A type of cyanine dye and its application

Abstract

This invention provides a series of cyanine-based near-infrared II fluorescent dye molecules with different side-group structures, exhibiting structures as shown in A-G. This invention uses the classic indocyanine green as the backbone framework, expanding the functionality and improving the hydrophilicity of the fluorescent small molecules by incorporating hydrophilic functional groups, enabling highly ordered assembly in aqueous solution. Notably, when the above-mentioned cyanine dyes are combined with FBS, the fluorescence intensity increases, exhibiting better photothermal effects, reducing liver and spleen accumulation, and enabling specific imaging of lymph nodes. During near-infrared II imaging, the influence of biological background signals is reduced, and the penetration depth is stronger, providing a novel technical means for the development of precision surgical navigation systems.

Description

Technical Field

[0001] This invention relates to the field of materials technology, and in particular to a cyanine dye and its applications. Background Technology

[0002] Fluorescence is a common luminescent phenomenon in nature. When a fluorescent material is irradiated with excitation light, its molecules or atoms absorb photons and transition from the ground state to an excited state, subsequently emitting longer-wavelength emitted light, which is fluorescence. In the mid-1990s, three scientists at Stanford University, Chris, Pamela Contage, and Beneroon, used a highly sensitive CCD camera to track optical signals inside live mice, thus opening the door to in vivo fluorescence imaging research in small animals.

[0003] Organic second near-infrared (NIR-II) windows possess excellent biocompatibility, deep tissue penetration, high imaging resolution, and low autofluorescence, thus showing great promise in optical imaging. Attempts to achieve NIR-II emission in organic dyes have primarily focused on molecular engineering strategies, such as creating large π-conjugated structures and mounting strong electron-donating (D) and electron-accepting (A) groups. However, due to the lack of suitable π-conjugated scaffolds, NIR-II emitting dyes are difficult to obtain. Therefore, there is an urgent need to explore alternative pathways for obtaining NIR-II dyes.

[0004] Cyanide dyes have a small molecular size, allowing them to easily penetrate cell membranes and tissue barriers. They also exhibit high fluorescence quantum yields, meaning a high proportion of absorbed photons are converted into emitted fluorescence, making them ideal for detecting low-fluorescence signals. Cyanide dyes typically emit fluorescence in the near-infrared band (750-900 nm), a wavelength that can penetrate deep tissues, reducing tissue absorption and scattering of the fluorescence signal. Cyanide dyes are relatively stable and have a long fluorescence lifetime, making them suitable for long-term experimental observation.

[0005] In summary, cyanine dyes possess advantages such as high permeability, high fluorescence quantum yield, long wavelength emission, and stability in fluorescence in vivo imaging, making them important tools in the fields of biomedical and chemical imaging.

[0006] By combining different excitation light sources and imaging wavelengths, this method can clearly display surrounding important structures (such as blood vessels and ureters) while tracing the lesion to be removed, effectively ensuring that adjacent vital organs are not damaged while completely and thoroughly clearing lymph nodes. Furthermore, for metastatic lymph nodes that cannot be surgically removed, photothermal therapy using nanoparticles can effectively treat lymph nodes with tumor metastases. The development of this dye provides new ideas, directions, and models for the research and development of precision surgical navigation systems. Its potential clinical application value can be demonstrated in fluorescence-guided surgery.

[0007] Current fluorescent dyes often suffer from poor photostability and require preparation before use in practical applications, which greatly limits the practical application range of cyanin-based fluorescent probes. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a cyanine dye and its application, which can be applied to fluorescence in vivo imaging diagnosis.

[0009] Based on this, the present invention designed and synthesized a series of cyanin-based near-infrared II fluorescent dye molecules AG with different side group structures:

[0010] Specifically, the present invention provides a cyanine dye containing a benzoindole structure, having the structure shown in Formula A or Formula B:

[0011]

[0012] This invention provides a cyanine dye containing a naphthalimide structure, having the structure shown in formula C or formula D:

[0013]

[0014] This invention provides a cyanine dye containing a binafenimide structure, having the structure shown in Formula E:

[0015]

[0016] This invention provides a cyanin dye containing an aristolochic acid lactam structure, having the structure shown in Formula F:

[0017]

[0018] This invention provides a benzoindole-based cyanine dye having the structure shown in Formula G:

[0019]

[0020] In compound AG, preferably, R1 is selected from ClO - I- BF4 - or PF6 - .

[0021] Preferably, R2 is selected from H or halogens;

[0022] Preferably, R3 is selected from any of the following structures:

[0023]

[0024] n is between 0 and 100. Preferably, it is any integer from 1 to 4.

