CN115286627A

CN115286627A - Azaindole hemicyanine dyes, and synthesis method and application thereof

Info

Publication number: CN115286627A
Application number: CN202210722300.5A
Authority: CN
Inventors: 杜健军; 潘静巍; 张晓雪; 刘圆; 樊江莉; 彭孝军
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2022-11-04

Abstract

The invention discloses azaindole hemicyanine fluorescent compounds, a synthesis method and application thereof, and relates to synthesis of azaindole hemicyanine fluorescent compounds and application thereof in the fields of biology and medicine. The dye takes azaindole as a parent, and simultaneously, the right end of a conjugated chain is connected with structures such as cyano-group, carboxyl or ester, and the like, so that the charge separation degree and the electron mobility of a conjugated system in a dye molecule are improved, and the dye has longer absorption and emission wavelengths. Meanwhile, an unsaturated six-membered ring is modified in a methine chain structure, so that the dye molecule is partially rigidized, and the light stability of the dye is improved. In addition, the dye has good biocompatibility and low cytotoxicity. Experiments prove that the dye has good photophysical properties and can be used in the fields of biological recognition imaging, nucleic acid marking, DNA sequencing, tumor photodynamic and photothermal therapy and the like.

Description

Azaindole hemicyanine dyes, and synthesis method and application thereof

Technical Field

The invention belongs to the technical field of organic dyes, and relates to azaindole hemicyanine fluorescent compounds, a synthetic method thereof, and application thereof in the fields of biology and medicine.

Background

Cyanine (Cyanine, cy for short) dyes are a class of important organic small molecule dyes, and the history thereof can trace back to the fifty generations in the nineteenth century. Since the first cyanine dye, namely, quinuclidine blue, was reported to be synthesized by Greville Williams in 1856, the related research on cyanine dyes has been rapidly developed, and various cyanine dyes with different structures and derivative materials thereof are continuously synthesized. People design and prepare organic dyes with special photophysical and photoelectric properties by regulating and controlling a large pi conjugated system and delocalized electron distribution in the cyanine dyes, thereby meeting different application requirements. The research and application of cyanine dye is widely regarded as the application of cyanine dye in the fields of biological recognition imaging, nucleic acid labeling, DNA sequencing and the like, and the aspects of tumor photodynamic and photothermal therapy and the like.

Because the absorption and emission wavelengths of many dyes are short, and the short-wavelength light is difficult to enter the biological tissue, and meanwhile, the autofluorescence of some components in the biological sample forms strong background interference, so that the efficiency in the aspects of deep-layer imaging, photodynamic and the like in the organism is greatly reduced. At present, the most important way to adjust the maximum absorption and emission wavelengths of cyanine dye molecules is by adjusting the length of the methine chain in the conjugated skeleton. However, in the study of cyanine dyes, it was found that the longer the conjugated methine chain of the molecule, the longer the maximum absorption and emission wavelengths of the dye, but the poorer the photostability of the dye. In practical photochemical applications, dyes need to have higher light stability, and the biggest problem of cyanine dyes is that the dyes are easy to generate a photobleaching phenomenon under illumination, and the application of cyanine dyes is limited due to the poorer light stability of cyanine dyes compared with other dyes.

Therefore, research and development of novel cyanine dyes which have long absorption and emission wavelengths, excellent light stability, selectivity to special cells and low toxicity are of great significance.

Disclosure of Invention

In order to solve the problems, the invention provides a near-infrared azaindole hemicyanine dye with long wavelength, a synthetic method thereof and application thereof in the fields of biology and medicine.

The dye of the invention has longer absorption and emission wavelength, better light stability, good biocompatibility and lower cytotoxicity, and can be used in the fields of biology and medicine.

