阅读说明：本技术 一种新型抗溶剂猝灭的近红外二区染料、及其制备方法和应用 (Novel anti-solvent quenching near-infrared two-region dye, and preparation method and application thereof ) 是由 郭志前 吴梦兰 赵紫君 张辽 张玉涛 李娟� 章旖烟 周业坤 朱为宏 于 2021-07-20 设计创作，主要内容包括：本发明提供了一种新型抗溶剂猝灭的近红外二区染料、及其制备方法和应用,是一类新型抗溶剂猝灭的含氮杂环的NIR-II染料。实验表明本发明的近红外二区染料具有水溶性优异,波长长,抗溶剂猝灭,光稳定性优良,穿透深度深以及蛋白质增强荧光发射等特点,通过尾静脉注射到小鼠中,可以看清小鼠全身的血管及肝脏。此外,染料的结构易于修饰,可以连接多种靶向基团和检测基团,应用范围广泛。本发明结构为式Ⅰ所示：(The invention provides a novel anti-solvent-quenching near-infrared two-region dye, and a preparation method and application thereof, and the dye is a novel anti-solvent-quenching NIR-II dye containing a nitrogen heterocycle. Experiments show that the near-infrared two-region dye has the characteristics of excellent water solubility, long wavelength, solvent quenching resistance, excellent light stability, deep penetration depth, protein enhanced fluorescence emission and the like, and can be injected into a mouse through tail vein to clearly see blood vessels and liver of the whole body of the mouse. In addition, the structure of the dye is easy to modify, and the dye can be connected with various targeting groups and detection groups, so that the application range is wide. The structure of the invention is shown in formula I:)

1. A novel near-infrared two-region dye with anti-solvent quenching is a novel NIR-II dye with anti-solvent quenching and containing nitrogen heterocycle, and the structure of the dye is shown as formula I:

in the formula I, R₁Is a group represented by formula II: sulfonic acid group (II-1) or N, N, N-trimethyl amine salt (II-2);

R₂any one of chlorine atoms or detection groups shown in a formula III;

R₃is hydrogen atom shown in formula IV or a bacterial cell wall targeting unit shown in IV-2 or a tumor targeting unit shown in IV-3;

2. the near infrared two-region dye according to claim 1, wherein R is₁Is sulfonic acid group.

3. The near infrared two-region dye according to claim 1, wherein the C-2 position of the quinoline unit is a phenyl group.

4. The method of preparing the anti-solvent-quenching near-infrared two-region dye according to claim 1, comprising the steps of:

the synthesis of the compound takes 4-methyl-2-chloroquinoline as a raw material, and quinoline salt intermediates are obtained through Suzuki cross-coupling reaction and nucleophilic substitution reaction; taking cyclohexanone as a raw material, and performing formylation and substitution reaction to obtain a Schiff base intermediate; or 1, 4-cyclohexanediol is used as a raw material, and a Schiff base intermediate with alkynyl is obtained through nucleophilic substitution reaction, formylation reaction and substitution reaction; then the quinoline salt intermediate and the Schiff base intermediate are subjected to condensation reaction to obtain the symmetrical heptamethine cyanine dye.

5. The near infrared two-region dye according to claim 1, wherein the absorption maximum wavelength is 995nm and the emission maximum wavelength is 1037 nm.

6. The near-infrared two-region dye according to claim 1, characterized by a protein-enhanced fluorescence emission in fetal bovine serum, as characterized by higher absorbance and fluorescence intensity than in DMSO, an absorption wavelength of 1008nm and an emission wavelength of 1027 nm.

7. The use of the near-infrared two-region dye according to claim 1, wherein the prepared dye is connected with a targeting group through a Click reaction, and is connected with a detection group through a nucleophilic substitution reaction to obtain the near-infrared two-region fluorescent probe, and the near-infrared two-region fluorescent probe is used for imaging deep tissues or blood vessels.

Technical Field

The invention belongs to the field of fine chemical engineering, and particularly relates to a novel anti-solvent-quenching near-infrared two-region dye, a preparation method and fluorescence imaging thereof.

Background

The fluorescence imaging technology has shown good application prospect in the aspects of real-time monitoring, rapid diagnosis, treatment and the like of diseases. Compared with near infrared first-zone (NIRI, 650-plus-900 nm) fluorescence imaging, the novel near infrared second-zone (NIRII, 1000-plus-1700 nm) fluorescence imaging technology can realize high-resolution imaging of deeper tissues due to small influence of tissue scattering and autofluorescence, and shows great application potential. At present, near-infrared two-region inorganic contrast agents and organic small-molecule fluorescent dyes have been developed, wherein the inorganic contrast agents comprise carbon nanotubes, rare earth element nanoparticles and quantum dots, and the materials contain heavy metal elements and are not easy to be discharged out of a body, so that potential biological toxicity exists. The near-infrared two-region organic small molecule dye has the advantages of easy modification of chemical structure, high biocompatibility and easy metabolism out of a body, thereby showing greater advantages to biological imaging.