[0025] Curved line Indicates the connection location.

[0026] This invention does not specifically limit the preparation method of the above-mentioned compound AG. Taking a specific compound A as an example, the reaction equation for its preparation process is as follows:

[0027]

[0028] In some implementations, the solvent in step (1) is selected from acetonitrile.

[0029] In some implementations, the reaction temperature in step (1) is 60-90°C.

[0030] In some implementations, the reaction time in step (1) is 3 hours to 2 days.

[0031] In some implementations, the solvent in step (2) is selected from ethanol.

[0032] In some implementations, the reaction temperature in step (2) is 90-110°C.

[0033] In some implementations, the reaction time in step (2) is 3 hours to 2 days.

[0034] In some implementation schemes, taking compound A as an example, the reaction equation for its preparation process is as follows:

[0035]

[0036] In some implementations, the solvent in step (1) is selected from acetonitrile.

[0037] In some implementations, the reaction temperature in step (1) is 60-90°C.

[0038] In some implementations, the reaction time in step (1) is 3 hours to 2 days.

[0039] In some implementations, the solvent in step (2) is selected from ethanol.

[0040] In some implementations, the reaction temperature in step (2) is 90-110°C.

[0041] In some implementations, the reaction time in step (2) is 3 hours to 2 days.

[0042] In some implementations, the solvent in step (3) is selected from methanol.

[0043] In some implementations, the reaction temperature in step (3) is 15-35°C.

[0044] In some implementations, the reaction time in step (3) is 3 hours to 2 days.

[0045] In some implementation schemes, taking compound A as an example, the reaction equation for its preparation process is as follows:

[0046]

[0047] In some implementations, the solvent in step (1) is selected from acetonitrile.

[0048] In some implementations, the reaction temperature in step (1) is 60-90°C.

[0049] In some implementations, the reaction time in step (1) is 3 hours to 2 days.

[0050] In some implementations, the solvent in step (2) is selected from ethanol.

[0051] In some implementations, the reaction temperature in step (2) is 90-110°C.

[0052] In some implementations, the reaction time in step (2) is 3 hours to 2 days.

[0053] In some implementations, the solvent in step (3) is selected from methanol.

[0054] In some implementations, the reaction temperature in step (3) is 15-35°C.

[0055] In some implementations, the reaction time in step (3) is 3 hours to 2 days.

[0056] In some implementation schemes, taking compound C as an example, the reaction equation for its preparation process is as follows:

[0057]

[0058] In some implementations, the solvent in step (1) is selected from acetonitrile.

[0059] In some implementations, the reaction temperature in step (1) is 60-90°C.

[0060] In some implementations, the reaction time in step (1) is 3 hours to 2 days.

[0061] In some implementations, the solvent in step (2) is selected from tetrahydrofuran.

[0062] In some implementations, the reaction temperature in step (2) is 35-60°C.

[0063] In some implementations, the reaction time in step (2) is 3 hours to 2 days.

[0064] In some embodiments, the solvent in step (3) is selected from N,N-dimethylformamide.

[0065] In some implementations, the reaction temperature in step (3) is 35-60°C.

[0066] In some implementations, the reaction time in step (3) is 3 hours to 2 days.

[0067] In some implementations, the solvent in step (4) is selected from ethanol.

[0068] In some implementations, the reaction temperature in step (4) is 90-110°C.

[0069] In some implementations, the reaction time in step (4) is 3 hours to 2 days.

[0070] In some implementations, the solvent in step (5) is selected from methanol.

[0071] In some implementations, the reaction temperature in step 5) is 15-35°C.

[0072] In some implementations, the reaction time in step (5) is 3 hours to 2 days.

[0073] In some implementation schemes, taking compound E as an example, the reaction equation for its preparation process is as follows:

[0074]

[0075] In some implementations, the solvent in step (1) is selected from acetonitrile.

[0076] In some implementations, the reaction temperature in step (1) is 60-90°C.

[0077] In some implementations, the reaction time in step (1) is 3 hours to 2 days.

[0078] In some implementations, the solvent in step (2) is selected from tetrahydrofuran.

[0079] In some implementations, the reaction temperature in step (2) is 35-60°C.

[0080] In some implementations, the reaction time in step (2) is 3 hours to 2 days.

[0081] In some embodiments, the solvent in step (3) is selected from N,N-dimethylformamide.

[0082] In some implementations, the reaction temperature in step (3) is 35-60°C.

[0083] In some implementations, the reaction time in step (3) is 3 hours to 2 days.

[0084] In some implementations, the solvent in step (4) is selected from ethanol.