The technical scheme of the application is as follows:

the first aspect of the application is to protect a class of azaindole hemicyanine fluorescent compounds, wherein the dye has a structure shown in a general formula I:

in the general formula I, the compound is shown in the specification,

R ₁ and R ₃ One selected from hydrogen, halogen, alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, aryl, alkylsulfonate, arylsulfonate, alkylsulfonate, or arylsulfonate; more preferably one selected from hydrogen, halogen, carboxyalkyl having 1 to 8 carbons, alkylsulfonate; most preferably selected from hydrogen.

R ₂ Selected from the group consisting of hydrogen, alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, alkylsulfonate, aryl, alkylsulfonate, arylsulfonate, alkylsulfonate, or arylsulfonate having 1-18 carbonsOne of (a) and (b); more preferably one selected from the group consisting of hydrogen, alkyl having 1 to 18 carbons, alkylsulfonate having 1 to 18 carbons; more preferably one selected from hydrogen, aryl, alkyl sulfonate having 1 to 8 carbons; most preferably selected from hydrogen, alkyl groups having 1-18 carbons.

R ₄ One selected from hydrogen, cyano, carboxyl and esters having 1 to 8 carbons; most preferably selected from cyano, carboxyl and esters having 1-8 carbons.

R ₅ One selected from halogen, alkoxy sulfonate with 1-18 carbon atoms, aryloxy sulfonate, alkoxy sulfonate, aryloxy sulfonate, alkyl imino with 1-18 carbon atoms and aryl imino; more preferably one selected from the group consisting of halogen, phenoxysulfonate, and an alkylimino group having 1 to 8 carbons; most preferably selected from halogens.

The second aspect of the application is to protect a synthetic method of azaindole hemicyanine fluorescent compounds, which comprises the following steps:

(1) In an organic solvent at 60-90 deg.C, containing R ₁ Substituted 2, 3-trimethyl-3H-pyrrolo [2,3-b ] s]Pyridine Y-1 with N-alkylating agent R ₂ Substituted halogenated alkane reacts for 3 to 24 hours and is converted into the compound containing N-R ₂ Y-2 of the substituted side chain. The molar ratio of compound Y-1 to N-alkylating agent is 1 to 10, more preferably 1. Recrystallizing the compound Y-2 by an organic solvent to obtain a pure product.

(2) Slowly and dropwise adding the corresponding phosphorus trihalide into a mixed solution of DMF and dichloromethane at the temperature of 0-30 ℃, wherein the volume ratio of DMF to dichloromethane is 1-5, stirring for 0.5-3h, cooling to room temperature, adding R ₃ Heating the substituted cyclohexanone for reflux reaction for 5h, and then carrying out neutralization reaction to obtain an intermediate product S-1.

(3) Adding the intermediate product Y-2 and the intermediate product S-1 into an organic solvent, heating to 80-130 ℃, carrying out reflux reaction for 1-3h, removing the solvent by rotary evaporation, and purifying by a silica gel column to obtain an intermediate product Y-3. The molar ratio of the intermediate product Y-2 to the intermediate product S-1 is 1-3.

(4) And dissolving the intermediate product Y-3 and a cyanogen compound in methanol, adding a small amount of piperidine as a catalyst, stirring at room temperature for 5 hours for reaction, and filtering to obtain an intermediate product Y-4. The molar ratio of the intermediate product Y-3 to the cyanogen compound is 1.

(5) Dissolving intermediate product Y-4 in organic solvent, adding corresponding R ₅ The side chain reacts with sodium carbonate at 25-80 ℃ to obtain the azaindole hemicyanine fluorescent compound I.

Further preferably, in the step (1), the organic solvent is at least one selected from the group consisting of ethanol, benzene, toluene and o-dichlorobenzene. The recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone and propanol.

Further preferably, in the step (3), the organic solvent is selected from one or more of benzene, toluene, o-dichlorobenzene, methanol, ethanol, propanol and butanol.

Further preferably, in the step (4) above, the cyano compound is selected from any one of acetonitrile, malononitrile, cyanoacetic acid or cyanoacetate having 1 to 8 carbons.

