CN117777751A

CN117777751A - Cyanine dye and application thereof

Info

Publication number: CN117777751A
Application number: CN202311780103.XA
Authority: CN
Inventors: 刘世勇; 张喻奔
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2023-12-21
Filing date: 2023-12-21
Publication date: 2024-03-29

Abstract

The invention provides a series of cyanine near-infrared two-region fluorescent dye molecules with different side group structures, which have structures shown as A-G. The invention adopts classical indocyanine green as a framework, by binding a functional group having hydrophilicity to the dye molecule, expands the functionality and improves the hydrophilicity of fluorescent small molecules, so that it can be assembled in water solution in a highly ordered manner. It is noted that, when the above cyanine dye is combined with FBS, the fluorescence intensity increases, has better photo-thermal effect, can reduce the enrichment of liver and spleen, can perform the specific imaging of lymph nodes, in the near infrared two-region imaging process, the biological background signal has smaller influence and stronger penetration depth, provides a novel technical means for the research and development of an accurate surgical operation navigation system.

Description

Cyanine dye and application thereof

Technical Field

The invention relates to the technical field of materials, in particular to a cyanine dye and application thereof.

Background

Fluorescence (Fluorescent) is a natural source is a common luminescence phenomenon. When the fluorescent substance is irradiated with excitation light, the internal molecules or atoms absorb photons from the ground state to the excited state, and then emits longer wavelength light, which is fluorescent light. Three scientists of Chris, pamela content and Beneron at the university of stenford in the middle 90 th century use a high-sensitivity CCD camera to track optical signals in a living mouse body, and pull open a prologue of living animal fluorescence imaging research.

The organic second near infrared (NIR-II) window has good biocompatibility, deep tissue permeability, high imaging resolution, and low autofluorescence, and thus has great promise in optical imaging. Attempts to achieve NIR-II emission in organic dyes have focused mainly on molecular engineering strategies by creating large pi-conjugated structures and installing strong electron donating (D) and electron accepting (a) groups. However, NIR-II emissive dyes are difficult to obtain due to the lack of a suitable pi-conjugated scaffold. Under such circumstances, there is an urgent need to explore an alternative route to obtain NIR-II dyes.

Cyanine dyes have a small molecular size and thus can readily cross cell membrane and tissue barriers. But also have a high fluorescence quantum yield, i.e. a high proportion of photons which after absorption are converted into emitted fluorescence, which makes them very suitable for detection of low fluorescence signals. Cyanine dyes generally emit fluorescence in the near infrared band (750-900 nm), and the light in this band can penetrate deep tissues, reducing absorption and scattering of fluorescence signals by the tissues. Cyanine dyes are relatively stable and have a long fluorescence lifetime, which makes them useful for long-term experimental observations.

In conclusion, the cyanine dyes have the advantages of high permeability, high fluorescence quantum yield, long wavelength emission, stability and the like in fluorescence living imaging, so that the cyanine dyes become important tools in the biomedical and chemical imaging fields.

By means of the mutual matching of different excitation light sources and different imaging wave bands, surrounding important structures (such as blood vessels and ureters) are clearly displayed while the focus part to be excised is tracked, and the adjacent important tissues and organs can be effectively prevented from being damaged on the premise of completely and thoroughly cleaning the lymph nodes. Meanwhile, for the metastatic lymph nodes which cannot be resected by operation, the lymph nodes with tumor metastasis can be effectively treated by utilizing nanoparticle photo-thermal treatment. The development of the dye can provide a new thought, a new direction and a new mode for the research and development of an accurate surgical navigation system. Can be used for fluorescence guided surgery and shows potential clinical application value.

The current fluorescent dye has the defects of poor light stability and current preparation in practical application, and the defects greatly limit the practical application range of the cyanine fluorescent probe.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a cyanine dye and application thereof, which can be applied to fluorescent living body imaging diagnosis.

Based on the above, the invention designs and synthesizes a series of cyanine near infrared two-region fluorescent dye molecules A-G with different side group structures:

specifically, the invention provides a cyanine dye containing a benzindole structure, which has a structure shown in a formula A or a formula B:

the invention provides a cyanine dye containing naphthalimide structure, which has a structure shown in a formula C or a formula D:

the invention provides a cyanine dye containing a binaphthyl imide structure, which has a structure shown in a formula E:

the invention provides a cyanine dye containing an aristololactam structure, which has a structure shown in a formula F:

the invention provides a nine-cyanine dye containing a benzindole structure, which has a structure shown in a formula G:

in the compounds A-G, preferably, the R ₁ Selected from ClO ^- 、I ^- 、BF ₄ ^- Or PF (physical pattern) ₆ ^- 。

Preferably, said R ₂ Selected from H or halogen;

preferably, said R ₃ Selected from any one of the following structures:

n is 0-100. Preferably any integer from 1 to 4.