[0085] In some implementations, the reaction temperature in step (4) is 90-110°C.

[0086] In some implementations, the reaction time in step (4) is 3 hours to 2 days.

[0087] In some implementations, the solvent in step (5) is selected from methanol.

[0088] In some implementations, the reaction temperature in step 5) is 15-35°C.

[0089] In some implementations, the reaction time in step (5) is 3 hours to 2 days.

[0090] In some implementation schemes, taking compound F as an example, the reaction equation for its preparation process is as follows:

[0091]

[0092] In some implementations, the solvent in step (1) is selected from acetonitrile.

[0093] In some implementations, the reaction temperature in step (1) is 60-90°C.

[0094] In some implementations, the reaction time in step (1) is 3 hours to 2 days.

[0095] In some implementations, the solvent in step (2) is selected from tetrahydrofuran.

[0096] In some implementations, the reaction temperature in step (2) is 35-60°C.

[0097] In some implementations, the reaction time in step (2) is 3 hours to 2 days.

[0098] In some embodiments, the solvent in step (3) is selected from N,N-dimethylformamide.

[0099] In some implementations, the reaction temperature in step (3) is 35-60°C.

[0100] In some implementations, the reaction time in step (3) is 3 hours to 2 days.

[0101] In some implementations, the solvent in step (4) is selected from ethanol.

[0102] In some implementations, the reaction temperature in step (4) is 90-110°C.

[0103] In some implementations, the reaction time in step (4) is 3 hours to 2 days.

[0104] In some implementations, the solvent in step (5) is selected from methanol.

[0105] In some implementations, the reaction temperature in step (5) is 15-35°C.

[0106] In some implementations, the reaction time in step (5) is 3 hours to 2 days.

[0107] In some implementation schemes, taking a specific compound G as an example, the reaction equation for its preparation process is as follows:

[0108]

[0109] In some implementations, the solvent in step (1) is selected from acetonitrile.

[0110] In some implementations, the reaction temperature in step (1) is 60-90°C.

[0111] In some implementations, the reaction time in step (1) is 3 hours to 2 days.

[0112] In some implementations, the solvent in step (2) is selected from ethanol.

[0113] In some implementations, the reaction temperature in step (2) is 90-110°C.

[0114] In some implementations, the reaction time in step (2) is 3 hours to 2 days.

[0115] Other compounds can be prepared using the same method described above, but with different reactants.

[0116] This invention provides a cyanine-based near-infrared II fluorescent probe nanoparticle assembly, which is prepared by mixing an organic solution of the above-mentioned cyanine dye with water via flash precipitation and then by supramolecular self-assembly.

[0117] The concentration of the organic solution of the cyanine dye is preferably 0.1–100 mg / mL.

[0118] The solvent for the organic solution is preferably dimethyl sulfoxide.

[0119] The organic solution of the cyanine dye can be mixed with water in any proportion. Preferably, the volume of the water is 0.1 to 100 times that of the organic solution of the cyanine dye.

[0120] In some implementations, the temperature for self-assembly in water is 15-35°C, preferably 25°C.

[0121] In some implementations, the stirring time is 10 seconds to 5 minutes, preferably 20 seconds.

[0122] The prepared nanoparticle assembly has the morphology of nanoparticles, wherein the diameter of the nanoparticles is preferably in the range of 0.5 to 10 μm and the length is preferably in the range of 0.1 to 100 μm.

[0123] The cyanine dyes prepared by this invention can be applied to advanced diagnostics and in vivo imaging. Their structure design is flexible, and their fluorescence properties can be adjusted by modifying the molecular structure and chemical properties. Moreover, they have higher in vivo precision in imaging applications.

[0124] Based on this, the present invention provides the application of the above-mentioned nanoparticle assembly as a fluorescent agent for in vivo imaging diagnostics.

[0125] Compared with existing technologies, this invention provides a series of cyanine-based near-infrared II fluorescent dye molecules with different side-group structures, exhibiting the structure shown in AG. This invention uses the classic indocyanine green as the backbone framework, expanding its functionality and improving the hydrophilicity of the fluorescent small molecules by incorporating hydrophilic functional groups, enabling highly ordered assembly in aqueous solution. Notably, when the aforementioned cyanine dyes are combined with FBS, the fluorescence intensity increases, resulting in better photothermal effects, reduced enrichment in the liver and spleen, and enabling specific imaging of lymph nodes. During near-infrared II imaging, the influence of biological background signals is less, and the penetration depth is stronger, providing a novel technical means for the development of precision surgical navigation systems. Attached Figure Description

[0126] Figure 1The matrix-assisted flight mass spectra of cyanine dyes (A) are shown;

[0127] Figure 2 The corresponding nuclear magnetic resonance spectra of cyanine dye (A) are shown;

[0128] Figure 3 The matrix-assisted flight mass spectra of cyanine dyes (B) are shown.

