阅读说明：本技术 一种水溶性双靶向近红外荧光探针及其制备方法 (Water-soluble double-targeting near-infrared fluorescent probe and preparation method thereof ) 是由 孙俊毅 张明明 宋忠孝 马凌志 于 2021-10-11 设计创作，主要内容包括：本发明公开了一种水溶性双靶向近红外荧光探针及其制备方法,属于近红外荧光探针领域。本发明的水溶性双靶向近红外荧光探针,以二苯乙烯为结构单元,修饰上具有拉电子能力的氰基,并在中心苯环两侧共轭链接具有推电子能力的吗啉基团,从而在荧光分子中形成A-π-D-π-A的结构,使分子具有明显的近红外荧光发射。本发明的近红外荧光探针,斯托克斯位移大、具有水溶性以及溶酶体和DNA双靶向性的特点,在生物成像和近红外荧光探针方面具有重要的应用前景。本发明的近红外荧光探针具有良好的水溶性,解决了绝大部分近红外分子疏水性的问题,可有效的保证探针在使用过程中的生物相容性及使用效果。(The invention discloses a water-soluble double-targeting near-infrared fluorescent probe and a preparation method thereof, belonging to the field of near-infrared fluorescent probes. The water-soluble double-targeting near-infrared fluorescent probe takes stilbene as a structural unit, modifies a cyano group with an electron withdrawing capability, and is conjugated and linked with morpholine groups with an electron pushing capability at two sides of a central benzene ring, so that an A-pi-D-pi-A structure is formed in a fluorescent molecule, and the molecule has obvious near-infrared fluorescence emission. The near-infrared fluorescent probe has the characteristics of large Stokes shift, water solubility and lysosome and DNA dual-targeting property, and has important application prospect in the aspects of biological imaging and near-infrared fluorescent probes. The near-infrared fluorescent probe has good water solubility, solves the problem of hydrophobicity of most near-infrared molecules, and can effectively ensure the biocompatibility and the use effect of the probe in the use process.)

1. A water-soluble double-target near-infrared fluorescent probe is characterized in that the structural formula is as follows:

wherein R is a cosolvent group, X^-Are counter ions.

2. The water-soluble double-targeted near-infrared fluorescent probe of claim 1, wherein the counter ion X is^-Is chloride ion, bromide ion, iodide ion or trifluoromethanesulfonate ion.

3. The water-soluble double-targeted near-infrared fluorescent probe according to claim 1, wherein R is methyl or

Wherein n is 1, 2 or 3.

4. A preparation method of a water-soluble double-targeting near-infrared fluorescent probe is characterized by comprising the following steps:

oxidizing 1, 4-dibromo-2, 5-dimethylbenzene to obtain 1, 4-dibromo-2, 5-difumaryl benzene;

reacting 1, 4-dibromo-2, 5-diformaldehyde benzene with 4-cyanopyridine hydrochloride to obtain a compound 2 with an A-pi-A structure;

introducing a morpholine group to a central benzene ring of the compound 2 to obtain a compound 3 with an A-pi-D-pi-A structure;

introducing a cosolvent group into the pyridine position of the compound 3 to obtain the water-soluble double-target near-infrared fluorescent probe.

5. The method for preparing the water-soluble double-targeting near-infrared fluorescent probe according to claim 4, wherein the specific operation for synthesizing the 1, 4-dibromo-2, 5-dialdehyde benzene is as follows:

adding 11-11.4 mL of concentrated sulfuric acid and 45-46.5 mmol of CrO to a suspension containing 11.4-11.8 mmol of 1, 4-dibromo-2, 5-xylene, 15-15.5 mL of acetic acid and 30-31 mL of acetic anhydride₃Stirring the obtained mixture at 0 ℃ for 8-9 h, pouring the mixture into ice water, filtering, carrying out reflux hydrolysis on the precipitate and a mixed solution of 15-15.5 mL of water, 15-15.5 mL of ethanol and 1.5-1.55 mL of sulfuric acid, and then recrystallizing by using dichloromethane/n-hexane to obtain the 1, 4-dibromo-2, 5-dihydroxy group.

6. The preparation method of the water-soluble double-targeting near-infrared fluorescent probe according to claim 4, characterized in that the specific operation of synthesizing the compound 2 is as follows:

dissolving 3.0-3.15 mmol of 1, 4-dibromo-2, 5-diformaldehyde and 6.6-6.93 mmol of 2- (pyridine-4-yl) acetonitrile hydrochloride in a mixed solution of 20-21 mL of ethanol and 20-21 mL of dichloromethane, adding 13.2-13.86 mmol of triethylamine, and stirring for reacting for 24-30 h at 40 ℃ to obtain a primary product;

the crude product was subjected to column chromatography to give compound 2.