Curved lineIndicating the connection location.

The preparation method of the compounds A-G is not particularly limited, and a specific compound of the compound A is taken as an example, and the reaction equation in the preparation process is as follows:

in some embodiments, the solvent in step (1) is selected from acetonitrile.

In some embodiments, the reaction temperature in step (1) is 60-90 ℃.

In some embodiments, the reaction time in step (1) is from 3 hours to 2 days.

In some embodiments, the solvent in step (2) is selected from ethanol.

In some embodiments, the reaction temperature in step (2) is from 90 to 110 ℃.

In some embodiments, the reaction time in step (2) is from 3 hours to 2 days.

In some embodiments, taking a specific compound of compound a as an example, the reaction equation for the preparation process is as follows:

in some embodiments, the solvent in step (1) is selected from acetonitrile.

In some embodiments, the reaction temperature in step (1) is 60-90 ℃.

In some embodiments, the reaction time in step (1) is from 3 hours to 2 days.

In some embodiments, the solvent in step (2) is selected from ethanol.

In some embodiments, the reaction time in step (2) is from 3 hours to 2 days.

In some embodiments, the solvent in step (3) is selected from methanol.

In some embodiments, the reaction temperature in step (3) is 15-35 ℃.

In some embodiments, the reaction time in step (3) is from 3 hours to 2 days.

in some embodiments, the solvent in step (1) is selected from acetonitrile.

In some embodiments, the reaction temperature in step (1) is 60-90 ℃.

In some embodiments, the reaction time in step (1) is from 3 hours to 2 days.

In some embodiments, the solvent in step (2) is selected from ethanol.

In some embodiments, the reaction time in step (2) is from 3 hours to 2 days.

In some embodiments, the solvent in step (3) is selected from methanol.

In some embodiments, the reaction temperature in step (3) is 15-35 ℃.

In some embodiments, the reaction time in step (3) is from 3 hours to 2 days.

In some embodiments, taking a specific compound of compound C as an example, the reaction equation for its preparation is as follows:

in some embodiments, the solvent in step (1) is selected from acetonitrile.

In some embodiments, the reaction temperature in step (1) is 60-90 ℃.

In some embodiments, the reaction time in step (1) is from 3 hours to 2 days.

In some embodiments, the solvent in step (2) is selected from tetrahydrofuran.

In some embodiments, the reaction temperature in step (2) is from 35 to 60 ℃.

In some embodiments, the reaction time in step (2) is from 3 hours to 2 days.

In some embodiments, the solvent in step (3) is selected from N, N-dimethylformamide.

In some embodiments, the reaction temperature in step (3) is from 35 to 60 ℃.

In some embodiments, the reaction time in step (3) is from 3 hours to 2 days.

In some embodiments, the solvent in step (4) is selected from ethanol.

In some embodiments, the reaction temperature in step (4) is from 90 to 110 ℃.

In some embodiments, the reaction time in step (4) is from 3 hours to 2 days.

In some embodiments, the solvent in step (5) is selected from methanol.

In some embodiments, the reaction temperature in step 5) is 15-35 ℃.

In some embodiments, the reaction time in step (5) is from 3 hours to 2 days.

In some embodiments, taking a specific compound of compound E as an example, the reaction equation for its preparation is as follows:

in some embodiments, the solvent in step (1) is selected from acetonitrile.

In some embodiments, the reaction temperature in step (1) is 60-90 ℃.

In some embodiments, the reaction time in step (1) is from 3 hours to 2 days.

In some embodiments, the solvent in step (2) is selected from tetrahydrofuran.

In some embodiments of the present invention, in some embodiments, the reaction temperature in the step (2) is 35-60 ℃.

In some embodiments, the reaction time in step (2) is from 3 hours to 2 days.

In some embodiments, the reaction temperature in step (3) is from 35 to 60 ℃.

In some embodiments, the reaction time in step (3) is from 3 hours to 2 days.

In some embodiments, the solvent in step (4) is selected from ethanol.