[0129] Figure 4 The matrix-assisted flight mass spectra of cyanine dyes (C) are shown.

[0130] Figure 5 The matrix-assisted flight mass spectra of cyanine dyes (E) are shown;

[0131] Figure 6 The matrix-assisted flight mass spectra of cyanine dyes (F) are shown.

[0132] Figure 7 The matrix-assisted flight mass spectra of cyanine dyes (G) are shown.

[0133] Figure 8 TEM image of the assembly formed by supramolecular self-assembly of the cyanine dye (A) prepared in this invention;

[0134] Figure 9 The ultraviolet absorption spectrum of cyanine dye (A) is shown;

[0135] Figure 10 The ultraviolet absorption spectrum of cyanine dye (B) is shown;

[0136] Figure 11 The ultraviolet absorption spectrum of cyanine dye (C) is shown;

[0137] Figure 12 The ultraviolet absorption spectra of cyanine dyes (D) are shown;

[0138] Figure 13 The ultraviolet absorption spectra of cyanine dyes (E) are shown;

[0139] Figure 14 The ultraviolet absorption spectra of cyanine dyes (F) are shown;

[0140] Figure 15 The in vivo fluorescence imaging results of the anthocyanin dyes prepared in this invention in mice are shown.

[0141] Figure 16 The results of in vivo lymphofluorescence imaging of mice with the cyanine dyes prepared according to the present invention are shown. Detailed Implementation

[0142] To further illustrate the present invention, the following detailed description of the cyanine dyes provided by the present invention and their applications is provided in conjunction with embodiments.

[0143] The raw materials used in this invention are described below:

[0144] p-Toluenesulfonyl chloride, tetraethylene glycol di-p-toluenesulfonate, sodium iodide, 1,1,2-trimethyl-1H-benzo[e]indole, N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride were purchased from Anaiji Chemical and used as is. Pentaethylene glycol and ketone acid were purchased from Bailingwei and used as is. Tripropylene glycol was purchased from Sinopharm Research and Development Co., Ltd. and used as is. 1,8-Naphthylimide was purchased from Bid Pharmaceutical and used as is. Perchloric acid was purchased from Sigma-Aldrich and used as is. Sodium carbonate (Na2CO3), anhydrous sodium acetate, ethyl acetate (EtOAc), tetrahydrofuran (THF), methanol, diethyl ether, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), toluene, acetone, and isopropanol were purchased from Sinopharm Chemical Reagent Co., Ltd. and used as is. Water was deionized (DI) using the Milli-Q SP reagent water system (Millipore) to achieve a resistivity of 18.4 MΩcm. Unless otherwise specified, all other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. and used as is.

[0145] Example 1: Cyanide Dye A

[0146]

[0147] The first step was to synthesize diethylene glycol monomethyl ether substituted derivative 1 (a diethylene glycol monomethyl ether derivative of 1,1,2-trimethyl-1H-benzo[e]indole), and the specific synthetic route is as follows:

[0148] Iododiethylene glycol monomethyl ether (1.15 g, 5 mmol) was dissolved in 15 mL of acetonitrile, and 1,1,2-trimethyl-1H-benzo[e]indole (1.25 g, 6 mmol) was added. The mixture was sonicated until completely dissolved, and the reaction was refluxed at 90 °C overnight. The solvent was evaporated, and the product was purified by column chromatography (DCM:methanol = 10:1) to give 1.01 g of a green oily product (purity 96.5%, yield 64.1%).

[0149] Step 2: Anthocyanin dye 2 is synthesized via the condensation reaction of methyl and aniline, and the specific reaction formula is as follows:

[0150]

[0151] Preparation method: 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 25 mL of toluene for azeotropic dehydration three times. Then, 15 mL of anhydrous ethanol was added, and the mixture was refluxed at 100 °C for 4 hours in the dark. The insoluble salt was removed by filtration, the solvent was evaporated by rotary evaporation, and the product was purified by column chromatography (DCM:methanol = 10:1) to obtain 214 mg of green solid product (purity 98.6%, yield 29.8%). Its structure was also confirmed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF), and the results are shown in... Figure 1 At the same time, nuclear magnetic resonance spectroscopy (NMR) was used. 1 H NMR also verified the correctness of its structure, such as Figure 2 As shown.