7. The preparation method of the water-soluble double-targeting near-infrared fluorescent probe according to claim 4, characterized in that the specific operation of synthesizing the compound 3 is as follows:

1.0 to 1.05mmol of compound 2, 10.0 to 10.5mmol of morpholine, 0.2 to 0.21mmol of RuPhos and 0.1 to 011mmol of Pd2 (d)ba)3, 3.0-3.15 mmol of Cs₂CO₃Adding 60-62 mL of methylbenzene into a reaction bottle, and heating for 48-50 h at 100-110 ℃ under an argon atmosphere to obtain a primary product;

the crude product was passed through column chromatography to afford compound 3.

8. The preparation method of the water-soluble double-targeting near-infrared fluorescent probe according to claim 4, characterized in that a cosolvent group is introduced into the pyridine position of the compound 3, and the specific operation of obtaining the water-soluble double-targeting near-infrared fluorescent probe is as follows:

dissolving 1.0-1.1 mmol of compound 3 in dichloromethane, adding 10.0-11.0 mmol of methyl iodide, carrying out reflux reaction for 12-16 h, filtering to obtain a primary product, and washing with dichloromethane to obtain the near-infrared fluorescent probe.

9. The preparation method of the water-soluble double-targeting near-infrared fluorescent probe according to claim 4, characterized in that a cosolvent group is introduced into the pyridine position of the compound 3, and the specific operation of obtaining the water-soluble double-targeting near-infrared fluorescent probe is as follows:

dissolving 1.0-1.1 mmol of compound 3 in dichloromethane, adding 10.0-12.0 mmol of 2-iodoethanol, carrying out reflux reaction for 12-16 h, filtering to obtain an initial product, and washing with dichloromethane to obtain the near-infrared fluorescent probe.

10. The preparation method of the water-soluble double-targeting near-infrared fluorescent probe according to claim 4, characterized in that a cosolvent group is introduced into the pyridine position of the compound 3, and the specific operation of obtaining the water-soluble double-targeting near-infrared fluorescent probe is as follows:

dissolving 1.0-1.1 mmol of compound 3 in dichloromethane, adding 10.0-11.5 mmol of 1-bromo-2- (2-methoxyethoxy) ethane, carrying out reflux reaction for 12-16 h, filtering to obtain a primary product, and washing with dichloromethane to obtain the near-infrared fluorescent probe.

Technical Field

The invention belongs to the field of near-infrared fluorescent probes, and particularly relates to a water-soluble double-targeting near-infrared fluorescent probe and a preparation method thereof.

Background

After the 21 st century, cancer has become one of the major diseases threatening human health. How to effectively achieve imaging of tumors is critical to the treatment of cancer. The fluorescence imaging technology is a non-invasive, ray-hazard-free, high-sensitivity, high-selectivity and real-time imaging visual detection technology, and therefore, the fluorescence imaging technology is widely applied to the field of biomedical imaging. Compared with fluorescent molecules with an emission range in a visible light region, the near-infrared fluorescent molecules have the advantages of deep penetration depth, low background noise, small tissue damage and the like, and gradually become the research focus in the field of fluorescence imaging.

The water solubility of the fluorescent probe is beneficial to realize the imaging of the fluorescent probe in a living body in consideration of the environment of the water phase of the living body system. If the probe has the function of targeting intracellular substructures, more accurate imaging and treatment processes can be realized. The design and synthesis of the molecule with near-infrared fluorescence imaging are the basis for realizing the near-infrared fluorescence probe. Research shows that most of the near infrared fluorescent molecules are realized by modifying the existing fluorescent molecules. Chinese invention patent CN106281306A discloses a near-infrared fluorescent molecule based on a cyanine structure, which can reversibly indicate the change of pH in cells in real time; chinese invention patent CN111778016A discloses a near-infrared fluorescent molecule based on BODIPY fluorescent dye, which realizes the synergistic effect of photoacoustic imaging, fluorescence imaging, photothermal therapy and immunotherapy; chinese invention patent CN109438425A discloses a near-infrared fluorescent molecule based on a rhodamine structure, which realizes certain water solubility. Chinese invention patents CN109970780A and CN112279832A respectively disclose a near infrared fluorescent molecule based on tetraphenylethylene and triamcinolone. The above near-infrared fluorescent molecules cannot realize effective water solubility, and have disadvantages in targeting intracellular substructures.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a water-soluble double-targeting near-infrared fluorescent probe and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

a water-soluble double-targeting near-infrared fluorescent probe has a structural formula as follows:

wherein R is a cosolvent group, X^-Are counter ions.