In some embodiments, the reaction time in step (4) is from 3 hours to 2 days.

In some embodiments, the solvent in step (5) is selected from methanol.

In some embodiments, the reaction temperature in step 5) is 15-35 ℃.

In some embodiments, the reaction time in step (5) is from 3 hours to 2 days.

In some embodiments, taking a specific compound of compound F as an example, the reaction equation for the preparation process is as follows:

in some embodiments, the solvent in step (1) is selected from acetonitrile.

In some embodiments, the reaction temperature in step (1) is 60-90 ℃.

In some embodiments, the reaction time in step (1) is from 3 hours to 2 days.

In some embodiments, the solvent in step (2) is selected from tetrahydrofuran.

In some embodiments, the reaction temperature in step (2) is from 35 to 60 ℃.

In some embodiments, the reaction time in step (2) is from 3 hours to 2 days.

In some embodiments, the reaction temperature in step (3) is from 35 to 60 ℃.

In some embodiments, the reaction time in step (3) is from 3 hours to 2 days.

In some embodiments, the solvent in step (4) is selected from ethanol.

In some embodiments, the reaction time in step (4) is from 3 hours to 2 days.

In some embodiments, the solvent in step (5) is selected from methanol.

In some embodiments, the reaction temperature in step (5) is 15-35 ℃.

In some embodiments, the reaction time in step (5) is from 3 hours to 2 days.

In some embodiments, taking a specific compound of compound G as an example, the reaction equation for the preparation process is as follows:

in some embodiments, the solvent in step (1) is selected from acetonitrile.

In some embodiments, the reaction temperature in step (1) is 60-90 ℃.

In some embodiments, the reaction time in step (1) is from 3 hours to 2 days.

In some embodiments, the solvent in step (2) is selected from ethanol.

In some embodiments, the reaction time in step (2) is from 3 hours to 2 days.

Other compounds can be prepared by the same method as described above, with the replacement of the reaction starting materials.

The invention provides a cyanine near infrared two-region fluorescent probe nanoparticle assembly which is prepared by mixing an organic solution of a cyanine dye with water in a flash precipitation mode and then performing supermolecule self-assembly.

Organic solutions of said cyanine dyes the concentration of (C) is preferably 0.1 to 100mg/mL.

Said organic solution the solvent is preferably dimethylsulfoxide.

The organic solution of the cyanine dye may be mixed with water in any ratio, and preferably the volume of the water is 0.1 to 100 times that of the organic solution of the cyanine dye.

In some embodiments, the temperature of self-assembly in water is 15-35 ℃, preferably 25 ℃.

In some embodiments, the time of agitation is from 10 seconds to 5 minutes, preferably 20 seconds.

The prepared nanoparticle assembly has the shape of nanoparticles, the diameter of the nanoparticles is preferably in the range of 0.5-10 mu m, and the length of the nanoparticles is preferably in the range of 0.1-100 mu m.

The cyanine dye prepared by the invention can be applied to advanced diagnosis and living body imaging, has flexible structural design, can adjust the fluorescence performance by adjusting the structure and chemical property of molecules, and has higher in vivo precision in imaging application.

Based on this, the present invention provides the use of the nanoparticle assemblies described above as fluorescent in vivo imaging diagnostic fluorescent agents.

Compared with the prior art, the invention provides a series of cyanine near-infrared two-region fluorescent dye molecules with different side group structures, and the cyanine near-infrared two-region fluorescent dye molecules have structures shown as A-G. The invention adopts classical indocyanine green as a framework frame, and combines hydrophilic functional groups onto dye molecules to expand the functionality of the dye molecules and improve the hydrophilicity of fluorescent small molecules, so that the dye molecules can be assembled in water solution in a highly ordered manner. It is worth noting that after the cyanine dye is combined with FBS, the fluorescence intensity is increased, the photo-thermal effect is better, the enrichment of livers and spleens can be reduced, the specific imaging of lymph nodes can be carried out, the influence of biological background signals is smaller in the near infrared two-region imaging process, the penetration depth is stronger, and a novel technical means is provided for the research and development of an accurate surgical operation navigation system.