Currently, most near-infrared two-region fluorophores are complex to synthesize, suffer from poor water solubility of the dye due to a large aromatic structure, are prone to aggregate in biological systems, and are difficult to interact with target molecules. Therefore, the development of the water-soluble near-infrared two-region fluorescent dye which is simple and convenient to synthesize, low in cost, long in wavelength and resistant to solvent quenching has important value for biological imaging.

Disclosure of Invention

The invention aims to provide a class of near-infrared two-region dyes containing nitrogen heterocycles, which are simple and convenient to synthesize, low in cost, long in wavelength, high in molar absorption coefficient and resistant to solvent quenching, and researches the photophysical properties and biological imaging of the dyes. The propyl sulfonic acid group is introduced to the N atom of the invention, the maximum absorption wavelength is at 995nm, and the maximum emission wavelength is at 1037 nm. The dye DQPS has the characteristic of enhancing fluorescence emission of protein in fetal calf serum, the absorbance and the fluorescence intensity of the dye DQPS are higher than those of the dye DQPS in DMSO, the absorption wavelength is 1008nm, and the emission wavelength is 1027 nm. The dye has excellent water solubility, and the water solution of the dye can be injected into a mouse body through a tail vein, so that the blood vessels and the liver of the whole body of an organism can be immediately and clearly observed, and the application prospect is good.

The invention is realized by the following scheme:

a novel near-infrared two-region dye with anti-solvent quenching is a novel NIR-II dye with anti-solvent quenching and containing nitrogen heterocycle, and the structure of the dye is shown as formula I:

in formula I;

R₁is a group represented by formula II: sulfonic acid group (II-1) or N, N, N-trimethyl amine salt (II-2);

R₂any one of chlorine atoms or detection groups shown in a formula III;

R₃is a hydrogen atom shown as a formula IV or a bacterial cell wall target shown as an IV-2A targeting unit to the tumor shown as unit IV-3;

the invention also provides a general preparation method of the novel anti-solvent quenching near-infrared two-region dye, which comprises the following steps:

the synthesis of the compound takes 4-methyl-2-chloroquinoline as a raw material, and quinoline salt intermediates are obtained through Suzuki cross-coupling reaction and nucleophilic substitution reaction; taking cyclohexanone as a raw material, and performing formylation and substitution reaction to obtain a Schiff base intermediate; or 1, 4-cyclohexanediol is used as a raw material, and a Schiff base intermediate with alkynyl is obtained through nucleophilic substitution, formylation reaction and substitution reaction; then the quinoline salt intermediate and the Schiff base intermediate are subjected to condensation reaction to obtain the symmetrical heptamethine cyanine dye. The reaction process is as follows:

the invention also provides application of the novel anti-solvent quenching near-infrared two-region dye, which is characterized in that the prepared dye is connected with a targeting group through a Click reaction, and is connected with a detection group through a nucleophilic substitution reaction to obtain a near-infrared two-region fluorescent probe, and the near-infrared two-region fluorescent probe is applied to imaging of deep tissues or blood vessels.

Drawings

FIG. 110. mu.M DQPS UV absorption spectrum in DMSO, dichloromethane, ethanol (see example 2 for details).

FIG. 210. mu.M fluorescence emission spectrum, λ, of DQPS in DMSO, dichloromethane, ethanol_ex1000nm (see example 2 for details).

FIG. 310. mu.M DPQS is a normalized spectrum of UV absorbance in both DMSO and fetal calf serum (see example 3 for details).

FIG. 410. mu.M DQPS is a normalized spectrum of fluorescence emission in dimethyl sulfoxide and fetal calf serum (see example 3 for details).

FIG. 5 application of DQPS dye in vivo fluorescence imaging in mice (see example 4 for details).

Detailed Description

In a preferred embodiment of the present invention:

R₁is sulfonic group shown in formula II or N, N, N-trimethyl amine salt;

R₂any one of chlorine atoms or detection groups shown in a formula III;

R₃is a hydrogen atom shown in a formula IV or any one targeting unit;

in a further preferred embodiment, R₁Is a sulfonic acid group;

the invention is further illustrated by the following examples, which are intended only for a better understanding of the contents of the invention. The examples given therefore do not limit the scope of protection of the invention:

example 1

The specific synthetic route is as follows:

synthesis of 12-phenyl-4-methylquinoline

A250 mL three-necked flask was charged with 2-chloro-4-methylquinoline (1.00g, 5.63mmol), Pd (PPh)₃)₂Cl₂(197mg, 0.281mmol), phenylboronic acid (755mg, 6.19mmol), K₂CO₃(2.33g, 16.89mmol), argon was replaced, and 45mL of tetrahydrofuran was added and the solution was yellow. The temperature is increased to 65 ℃ and the reaction is carried out for 36 h. Cooling, adding 120mL ethyl acetate, washing with 50mL water for 3 times, drying the organic layer with anhydrous sodium sulfate, filtering, and spin-drying. The column was purified by passing PE, EA 10:1 to give 1.0376g of a colorless oil in 84% yield.