Further, the counter ion X shown^-Is chloride ion, bromide ion, iodide ion or trifluoromethanesulfonate ion.

Further, R is methyl or

Wherein n is 1, 2 or 3.

A preparation method of a water-soluble double-targeting near-infrared fluorescent probe comprises the following steps:

oxidizing 1, 4-dibromo-2, 5-dimethylbenzene to obtain 1, 4-dibromo-2, 5-difumaryl benzene;

reacting 1, 4-dibromo-2, 5-diformaldehyde benzene with 4-cyanopyridine hydrochloride to obtain a compound 2 with an A-pi-A structure;

introducing a morpholine group to a central benzene ring of the compound 2 to obtain a compound 3 with an A-pi-D-pi-A structure;

introducing a cosolvent group into the pyridine position of the compound 3 to obtain the water-soluble double-target near-infrared fluorescent probe.

Further, the specific operation for synthesizing the 1, 4-dibromo-2, 5-dialdehyde phenyl is as follows:

adding 11-11.4 mL of concentrated sulfuric acid and 45-46.5 mmol of CrO to a suspension containing 11.4-11.8 mmol of 1, 4-dibromo-2, 5-xylene, 15-15.5 mL of acetic acid and 30-31 mL of acetic anhydride₃Stirring the obtained mixture at 0 ℃ for 8-9 h, pouring the mixture into ice water, filtering, carrying out reflux hydrolysis on the precipitate and a mixed solution of 15-15.5 mL of water, 15-15.5 mL of ethanol and 1.5-1.55 mL of sulfuric acid, and then recrystallizing by using dichloromethane/n-hexane to obtain the 1, 4-dibromo-2, 5-dihydroxy group.

Further, the specific operation for synthesizing the compound 2 is as follows:

dissolving 3.0-3.15 mmol of 1, 4-dibromo-2, 5-diformaldehyde and 6.6-6.93 mmol of 2- (pyridine-4-yl) acetonitrile hydrochloride in a mixed solution of 20-21 mL of ethanol and 20-21 mL of dichloromethane, adding 13.2-13.86 mmol of triethylamine, and stirring for reacting for 24-30 h at 40 ℃ to obtain a primary product;

the crude product was subjected to column chromatography to give compound 2.

Further, the specific operation for synthesizing the compound 3 is as follows:

1.0 to 1.05mmol of compound 2, 10.0 to 10.5mmol of morpholine, 0.2 to 0.21mmol of RuPhos, 0.1 to 011mmol of Pd2(dba)3 and 3.0 to 3.15mmol of Cs₂CO₃Adding 60-62 mL of methylbenzene into a reaction bottle, and heating for 48-50 h at 100-110 ℃ under an argon atmosphere to obtain a primary product;

the crude product was passed through column chromatography to afford compound 3.

Further, a cosolvent group is introduced into the pyridine position of the compound 3 to obtain the water-soluble double-target near-infrared fluorescent probe, and the specific operation is as follows:

dissolving 1.0-1.1 mmol of compound 3 in dichloromethane, adding 10.0-11.0 mmol of methyl iodide, carrying out reflux reaction for 12-16 h, filtering to obtain a primary product, and washing with dichloromethane to obtain the near-infrared fluorescent probe.

Further, a cosolvent group is introduced into the pyridine position of the compound 3 to obtain the water-soluble double-target near-infrared fluorescent probe, and the specific operation is as follows:

dissolving 1.0-1.1 mmol of compound 3 in dichloromethane, adding 10.0-12.0 mmol of 2-iodoethanol, carrying out reflux reaction for 12-16 h, filtering to obtain an initial product, and washing with dichloromethane to obtain the near-infrared fluorescent probe.

Further, a cosolvent group is introduced into the pyridine position of the compound 3 to obtain the water-soluble double-target near-infrared fluorescent probe, and the specific operation is as follows:

dissolving 1.0-1.1 mmol of compound 3 in dichloromethane, adding 10.0-11.5 mmol of 1-bromo-2- (2-methoxyethoxy) ethane, carrying out reflux reaction for 12-16 h, filtering to obtain a primary product, and washing with dichloromethane to obtain the near-infrared fluorescent probe.