Drawings

FIG. 1 shows a corresponding matrix assisted flight mass spectrum of a cyanine dye (A);

FIG. 2 shows the nuclear magnetic resonance spectrum of the cyanine dye (A);

FIG. 3 shows a corresponding matrix assisted flight mass spectrum of the cyanine dye (B);

FIG. 4 shows a corresponding matrix assisted flight mass spectrum of a cyanine dye (C);

FIG. 5 shows a corresponding matrix assisted flight mass spectrum of a cyanine dye (E);

FIG. 6 shows a corresponding matrix assisted flight mass spectrum of a cyanine dye (F);

FIG. 7 shows a corresponding matrix assisted flight mass spectrum of a cyanine dye (G);

FIG. 8 is a TEM photograph of an assembly of the cyanine dye (A) prepared according to the present invention formed by supramolecular self-assembly;

FIG. 9 shows the ultraviolet absorption spectrum of the cyanine dye (A);

fig. 10 shows the ultraviolet absorption spectrum of the cyanine dye (B);

FIG. 11 shows the ultraviolet absorption spectrum of the cyanine dye (C);

FIG. 12 shows the ultraviolet absorption spectrum of the cyanine dye (D);

FIG. 13 shows the ultraviolet absorption spectrum of the cyanine dye (E);

FIG. 14 shows the ultraviolet absorption spectrum of the cyanine dye (F);

FIG. 15 shows in vivo fluorescence imaging results of mice of cyanine dyes prepared in accordance with the present invention;

FIG. 16 shows in vivo lymphatic fluorescence imaging results of mice of cyanine dyes prepared in accordance with the present invention.

Detailed Description

In order to further illustrate the present invention, the cyanine dyes and their applications provided in the present invention are described in detail below with reference to examples.

The raw materials used in the invention are described as follows:

para-toluenesulfonyl chloride, tetraethylene glycol di-para-toluenesulfonate, sodium iodide, 1, 2-trimethyl-1H-benzo [ e ]]Indole, N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene]The aniline hydrochloride was purchased from Annaiji chemistry and used as such. Pentaethylene glycol, croconic acid was purchased from carbofuran and used as received. Tripropylene glycol is purchased from the chinese scientific research and used as such. 1, 8-naphthalimide was purchased from Pichia medicine and used as received. Perchloric acid was purchased from Sigma-Aldrich and used as received. Sodium carbonate (Na) ₂ CO ₃ ) Anhydrous sodium acetate, ethyl acetate (EtOAc), tetrahydrofuran (THF), methanol, diethyl ether, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), toluene, acetone, isopropanol were purchased from national medicine control chemical company and used as received. The use of Milli-Q SP reagent water system (Millipor)e) Deionized (DI) water to achieve a resistivity of 18.4mΩ cm. Unless otherwise indicated, all other reagents were purchased from national pharmaceutical group chemical company limited, and as is and (3) using.

EXAMPLE 1 cyanine dye A

In the first step, diethylene glycol monomethyl ether substituted derivative 1 (diethylene glycol monomethyl ether derivative of 1, 2-trimethyl-1H-benzo [ e ] indole) is synthesized by the following synthetic route:

iododiglycol monomethyl ether (1.15 g,5 mmol) was dissolved in 15mL acetonitrile, 1, 2-trimethyl-1H-benzo [ e ] indole (1.25 g,6 mmol) was added, sonicated to complete dissolution, and the reaction was refluxed at 90℃for continued overnight reaction. The solvent was evaporated and purified by column chromatography (DCM: methanol=10:1) to give 1.01g (96.5% purity, 64.1% yield) of the product as a green oil.

And a second step of: the cyanine dye 2 is synthesized through the condensation reaction of methyl and aniline, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 1 (312 mg,1 mmol), N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene]Aniline hydrochloride (172.5 mg,0.5 mmol) and anhydrous sodium acetate (40.5 mg,0.5 mmol) were added to 25mL of toluene to azeotropically remove water three times, then 15mL of anhydrous ethanol was added thereto, and the mixture was reacted under reflux at 100℃for 4 hours under dark conditions, insoluble salts were removed by filtration, and the evaporated solvent was purified by spin-drying (DCM: methanol=10:1) to give 214mg (purity 98.6%, yield 29.8%) of a green solid product. The structure was also demonstrated by matrix assisted-flight mass spectrometry (MALDI-TOF), and the results are shown in FIG. 1. At the same time, nuclear magnetic resonance spectrum is used ¹ H NMR) also verifies the correctness of its structure, as shown in fig. 2.

