine conjugated chain prepared by the preparation method as claimed in any one of claims 4 to 9, characterized in that: The near-infrared small molecule dye is used for tumor targeting imaging, in vivo vascular imaging, and fluorescence imaging.

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