Compared with the prior art, the invention has the following beneficial effects:

the water-soluble double-targeting near-infrared fluorescent probe takes stilbene as a structural unit, modifies a cyano group with an electron withdrawing capability, and is conjugated and linked with morpholine groups with an electron pushing capability at two sides of a central benzene ring, so that an A-pi-D-pi-A structure is formed in a fluorescent molecule, and the molecule has obvious near-infrared fluorescence emission. The near-infrared fluorescent probe has the characteristics of large Stokes shift, water solubility and lysosome and DNA dual-targeting property, and has important application prospect in the aspects of biological imaging and near-infrared fluorescent probes. The near-infrared fluorescent probe has good water solubility, solves the problem of hydrophobicity of most near-infrared molecules, and can effectively ensure the biocompatibility and the use effect of the probe in the use process. The near-infrared fluorescent probe has the effect of targeting lysosomes and DNA, is favorable for realizing the effect of accurately imaging the cell substructure, and provides possibility for realizing accurate treatment. The near-infrared fluorescent probe disclosed by the invention is beneficial to realizing the effect of enhancing the fluorescence induced by the accumulation of the stilbene in the target lysosome and DNA parts, so that the near-infrared fluorescent imaging effect is improved.

The preparation method of the water-soluble double-targeting near-infrared fluorescent probe has the advantages of simple synthetic route and simple synthetic steps.

Drawings

FIG. 1 is a drawing of Compound 1 of example 1¹H NMR nuclear magnetic map;

FIG. 2 is a drawing of Compound 2 of example 1¹H NMR nuclear magnetic map;

FIG. 3 is a drawing of Compound 3 of example 1¹H NMR nuclear magnetic map;

FIG. 4 shows a diagram of a near-infrared fluorescent probe in example 1¹H NMR nuclear magnetic map;

FIG. 5 is a graph showing the UV-VIS absorption and fluorescence spectra of the NIR fluorescent probe of example 1.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The second ethylene is a structural unit for constructing a fluorescent molecule, and functional groups with electron pushing and pulling properties are introduced into the structure, so that the fluorescent molecule with a D-pi-A structure can be constructed, and the fluorescence emission red of the fluorescent molecule is shifted to a near infrared region. According to the invention, through structural design, a morpholine group with good water solubility is introduced into cyano-diene ethylene, and the morpholine group is used as an electron donor, so that a fluorescent molecule has the property of targeting lysosomes. The pyridine salt structure introduced at the pyridine position enables the fluorescent molecule to have the property of targeting DNA, and the introduced water-soluble group is beneficial to further enhancing the water solubility of the fluorescent molecule. The aggregation of near-infrared fluorescent molecules at lysosomes, DNA and other parts in cells is beneficial to realizing the effect of fluorescence enhancement induced by stilbene aggregation, thereby improving the near-infrared fluorescence imaging effect. The near-infrared fluorescent molecule with good water solubility and targeted lysosome and DNA characteristics constructed by the invention has very important significance for promoting the application of a near-infrared fluorescent probe.

The invention is described in further detail below with reference to the accompanying drawings:

example 1

A preparation method of a water-soluble double-targeting near-infrared fluorescent probe comprises the following steps:

1) synthesis of 1, 4-dibromo-2, 5-diyl

To a suspension of 11.4mmol of 1, 4-dibromo-2, 5-xylene, 15mL of acetic acid and 30mL of acetic anhydride was slowly added dropwise 11mL of concentrated sulfuric acid and 45mmol of CrO₃And (3) stirring the obtained mixture at 0 ℃ for 8h, pouring the mixture into ice water, filtering, carrying out reflux hydrolysis on the obtained white solid and a mixed solution of 15mL of water, 15mL of ethanol and 1.5mL of sulfuric acid overnight, and then recrystallizing by dichloromethane/n-hexane to obtain a light yellow solid product, namely the 1, 4-dibromo-2, 5-dihydroxy-group, wherein the synthetic route is shown as the following formula.