EXAMPLE 2 cyanine dye B

In the first step, diethylene glycol monomethyl ether substituted derivative 1 (diethylene glycol derivative of 1, 2-trimethyl-1H-benzo [ e ] indole) is synthesized, and the specific reaction formula is as follows:

the preparation method comprises the following steps: 1, 2-trimethyl-1H-benzo [ e ] indole (9.13 g,43.64 mmol) and iodo-diethylene glycol mono-trityl (10 g,21.82 mmol) were added to a 250mL round bottom flask, dissolved in 150mL anhydrous acetonitrile and stirred at 90℃under reflux. After the solvent was removed by rotary evaporation, the reaction mixture was purified by column chromatography (DCM: methanol=50:1) to give 117.4g of intermediate (purity 97.4%, yield 74.13%).

And a second step of: intermediate 2 is synthesized by the condensation reaction of methyl and aniline, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 1 (540 mg,1 mmol), N- [ (3- (anilinomethylene) -2-chloro-1-cyclopenten-1-yl) methylene ] aniline hydrochloride (172.5 mg,0.5 mmol) and anhydrous sodium acetate (40.5 mg,0.5 mmol) were added to azeotropically remove water three times with 25mL toluene, then 15mL absolute ethanol was added thereto, the reaction was refluxed at 100℃for 4 hours under dark conditions, insoluble salts were removed by filtration, and the evaporated solvent was purified by column chromatography (DCM: methanol=10:1) to give 687mg (purity 95.5%, yield 56.4%) of a green solid product.

And a third step of: the trityl protection of the p-toluenesulfonic acid is removed, and a final product B is synthesized, wherein the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 2 (100 mg,0.083 mmol) was placed in a 50mL round bottom flask, 9mL of dichloromethane was added to dissolve, 1mL of methanol was further added, p-toluene sulfonic acid (15.7 mg,0.083 mmol) was dissolved in 1mL of dichloromethane and slowly added dropwise to the system, and stirred at room temperature for 12h. Sedimentation in diethyl ether, dissolution of the solid with DCM, column chromatography (200-300 mesh, DCM: meoh=6:1) and collection of the target component gave 43mg (purity 98.4, yield 70.7%) of brown solid. The structure was also confirmed by matrix assisted-flight mass spectrometry (MALDI-TOF), and the results are shown in FIG. 3.

EXAMPLE 3 cyanine dye C

In the first step, diethylene glycol monomethyl ether substituted derivative 1 is synthesized by the following specific synthetic route:

the preparation method comprises the following steps: naphthalimide (10 g,18.3 mmol) was dissolved in 30mL of N, N-dimethylformamide, stirred for 5 minutes under ice bath, and sodium hydride (0.44 g,18.3 mmol) was added to the reaction system, and stirred for 10 minutes under ice bath. 13-iodo-1, 1-triphenyl-2, 5,8, 11-tetraoxytridecane (11.99 g,21.96 mmol) was slowly added dropwise to the reaction system, and reacted overnight at room temperature. The solvent was evaporated and purified by column chromatography (DCM: methanol=10:1) to give product 1 as a yellow oil, 8.8g (purity 94.8%, yield 81.9%).

And a second step of: the methylated naphthalimide derivative is synthesized by a Grignard reagent, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 1 (1.2 g,2.04 mmol) was placed in a 100mL two-necked round bottom flask, 20mL of anhydrous tetrahydrofuran was added to dissolve, 3M methyl magnesium chloride (3.06 mL,9.19 mmol) was added, and stirring was performed at 60℃for 1 hour under nitrogen atmosphere, cooled to room temperature, 25wt% aqueous tetrafluoroboric acid solution 3.2mL was added, and stirring was continued at room temperature for 10 minutes. Dichloromethane was added for dissolution, extracted twice with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography (200-300 mesh, PE: ea=6:1), and the objective component was collected to give 0.38g (purity 84%, yield 31.7%) of a dark green viscous liquid.

And a third step of: preparation of cyclohexene chloride containing aldehyde group and enol structure, wherein the specific reaction formula is as follows:

the preparation method comprises the following steps: 40mL of methylene chloride and 40mL of N, N-dimethylformamide were added to a 250mL round bottom flask, phosphorus oxychloride (45.81 g,305.65 mmol) was added under ice-bath stirring, cyclohexanone (10 g,101.88 mmol) was added after stirring in an ice bath for 20 minutes, and the mixture was refluxed at 60℃for 3 hours. Cooling in ice bath, and recrystallizing in a refrigerator at-20deg.C to obtain yellow solid. Suction filtration, washing the solid with ice water for 3 times, and drying to obtain 7.5g (purity 91.2%, yield 42.3%) of yellow solid.