[0152] Example 2: Cyanide Dye B

[0153] The first step is to synthesize diethylene glycol monomethyl ether substituted derivative 1 (a diethylene glycol derivative of 1,1,2-trimethyl-1H-benzo[e]indole), the specific reaction formula of which is as follows:

[0154]

[0155] Preparation method: 1,1,2-trimethyl-1H-benzo[e]indole (9.13 g, 43.64 mmol) and diethylene glycol monotriphenylmethyl iodide (10 g, 21.82 mmol) were added to a 250 mL round-bottom flask, dissolved in 150 mL of anhydrous acetonitrile, and refluxed at 90 °C with stirring overnight. After rotary evaporation to remove the solvent from the reaction mixture, it was purified by column chromatography (DCM:methanol = 50:1) to give 117.4 g of intermediate (purity 97.4%, yield 74.13%).

[0156] Step 2: Intermediate 2 is synthesized via a condensation reaction of methyl and aniline, the specific reaction formula of which is as follows:

[0157]

[0158] Preparation method: 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 25 mL of toluene for azeotropic dehydration three times. Then, 15 mL of anhydrous ethanol was added, and the mixture was refluxed at 100 °C for 4 hours in the dark. The insoluble salt was removed by filtration, the solvent was evaporated by rotary evaporation, and the product was purified by column chromatography (DCM: methanol = 10:1) to obtain 687 mg of green solid product (purity 95.5%, yield 56.4%).

[0159] Step 3: Deprotect the triphenylmethyl group from p-toluenesulfonic acid to synthesize the final product B. The specific reaction formula is as follows:

[0160]

[0161] Preparation method: Intermediate 2 (100 mg, 0.083 mmol) was placed in a 50 mL round-bottom flask, dissolved in 9 mL of dichloromethane, and then 1 mL of methanol was added. P-Toluenesulfonic acid (15.7 mg, 0.083 mmol) dissolved in 1 mL of dichloromethane was slowly added dropwise to the system, and the mixture was stirred at room temperature for 12 h. The mixture was precipitated in diethyl ether, and the solid was dissolved in DCM and subjected to column chromatography (200-300 mesh, DCM:MeOH = 6:1). The target fraction was collected to obtain 43 mg of a brown solid (purity 98.4%, yield 70.7%). Its structure was also confirmed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF), and the results are shown in [data missing]. Figure 3 .

[0162] Example 3: Cyanide Dye C

[0163] The first step is to synthesize derivative 1, which is substituted with diethylene glycol monomethyl ether. The specific synthetic route is as follows:

[0164]

[0165] Preparation method: Naphthalimide (10 g, 18.3 mmol) was dissolved in 30 mL of N,N-dimethylformamide and stirred in an ice bath for 5 minutes. Sodium hydride (0.44 g, 18.3 mmol) was added to the reaction system and stirred in an ice bath for 10 minutes. 13-Iodo-1,1,1-triphenyl-2,5,8,11-tetraoxothiadeane (11.99 g, 21.96 mmol) was slowly added dropwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature. The solvent was evaporated, and the product was purified by column chromatography (DCM:methanol = 10:1) to give 8.8 g of a yellow oil (purity 94.8%, yield 81.9%).

[0166] Step 2: Synthesize the methylated naphthimide derivative using Grignard reagents, the specific reaction formula of which is as follows:

[0167]

[0168] Preparation method: Intermediate 1 (1.2 g, 2.04 mmol) was placed in a 100 mL two-necked round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and 3M magnesium methyl chloride (3.06 mL, 9.19 mmol) was added. The mixture was stirred at 60 °C for 1 hour under a nitrogen atmosphere, cooled to room temperature, and 3.2 mL of 25 wt% tetrafluoroboric acid aqueous solution was added. The mixture was stirred at room temperature for another 10 minutes. Dichloromethane was added to dissolve the mixture, and it was extracted twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and subjected to column chromatography (200-300 mesh, PE:EA = 6:1). The target fraction was collected to obtain 0.38 g of a dark green viscous liquid (purity 84%, yield 31.7%).

[0169] Step 3: Prepare cyclohexene chloride containing aldehyde and enol groups, the specific reaction formula is as follows:

[0170]

[0171] Preparation method: 40 mL of dichloromethane and 40 mL of N,N-dimethylformamide were added to a 250 mL round-bottom flask. Phosphorus oxychloride (45.81 g, 305.65 mmol) was added with stirring in an ice bath. After stirring in an ice bath for 20 minutes, cyclohexanone (10 g, 101.88 mmol) was added, and the mixture was refluxed at 60 °C for 3 hours. The mixture was cooled in an ice bath and recrystallized at -20 °C to obtain a yellow solid. The solid was filtered, washed three times with ice water, and dried to obtain 7.5 g of a yellow solid (purity 91.2%, yield 42.3%).