Example 1 preparation of 1, 4-dibromo-2, 5-diyl Compound 1¹The NMR nuclear magnetic pattern is shown in FIG. 1,¹H NMR(400MHz,CDCl₃,295K)δ10.39(s,2H),8.19(s,2H)。

2) synthesis of Compound 2

3.0mmol of 1, 4-dibromo-2, 5-diyl and 6.6mmol of 2- (pyridin-4-yl) acetonitrile hydrochloride were dissolved in a mixed solution of 20mL of ethanol and 20mL of dichloromethane, and 13.2mmol of triethylamine was added thereto. The reaction was stirred at 40 ℃ for 24 h. And (3) carrying out column chromatography (the volume ratio of n-hexane to ethyl acetate is 3: 2-1: 1) on the primary product to obtain a yellow solid compound 2, wherein the synthetic route of the compound 2 is shown as the following formula.

Preparation of Compound 2 obtained in example 1¹The H NMR nuclear magnetic diagram is shown in fig. 2, and 1H NMR (400MHz, cdcl3,295k) δ 8.78(dd, J ═ 4.6,1.6Hz,4H),8.40(s,2H),7.96(s,2H),7.62(dd, J ═ 4.6,1.6Hz, 4H).

3) Synthesis of Compound 3

Mixing 1.0mmol of compound 2, 10.0mmol of morpholine, 0.2mmol of RuPhos and 0.1mmol of Pd₂(dba)₃、3.0mmol Cs₂CO₃And 60mL of fresh toluene with water removed were added to the reaction flask and heated at 100 ℃ under an argon atmosphere for 48 h. And (3) carrying out column chromatography (the volume ratio of ethyl acetate to dichloromethane is 2: 1-1: 1) on the primary product to obtain a red solid compound 3, wherein the synthetic route of the compound 3 is shown as the following formula.

Preparation of the resulting Compound 3¹The H NMR nuclear magnetic diagram is shown in fig. 3, and 1H NMR (400MHz, cd2cl2,295k) δ 8.73(d, J ═ 5.2Hz,4H),8.24(s,2H),7.94(s,2H),7.65(d, J ═ 6.1Hz,4H),3.90-3.81(m,8H),3.06-2.98(m, 8H).

4) Synthesis of near-infrared fluorescent probe

Dissolving 1.0-1.1 mmol of compound 3 in dichloromethane, adding 10.0-11.0 mmol of iodomethane (1.42-1.56 g), refluxing for 12-16 h, filtering to obtain a primary product, washing with dichloromethane to obtain a black solid near-infrared fluorescent probe, wherein the synthesis path of the near-infrared fluorescent probe is shown in the following formula.

Example 1 near Infrared fluorescent Probe¹The H NMR nuclear magnetic map is shown in fig. 4, and 1H NMR (400MHz, D2O,295K) δ 8.77(D, J ═ 6.4Hz,4H),8.60(s,2H),8.26(D, J ═ 6.4Hz,4H),7.89(s,2H),4.33(s,6H),3.82(s,8H),3.00(s, 8H).

The ultraviolet-visible spectrum of the near-infrared fluorescent probe 4a was measured using a lamb 950 absorption spectrophotometer. The fluorescence emission spectrum of the near-infrared fluorescent probe 4a was measured using an Edinburgh FLS920 spectrofluorometer.

Referring to fig. 5, fig. 5 is a graph of the ultraviolet-visible absorption and fluorescence spectra of the near-infrared fluorescent probe 4a in example 1, and as can be seen from the fluorescence emission spectrum result of the near-infrared fluorescent probe 4a, the emission wavelength reaches 735nm, and is in the near-infrared emission region, and can be effectively used for near-infrared fluorescence imaging.

Example 2

A preparation method of a water-soluble double-targeting near-infrared fluorescent probe comprises the following steps:

1) synthesis of 1, 4-dibromo-2, 5-diyl (1)

To a suspension of 11.8mmol1, 4-dibromo-2, 5-xylene, 15.5mL acetic acid and 31mL acetic anhydride was slowly added dropwise 11.4mL concentrated sulfuric acid and 46.5mmol CrO₃And (3) granules. The resulting mixture was stirred at 0 ℃ for 9h, poured into ice water and filtered. The obtained white solid is hydrolyzed by refluxing with a mixed solution of 15.5mL of water, 15.5mL of ethanol and 1.55mL of sulfuric acid overnight, and then recrystallized by dichloromethane/n-hexane to obtain a light yellow solid product of 1, 4-dibromo-2, 5-diamino.

2) Synthesis of Compound 2

3.1mmol of 1, 4-dibromo-2, 5-diyl and 6.8mmol of 2- (pyridin-4-yl) acetonitrile hydrochloride were dissolved in a mixed solution of 20.5mL of ethanol and 20.5mL of dichloromethane, and 13.5mmol of triethylamine was added thereto. The reaction was stirred at 40 ℃ for 28 h. And (3) subjecting the primary product to column chromatography (the volume ratio of n-hexane to ethyl acetate is 3: 2-1: 1) to obtain a yellow solid compound 2.