Fourth step: intermediate 4 is synthesized to prepare naphthalimide cyanine dye containing trityl protection, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 2 (0.89 g,1.52 mmol), 3 (0.12 g,0.69 mmol) and anhydrous sodium acetate (0.057 g,0.69 mmol) were placed in a 100mL two port round bottom flask and 30mL acetic anhydride was added to react for 2 hours at room temperature, the solid was precipitated in diethyl ether, dissolved in dichloromethane, chromatographed (200-300 mesh, DCM: meOH=10:1), and the target component was collected as 82mg of brown gummy solid (96.6% purity, 41.21% yield).

Fifth step: the final target product 5 naphthalimide cyanine dye is synthesized, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 4 (100 mg,0.076 mmol) was placed in a 50mL round bottom flask, 9mL of dichloromethane was added to dissolve, 1mL of methanol was further added, p-toluenesulfonic acid (14 mg,0.076mmol dissolved in 1mL of dichloromethane was slowly added dropwise to the system, stirred at room temperature for 12h, settled in diethyl ether, the solid was dissolved in DCM, column chromatographed (200-300 mesh, DCM: meOH=6:1), and the target component was collected to give 32mg (purity 98.6%, yield 51.07%) of a brown solid, the structure of which was also demonstrated by matrix assisted flight mass spectrometry (MALDI-TOF), the results of which are shown in FIG. 4.

EXAMPLE 4 cyanine dyes E

In the first step, the first step is to provide, synthesizing tetraethylene glycol monomethyl ether substituted derivative 1, specific Synthesis the route is as follows:

the preparation method comprises the following steps: dinaphthalimide (0.5 g,1.69 mmol) was dissolved in 5mL of N, N-dimethylformamide, stirred under ice bath for 5 minutes, and sodium hydride (0.5 g,1.69 mmol) was added to the reaction system and stirred under ice bath for 10 minutes. 13-iodo-1, 1-triphenyl-2, 5,8, 11-tetraoxytridecane (1.11 g,2.03 mmol) was slowly added dropwise to the reaction system, and reacted overnight at room temperature. The solvent was evaporated and purified by column chromatography (DCM: methanol=10:1) to give product 1 as a yellow oil, quality 0.95g (purity 96.6%, yield 79.3%).

And a second step of: the methylated bisnaphthalimide derivative is synthesized by a Grignard reagent, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 1 (0.5 g,0.7 mmol) was placed in a 100mL two-necked round bottom flask, 20mL of anhydrous tetrahydrofuran was added to dissolve, 3M methyl magnesium chloride (1.05 mL,3.15 mmol) was added, and stirring was continued at room temperature for 10 minutes at 60℃for 1 hour under nitrogen atmosphere, cooled to room temperature, and 25wt% aqueous tetrafluoroboric acid solution 1.1mL was added. Dichloromethane was added for dissolution, extracted twice with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography (200-300 mesh, PE: ea=6:1), and the objective component was collected to give 0.16g (purity 81%, yield 32.6%) of a dark green viscous liquid.

And a third step of: the intermediate 3 is synthesized to prepare the bis-naphthalimide cyanine dye containing trityl protection, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 2 (0.16 g,0.22 mmol), N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene ] anilide hydrochloride (0.08 g,0.11 mmol), sodium acetate (0.009 g,0.11 mmol) was placed in a 50mL two-necked round bottom flask, 10mL of an N-butanol/toluene mixed solvent was added, and the mixture was reacted at 120℃for 3 hours under nitrogen atmosphere, cooled to room temperature, and settled 2 times in diethyl ether. Column chromatography (200-300 mesh, DCM: meoh=10:1-5:1) afforded the target component as a black solid 107mg (96.9% purity, 31.21% yield).

Fourth step: the final target product 4 is synthesized by the bis-naphthalimide cyanine dye, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 3 (100 mg,0.06 mmol) was placed in a 50mL round bottom flask, 9mL of dichloromethane was added to dissolve, 1mL of methanol was further added, p-toluene sulfonic acid (11.4 mg,0.06 mmol) was dissolved in 1mL of dichloromethane and slowly added dropwise to the system, and stirring was performed at room temperature for 12h. Sedimentation in diethyl ether, dissolution of the solid with DCM, column chromatography (200-300 mesh, DCM: meoh=6:1) and collection of the target component gave 36mg (purity 98.8%, yield 53.71%) of brown solid. The structure was also confirmed by matrix assisted-flight mass spectrometry (MALDI-TOF), and the results are shown in FIG. 5.