[0172] Step 4: Synthesis of intermediate 4 to prepare naphthalimide anthocyanin dyes protected by triphenylmethyl groups. The specific reaction formula is as follows:

[0173]

[0174] Preparation method: Intermediates 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 100 mL two-necked round-bottom flask, and 30 mL of acetic anhydride was added. The mixture was reacted at room temperature for 2 hours, and then precipitated in diethyl ether. The solid was dissolved in dichloromethane and subjected to column chromatography (200-300 mesh, DCM:MeOH = 10:1). The target component was collected to obtain 82 mg of brown colloidal solid (purity 96.6%, yield 41.21%).

[0175] Step 5: Synthesis of the final target product, 5-naphthalimide anthocyanin dye, with the specific reaction formula as follows:

[0176]

[0177] Preparation method: Intermediate 4 (100 mg, 0.076 mmol) was placed in a 50 mL round-bottom flask, dissolved in 9 mL of dichloromethane, and then 1 mL of methanol was added. P-Toluenesulfonic acid (14 mg, 0.076 mmol dissolved in 1 mL of dichloromethane) was slowly added dropwise to the system, and the mixture was stirred at room temperature for 12 h. The mixture was precipitated in diethyl ether, and the solid was dissolved in DCM and subjected to column chromatography (200-300 mesh, DCM:MeOH = 6:1). The target fraction was collected to obtain 32 mg of a brown solid (purity 98.6%, yield 51.07%). Its structure was also confirmed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF), and the results are shown in [data missing]. Figure 4 .

[0178] Example 4: Cyanide Dye E

[0179] The first step is to synthesize tetraethylene glycol monomethyl ether substituted derivative 1. The specific synthetic route is as follows:

[0180]

[0181] Preparation method: Dissolve 0.5 g (1.69 mmol) of bis(naphthyl)imide in 5 mL of N,N-dimethylformamide and stir for 5 minutes in an ice bath. Add sodium hydride (0.5 g, 1.69 mmol) to the reaction system and stir for 10 minutes in an ice bath. Slowly add 13-iodo-1,1,1-triphenyl-2,5,8,11-tetraoxothiadeane (1.11 g, 2.03 mmol) to the reaction system and react overnight at room temperature. Evaporate the solvent and purify by column chromatography (DCM:methanol = 10:1) to obtain a yellow oily product 1, weighing 0.95 g (purity 96.6%, yield 79.3%).

[0182] Step 2: Synthesize the methylated bis(naphthyl)imide derivative using Grignard reagents. The specific reaction formula is as follows:

[0183]

[0184] Preparation method: Intermediate 1 (0.5 g, 0.7 mmol) was placed in a 100 mL two-necked round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and 3M magnesium methyl chloride (1.05 mL, 3.15 mmol) was added. The mixture was stirred at 60 °C for 1 hour under a nitrogen atmosphere, cooled to room temperature, and 1.1 mL of 25 wt% tetrafluoroboric acid aqueous solution was added. The mixture was stirred at room temperature for another 10 minutes. Dichloromethane was added to dissolve the mixture, and it was extracted twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and subjected to column chromatography (200-300 mesh, PE:EA = 6:1). The target fraction was collected to obtain 0.16 g of a dark green viscous liquid (purity 81%, yield 32.6%).

[0185] Step 3: Synthesis of intermediate 3 to prepare triphenylmethyl-protected bis(naphthyl)imide anthocyanin dyes. The specific reaction formula is as follows:

[0186]

[0187] Preparation method: Intermediate 2 (0.16 g, 0.22 mmol), N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride (0.08 g, 0.11 mmol), and sodium acetate (0.009 g, 0.11 mmol) were placed in a 50 mL two-necked round-bottom flask, and 10 mL of n-butanol / toluene mixed solvent was added. The mixture was reacted at 120 °C for 3 h under a nitrogen atmosphere, cooled to room temperature, and precipitated twice in diethyl ether. Column chromatography (200-300 mesh, DCM:MeOH = 10:1-5:1) was performed, and the target component was collected to obtain 107 mg of black solid (purity 96.9%, yield 31.21%).