¹H-NMR(400MHz,CDCl₃,ppm):δ＝2.78(s,3H,-CH₃),7.44-7.48(m,1H,Phenyl-H),7.51-7.58(m,3H,Phenyl-H),7.73(t,2H,Phenyl-H),8.01(d,J＝8.3Hz,1H,Phenyl-H),8.15(s,1H,Phenyl-H),8.16(s,1H,Phenyl-H),8.22(d,J＝8.3Hz,1H,Phenyl-H).¹³C-NMR(100MHz,CDCl₃,ppm):δ＝19.07,119.81,123.65,126.06,127.27,127.57,128.82,129.23,129.37,130.30,139.83,144.87,148.12,157.10.HRMS(ESI-MS):m/z:calcd for[C₁₆H₁₄N]⁺:220.1126,found:220.1114.

Synthesis of 2N-propyl sulfonic group-2-phenyl-4-methylquinoline

In a 25mL single-neck flask, 2-phenyl-4-methylquinoline (585mg,2.67mmol), 1, 3-cyclopropylsultone (325mg,2.67mmol), and 1.67mL of dichlorobenzene were added, and the mixture was heated to 150 ℃ in an oil bath and reacted for 15 hours. After the reaction is finished, cooling, performing suction filtration, washing a filter cake by using dichloromethane, and performing vacuum drying to obtain 360mg of purple pink solid with the yield of 39%. HRMS (ESI-MS) m/z calcd for [ C [ ]₁₆H₁₄N]⁺:342.1164,found:342.1159.

Synthesis of 3 DQPS

10mL Schlenk's tube was dehydrated, N-propylsulfo-2-phenyl-4-methylquinoline (100mg, 0.293mmol) was added, Schiff's base (48mg, 0.133mmol) was added, 3.2mL of acetic anhydride was added and dissolved, anhydrous NaOAc (33mg, 0.399mmol) was added, argon was replaced, and the temperature was raised to 70 ℃ for 12 hours. After cooling, the solution was dissolved in 20mL of water, washed three times with 10mL of dichloromethane, and the aqueous layer was rotary evaporated and lyophilized to obtain 53mg of a solid with a yield of 48%.

¹H-NMR(400MHz,CD₃OD,ppm):δ＝1.29(t,4H,-CH₂),1.78-1.89(m,2H,-CH₂),2.10-2.17(m,4H,-CH₂),2.58(t,4H,-CH₂),4.22-4.42(m,4H,-CH₂),6.79-6.59(m,4H,alkene-H),7.52-7.60(m,12H,Phenyl-H),7.71(t,J＝7.5Hz,4H,Phenyl-H),7.90(d,J＝8.5Hz,2H,Phenyl-H),8.23(d,J＝8.5Hz,2H,Phenyl-H).HRMS(ESI-MS):m/z:calcd for[C₄₆H₄₂N₂O₆S₂Cl]⁺:817.2173,found:817.2167.

Example 2

Ultraviolet absorption and fluorescence emission spectra of DQPS dyes

The dye DQPS of the invention example 1 is respectively dissolved in a certain amount of analytically pure dimethyl sulfoxide (DMSO) to prepare 1.0X 10^-3M, mother liquor. 30 mu L of the mother liquor is respectively added into 3mL of dimethyl sulfoxide, dichloromethane and ethanol, and the mixture is uniformly mixed and transferred into an optical quartz cuvette (10 multiplied by 10mm) to test ultraviolet absorption and fluorescence emission spectra. As shown in FIG. 1, the maximum absorption wavelength of DQPS is around 995nm, and as shown in FIG. 2, when excited at 1000nm, the near-infrared region II emission peak of DQPS is around 1037 nm. Most of the previously published near infrared dibenzcyanine dyes have better molar extinction coefficients and fluorescence intensities in low-polarity dichloromethane than other highly polar solvents. However, the molar extinction coefficient and fluorescence intensity of the DQPS dye of the present invention are higher in dimethyl sulfoxide than in dichloromethane, and thus DQPS has a certain anti-solvent quenching ability.

Example 3

Ultraviolet absorption, fluorescence emission spectra of DQPS in Fetal Bovine Serum (FBS)

mu.L of the mother liquor of DQPS from example 2 were added to 3mL DMSO and FBS, respectively, mixed well and transferred to an optical quartz cuvette (10X 10mm) for UV absorption and fluorescence emission spectroscopy. The absorbance of DQPS in FBS was increased by 7.6% compared to DMSO, as shown in FIG. 3. In addition, the fluorescence intensity of DQPS in FBS is increased by 10% compared with that in DMSO, and the protein has the characteristic of enhanced fluorescence emission, as shown in FIG. 4. It is presumed that DQPS is expected to be a blood vessel imaging agent excellent in photophysical properties.

Example 4

Near-infrared two-zone living body fluorescence imaging of DQPS dye on mouse

Near-infrared two-zone fluorescence imaging of DQPS (100 μ M in PBS, tail vein injection) in mice, as shown in fig. 5, excitation light: 808nm, exposure time: 200ms, filter plate: 1050 nm. On live mice, blood vessels and livers of the mice were immediately visible after injection of water-soluble DQPS, and the fluorescence intensity was strong.