3) Synthesis of Compound 3

Mixing 1.05mmol of compound 2, 10.5mmol of morpholine, 0.21mmol of RuPhos and 011mmol of Pd₂(dba)₃、3.15mmol Cs₂CO₃And 62mL of fresh toluene with water removed was added to the reactionIn a bottle and heated at 110 ℃ for 50h under an argon atmosphere. And (3) subjecting the primary product to column chromatography (the volume ratio of ethyl acetate to dichloromethane is 2: 1-1: 1) to obtain a red solid compound 3.

4) Synthesis of near-infrared fluorescent probe

1.0mmol of compound 3 was dissolved in dichloromethane, then 10.0mmol of 2-iodoethanol was added and the reaction was refluxed for 12 h. Filtering to obtain a primary product, and washing by using dichloromethane to obtain a black solid near-infrared fluorescent probe.

Example 3

A preparation method of a water-soluble double-targeting near-infrared fluorescent probe comprises the following steps:

1) synthesis of 1, 4-dibromo-2, 5-diyl (1)

To a suspension of 11.6mmol of 1, 4-dibromo-2, 5-xylene, 15.2mL of acetic acid and 30.5mL of acetic anhydride, 11.2mL of concentrated sulfuric acid and 46mmol of CrO were slowly added dropwise₃(4.5-4.65 g, 45-46.5 mmol) of particles. The resulting mixture was stirred at 0 ℃ for 8.5h, poured into ice water and filtered. The obtained white solid is hydrolyzed by refluxing with a mixed solution of 15.2mL of water, 15.2mL of ethanol and 1.52mL of sulfuric acid overnight, and then recrystallized by dichloromethane/n-hexane to obtain a light yellow solid product of 1, 4-dibromo-2, 5-diamino.

2) Synthesis of Compound 2

3.15mmol of 1, 4-dibromo-2, 5-diyl 6.93mmol of 2- (pyridin-4-yl) acetonitrile hydrochloride was dissolved in a mixed solution of 21mL of ethanol and 21mL of dichloromethane, and 13.86mmol of triethylamine was added thereto. The reaction was stirred at 40 ℃ for 30 h. And (3) subjecting the primary product to column chromatography (the volume ratio of n-hexane to ethyl acetate is 3: 2-1: 1) to obtain a yellow solid compound 2.

3) Synthesis of Compound 3

Mixing 1.02mmol of compound 2, 10.2mmol of morpholine, 0.21mmol of RuPhos and 011mmol of Pd₂(dba)₃、3.12mmol Cs₂CO₃And 61mL of fresh toluene with water removed were added to the reaction flask and heated at 105 ℃ for 49h under an argon atmosphere. And (3) subjecting the primary product to column chromatography (the volume ratio of ethyl acetate to dichloromethane is 2: 1-1: 1) to obtain a red solid compound 3.

4) Synthesis of near-infrared fluorescent probe

1.0mmol of compound 3 was dissolved in dichloromethane, then 10.0mmol of 1-bromo-2- (2-methoxyethoxy) ethane was added and the reaction was refluxed for 12 h. Filtering to obtain a primary product, and washing by using dichloromethane to obtain a black solid near-infrared fluorescent probe.

Example 4

Mixing 1.05mmol of compound 2, 10.5mmol of morpholine, 0.21mmol of RuPhos, 0.11mmol of Pd2(dba)3 and 3.15mmol of Cs₂CO₃And 62mL of fresh toluene with water removed were added to the reaction flask and heated at 110 ℃ for 50h under an argon atmosphere. And (3) subjecting the primary product to column chromatography (the volume ratio of ethyl acetate to dichloromethane is 2: 1-1: 1) to obtain a red solid compound 3.

Example 5

1.1mmol of compound 3 was dissolved in dichloromethane, then 12.0mmol of 2-iodoethanol was added and the reaction was refluxed for 16 h. Filtering to obtain a primary product, and washing by using dichloromethane to obtain a black solid near-infrared fluorescent probe.

Example 6

1.1mmol of compound 3 was dissolved in dichloromethane, then 11.5mmol of 1-bromo-2- (2-methoxyethoxy) ethane was added and the reaction refluxed for 16 h. Filtering to obtain a primary product, and washing by using dichloromethane to obtain a black solid near-infrared fluorescent probe.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.