[0188] Step 4: Synthesis of the final target product, 4-pinamidimide anthocyanin dye, with the specific reaction formula as follows:

[0189]

[0190] Preparation method: Intermediate 3 (100 mg, 0.06 mmol) was placed in a 50 mL round-bottom flask, dissolved in 9 mL of dichloromethane, and then 1 mL of methanol was added. P-Toluenesulfonic acid (11.4 mg, 0.06 mmol) dissolved in 1 mL of dichloromethane was slowly added dropwise to the system, and the mixture was stirred at room temperature for 12 h. The mixture was precipitated in diethyl ether, and the solid was dissolved in DCM and subjected to column chromatography (200-300 mesh, DCM:MeOH = 6:1). The target fraction was collected to obtain 36 mg of a brown solid (purity 98.8%, yield 53.71%). Its structure was also confirmed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF), and the results are shown in [data missing]. Figure 5 .

[0191] Example 5: Cyanide Dye F

[0192] The first step is to synthesize tetraethylene glycol monomethyl ether substituted derivative 1. The specific synthetic route is as follows:

[0193]

[0194] Preparation method: Aristolochic acid (0.5 g, 1.7 mmol) was dissolved in 5 mL of N,N-dimethylformamide and stirred in an ice bath for 5 minutes. Sodium hydride (0.041 g, 1.7 mmol) was added to the reaction system and stirred in an ice bath for 10 minutes. 13-Iodo-1,1,1-triphenyl-2,5,8,11-tetraoxothiadeane (0.93 g, 1.7 mmol) was slowly added dropwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature. The solvent was evaporated, and the product was purified by column chromatography (DCM:methanol = 10:1) to give 0.93 g of a yellow oily product (purity 97.6%, yield 77.3%).

[0195] Step 2: Synthesize methylated aristolochic acid lactam derivatives using Grignard reagents. The specific reaction formula is as follows:

[0196]

[0197] Preparation method: Intermediate 1 (0.93 g, 1.31 mmol) was placed in a 100 mL two-necked round-bottom flask, dissolved in 20 mL of anhydrous tetrahydrofuran, and 3M magnesium methyl chloride (1.96 mL, 5.9 mmol) was added. The mixture was stirred at 60 °C for 1 hour under a nitrogen atmosphere, cooled to room temperature, and 1.1 mL of 25 wt% tetrafluoroboric acid aqueous solution was added. The mixture was stirred at room temperature for another 10 minutes. Dichloromethane was added to dissolve the mixture, and it was extracted twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and subjected to column chromatography (200-300 mesh, PE:EA = 6:1). 0.5 g of the target component was collected as a dark green viscous liquid (purity 88%, yield 32.1%).

[0198] Step 3: Synthesis of intermediate 3 to prepare aristolochic acid dyes containing triphenylmethyl-protected aristolochic acid lactam. The specific reaction formula is as follows:

[0199]

[0200] Preparation method: Intermediate 2 (0.5 g, 0.42 mmol), N-[(3-(anilinemethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride (0.150 g, 0.42 mmol), and sodium acetate (0.034 g, 0.42 mmol) were placed in a 50 mL two-necked round-bottom flask, and 10 mL of n-butanol / toluene mixed solvent was added. The mixture was reacted at 120 °C for 3 h under a nitrogen atmosphere, cooled to room temperature, and precipitated twice in diethyl ether. Column chromatography (200-300 mesh, DCM:MeOH = 10:1-5:1) was performed, and the target component was collected to obtain 249 mg of black solid (purity 97.9%, yield 36.11%).

[0201] Step 4: Synthesis of the final target product, aristolochic acid dye, with the specific reaction formula as follows:

[0202]

[0203] Preparation method: Intermediate 3 (249 mg, 0.15 mmol) was placed in a 50 mL round-bottom flask, dissolved in 9 mL of dichloromethane, and then 1 mL of methanol was added. p-Toluenesulfonic acid (28.5 mg, 0.15 mmol) dissolved in 1 mL of dichloromethane was slowly added dropwise to the system, and the mixture was stirred at room temperature for 12 h. The mixture was precipitated in diethyl ether, and the solid was dissolved in DCM and subjected to column chromatography (200-300 mesh, DCM:MeOH = 6:1). The target fraction was collected to obtain 140 mg of a brown solid (purity 98.3%, yield 56.91%). Its structure was also confirmed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF), and the results are shown in [data missing]. Figure 6 .

[0204] Example 6: Cyanide Dye G

[0205] The precursor of the quinacrine dye G is synthesized using the same method as dye A.