Example 5 cyanine dyes F

In the first step, tetraethylene glycol monomethyl ether substituted derivative 1 is synthesized, and the specific synthetic route is as follows:

the preparation method comprises the following steps: aristolochia lactam (0.5 g,1.7 mmol) was dissolved in 5mL of N, N-dimethylformamide, stirred for 5 minutes in an ice bath, and sodium hydride (0.041 g,1.7 mmol) was added to the reaction system and stirred for 10 minutes in an ice bath. 13-iodo-1, 1-triphenyl-2, 5,8, 11-tetraoxytridecane (0.93 g,1.7 mmol) was slowly added dropwise to the reaction system, and reacted overnight at room temperature. The solvent was evaporated and purified by column chromatography (DCM: methanol=10:1) to give product 1 as a yellow oil, 0.93g (purity 97.6%, yield 77.3%).

And a second step of: the methylated aristololactam derivative is synthesized by Grignard reagent, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 1 (0.93 g,1.31 mmol) was placed in a 100mL two-necked round bottom flask, 20mL of anhydrous tetrahydrofuran was added to dissolve, 3M methyl magnesium chloride (1.96 mL,5.9 mmol) was added, and stirring was performed at 60℃for 1 hour under a nitrogen atmosphere, cooled to room temperature, 25wt% aqueous tetrafluoroboric acid solution 1.1mL was added, and stirring was continued at room temperature for 10 minutes. Dichloromethane was added for dissolution, extracted twice with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography (200-300 mesh, PE: ea=6:1), and the objective component was collected to give 0.5g (purity 88%, yield 32.1%) of a dark green viscous liquid.

And a third step of: intermediate 3 is synthesized to prepare the aristololactam cyanine dye containing trityl protection, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 2 (0.5 g,0.42 mmol), N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene ] anilide hydrochloride (0.150 g,0.42 mmol), sodium acetate (0.034 g,0.42 mmol) was placed in a 50mL two port round bottom flask, 10mL of an N-butanol/toluene mixed solvent was added, reacted under nitrogen atmosphere at 120℃for 3h, cooled to room temperature, and settled 2 times in diethyl ether. Column chromatography (200-300 mesh, DCM: meoh=10:1-5:1) collected the target component yielded 249mg (purity 97.9%, yield 36.11%) of a black solid.

Fourth step: the final target product 4 is synthesized by the aristololactam cyanine dye, and the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 3 (249 mg,0.15 mmol) was placed in a 50mL round bottom flask, 9mL of dichloromethane was added to dissolve, 1mL of methanol was further added, p-toluene sulfonic acid (28.5 mg,0.15 mmol) was dissolved in 1mL of dichloromethane and slowly added dropwise to the system, and stirring was performed at room temperature for 12h. Sedimentation in diethyl ether, dissolution of the solid with DCM, column chromatography (200-300 mesh, DCM: meoh=6:1) and collection of the target component gave 140mg (purity 98.3%, yield 56.91%) of a brown solid. The structure was also confirmed by matrix assisted-flight mass spectrometry (MALDI-TOF), and the results are shown in FIG. 6.

EXAMPLE 6 cyanine dye G

The precursor of the nine-methyl cyanine dye G is the same as the synthesis method of the dye A

And a second step of: synthesizing the cyanine dye 2 through condensation reaction of methyl and aniline, the specific reaction formula is as follows:

the preparation method comprises the following steps: intermediate 1 (312 mg,1 mmol), pimelic aldehyde diphenylamine hydrochloride (155 mg,0.5 mmol) and anhydrous sodium acetate (40.5 mg,0.5 mmol) were azeotropically dehydrated three times by adding 25mL of toluene, then 15mL of anhydrous ethanol was added thereto, and reacted under reflux at 100 ℃ for 4 hours in dark condition, insoluble salts were removed by filtration, and the solvent was distilled off by spin-drying, and purified by column chromatography (DCM: methanol=10:1) to give 226mg (purity 98.9%, yield 31.8%) of a green solid product. The structure was also confirmed by matrix assisted-flight mass spectrometry (MALDI-TOF), and the results are shown in FIG. 7.