[0206] Step 2: Anthocyanin dye 2 is synthesized via the condensation reaction of methyl and aniline, and the specific reaction formula is as follows:

[0207]

[0208] Preparation method: Intermediate 1 (312 mg, 1 mmol), heptaenal diphenylamine hydrochloride (155 mg, 0.5 mmol), and anhydrous sodium acetate (40.5 mg, 0.5 mmol) were added to 25 mL of toluene for azeotropic dehydration three times. Then, 15 mL of anhydrous ethanol was added, and the mixture was refluxed at 100 °C for 4 hours in the dark. The insoluble salt was removed by filtration, and the solvent was evaporated by rotary evaporation. The product was purified by column chromatography (DCM:methanol = 10:1) to obtain 226 mg of green solid product (purity 98.9%, yield 31.8%). Its structure was also confirmed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF), and the results are shown in... Figure 7 .

[0209] Example 7: Preparation of Cyanide Near-Infrared II Assembly with Protein Regulatory Function

[0210] 2 mg of anthocyanin dye A was dissolved in 2 mL of dimethyl sulfoxide to obtain an organic solution. 100 μL of this solution was flash-precipitated into 1900 μL of deionized water under high-speed stirring for 30 s, and then allowed to stand at room temperature in the dark for 4 hours. The morphology of the assembly was observed using transmission electron microscopy, and the results are shown in [Figure number missing]. Figure 8 . Figure 8 In the figures, a)-d) represent the microstructures of the assemblies prepared using cyanine dye A, observed at a scale of 0.5 μm. Figure 8 It can be seen that the prepared assembly has a nanomicelle structure.

[0211] Example 8: Ultraviolet absorption spectra of cyanin-based near-infrared II dyes with protein regulation functions and their assemblies.

[0212] 2 mg of each cyanine dye (AG) was dissolved in 2 mL of dimethyl sulfoxide to obtain an organic solution. 100 μL of the prepared solution was flash-precipitated into 1900 μL of deionized water, which was being stirred at high speed on a stirrer for 30 s. The solution was then allowed to stand at room temperature in the dark for 4 hours. The UV absorption spectrum was measured using a UV-Vis spectrometer, and the results are as follows: Figure 9-14 As shown. By Figure 9-14 It can be seen that near-infrared II imaging diagnostic reagents with different wavelength structures have been successfully prepared, and their ultraviolet absorption wavelength can reach 1078m, which can be used for near-infrared II fluorescence imaging.

[0213] Example 9: Fluorescence Imaging Study of Near-Infrared II Assemblies of Cyanide with Protein Regulatory Functions

[0214] Nude mice (provided by the Animal Center of Anhui Medical University) were used for animal experiments. Female nude mice were randomly selected and anesthetized by inhalation of isoflurane. In vivo imaging of the mice was performed using a live imaging system. Cyanide dye A assemblages were injected via the tail vein at a dose of 0.5 mL / kg. Results are as follows: Figure 15 As shown, the assembly prepared in this application exhibits strong fluorescence signals in various parts of the mouse. The two figures below show the liver and spleen still exhibiting high fluorescence intensity after 96 hours of in vivo imaging and dissection, as observed under LP900 and LP1000 filters, respectively.

[0215] Example 10: Study on lymphocyte-specific fluorescence imaging of near-infrared II assemblies of cyanin species with protein regulatory functions.

[0216] Nude mice (provided by the Animal Center of Anhui Medical University) were used for animal experiments. Female nude mice were randomly selected and anesthetized by inhalation of isoflurane. In vivo imaging of the mice was performed using a near-infrared II in vivo imager equipped with an infrared camera. Cyanide dye A assemblages were injected via the footpads at a dose of 0.5 mL / kg. Results are as follows: Figure 16 As shown, the assembly prepared in this application exhibits strong fluorescence signals in various parts of the mouse.

[0217] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A cyanin dye containing a binafenimide structure, having the structure shown in Formula E: Formula E; R1 is selected from ClO - I - BF4 - or PF6 - ; R3 is selected from any of the following structures: ； n is between 0 and 100.

2. A cyanin dye containing an aristolochic acid lactam structure, having the structure shown in Formula F: Formula F; R1 is selected from ClO - I - BF4 - or PF6 - ; R3 is selected from any of the following structures: ； n is between 0 and 100.

3. A nanoparticle assembly, prepared by mixing an organic solution of the cyanine dye described in any one of claims 1 to 2 with water via flash precipitation, and then by supramolecular self-assembly.

4. The nanoparticle assembly according to claim 3, characterized in that, The concentration of the organic solution of the cyanine dye is 0.1~100 mg / mL; The volume of the water is 0.1 to 100 times that of the organic solution of the cyanine dye.

5. The nanoparticle assembly according to claim 3, characterized in that, The nanoparticle assembly has a diameter of 0.5~10 μm and a length of 0.1~100 μm.

6. The application of the nanoparticle assembly according to any one of claims 3 to 5 as a fluorescent agent for in vivo imaging diagnostics.

Images