Example 7 preparation of cyanine near-infrared two-component preparation with protein regulatory function

2mg of cyanine dye A was dissolved in 2mL of dimethyl sulfoxide to obtain an organic solution. 100. Mu.l of this solution was taken, flash-sinking into 1900. Mu.l of deionized water stirred at high speed on a stirrer, stirred for 30s, and then allowed to stand at room temperature for 4 hours under dark conditions for use. The morphology of the assembly was observed by a transmission electron microscope, and the result is shown in fig. 8. In FIG. 8, a) -d) are the microstructures observed at the 0.5 μm scale of the assemblies prepared by means of the cyanine dyes A. From fig. 8, it can be seen that the prepared assembly has a structure of nano micelle.

Example 8 cyanine near infrared two-region dye with protein control function and ultraviolet absorption Spectrum of its Assembly

The cyanine dyes A to G were each taken at 2mg and dissolved in 2mL of dimethyl sulfoxide to obtain an organic solution. 100 microliters of the configured solution was flash-settled into 1900 microliters of deionized water stirred at high speed on a stirrer, stirred for 30s and then left to stand at room temperature for 4 hours under dark conditions for later use. The ultraviolet absorption spectrum was measured by an ultraviolet absorption spectrometer, and the results are shown in FIGS. 9 to 14. From fig. 9-14, it can be seen that near infrared two-region imaging diagnosis and treatment reagents with different wavelength structures are successfully prepared, the ultraviolet absorption wavelength can reach 1078m, and the near infrared two-region imaging diagnosis and treatment reagent can be used for near infrared two-region fluorescence imaging.

Example 9 fluorescence imaging study of cyanine near-infrared two-region Assembly with protein Regulation function

Nude mice (supplied by university of Anhui medical science animal center) were selected for animal experiments. Female nude mice were randomly selected and anesthetized by inhalation of isoflurane. In vivo imaging of mice was performed using a living imager. The cyanine dye a assembly was injected at a dose of 0.5mL/kg via the tail vein. As shown in FIG. 15, it was found that the assembly prepared in the present application had strong fluorescence signals at various sites in the mouse body. The following two figures show that after 96 hours of in vivo imaging of mice, dissection was performed, and that after 96 hours, higher fluorescence intensity was still observed in the liver and spleen under the filters of LP900 and LP1000, respectively.

EXAMPLE 10 cyanine near-infrared two-region Assembly lymph-specific fluorescence imaging study with protein Regulation function

Nude mice (supplied by university of Anhui medical science animal center) were selected for animal experiments. Female nude mice were randomly selected and anesthetized by inhalation of isoflurane. Living imaging of mice was performed using a near infrared two-zone living imager equipped with an infrared camera. The cyanine dye a assembly was injected through the footpad at a dose of 0.5 mL/kg. As shown in FIG. 16, it was found that the assembly prepared in the present application had strong fluorescence signals at various sites in the mouse body.

The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

Claims

1. A cyanine dye containing a benzindole structure, having a structure represented by formula a or formula B:

2. a cyanine dye containing naphthalimide structure, having a structure represented by formula C or formula D:

3. a cyanine dye containing a binaphthyl imide structure, having a structure represented by formula E:

4. a cyanine dye containing an aristololactam structure, having a structure represented by formula F:

5. a nine-cyanine dye containing a benzindole structure, which has a structure shown in a formula G:

6. the cyanine dye of any of claims 1 to 5, wherein R ₁ Selected from ClO ^- 、I ^- 、BF ₄ ^- Or PF (physical pattern) ₆ ^- ；

R ₂ Selected from H or halogen;

R ₃ selected from any one of the following structures:

n is 0-100.

7. A nanoparticle assembly prepared by mixing the organic solution of the cyanine dye according to any one of claims 1 to 6 with water by flash precipitation and then self-assembling by supermolecules.

8. Nanoparticle assembly according to claim 7, wherein the concentration of the organic solution of the cyanine dye is between 0.1 and 100mg/mL;

the volume of the water is 0.1-100 times of that of the organic solution of the cyanine dye.

9. The nanoparticle assembly of claim 7, wherein the nanoparticle assembly has a diameter of 0.5 to 10 μm and a length of 0.1 to 100 μm.

10. Use of the nanoparticle assembly of any one of claims 7 to 9 as a fluorescent in vivo imaging diagnostic fluorescent agent.