Further preferably, in the step (4), the organic solvent is selected from one or more of methanol, ethanol, acetonitrile and ethyl acetate.

Further preferably, in the step (5), the organic solvent is selected from one or more of methanol, ethanol, acetonitrile and dichloromethane.

It is further preferred that in step (5) above, if the group R ₅ And selecting halogen to obtain the azaindole hemicyanine fluorescent compound I.

The third aspect of the application is to protect the application of azaindole hemicyanine fluorescent compounds, and the azaindole hemicyanine fluorescent compounds are used as hemicyanine dyes in the technical fields of cell imaging, protein labeling, antibody specificity recognition, nucleic acid labeling, DNA sequencing, preparation of tumor photodynamic and photothermal therapy preparations and the like.

Further preferably, the azaindole hemicyanine fluorescent compound provided by the invention has a fluorescence imaging emission wavelength of 600-950nm in detection application.

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

(1) The azaindole hemicyanine dye provided by the invention has longer absorption and emission wavelengths.

The compound 1 prepared in the embodiment of the application has near infrared light absorption, the maximum absorption and emission wavelengths are 711nm and 733nm respectively, and compared with the maximum absorption and emission wavelengths 630nm and 693nm of the traditional indole hemicyanine dye compound 3 of the same type, the red shift is 81nm and 40nm respectively. Compared with the traditional indole hemicyanine dye, the aza-indole hemicyanine dye has the advantages that the maximum emission wavelength is large and red-shifted, so that the aza-indole hemicyanine dye is more suitable for being applied to scenes of long-wavelength absorption and emission, and can be well applied to the fields of deep-layer in-vivo imaging and tumor treatment.

(2) Compared with the traditional hemicyanine dye, the dye provided by the invention has better light stability. In the embodiment of the invention, the light stability of the compound 1 and the compound 2 with the concentration of 4 mu mol/L under the irradiation of a xenon lamp light source is measured in ethanol, and the absorption intensity is not obviously reduced within 2h, which shows that the azaindole hemicyanine dye has good light stability.

(3) The dye provided by the invention has good biocompatibility. In the embodiment of the invention, after MCF-7 cells are cultured for 24 hours by using the compound 1 and the compound 2 with the maximum concentration of 32 mu mol/L, the cells still have better survival rate, which indicates that the azaindole hemicyanine dye has very good biocompatibility and can not generate toxic or side effect on the cells within the working concentration range.

Drawings

FIG. 1 is a high resolution mass spectrum of Compound 1;

FIG. 2 is a mass spectrum of Compound 2;

FIG. 3 is a high resolution mass spectrum of Compound 3;

FIG. 4 is a graph of normalized absorption spectra of compounds 1-3 in ethanol;

FIG. 5 is a graph of normalized fluorescence spectra of compounds 1-3 in ethanol;

FIG. 6 is the photostability of

Compounds

1 and 2 in ethanol over 2 h;

FIG. 7 is a graph of the MTT assay for

compounds

1 and 2.

Detailed Description

The present invention will be described in further detail below.

Unless otherwise indicated, the terms used herein have the following meanings.

X is used herein to denote the term "halogen" and includes fluorine, chlorine, bromine and iodine.

The term "MTT" as used herein refers to a method for detecting cell survival and growth.

The term "alkyl" as used herein includes straight chain and branched chain alkyl groups.

The instruments and equipment used in the examples:

in the column chromatography process, 200-300 mesh and 100-200 mesh column chromatography silica gel purchased from Qingdao Meigaoji Co Ltd and 20-40 mesh analytical pure quartz sand purchased from Tianda chemical reagent factory are adopted.

In the process of detecting the compound, the mass spectrometer uses a Synapt G2-Si HDMS high-resolution mass spectrometer of waters corporation in America, and adopts a double-spray-needle electrospray ion source to carry out positive and negative mode detection on the compound.

The absorption, emission spectra and light stability of the dyes were measured using the Cary 60 uv-vis spectrophotometer and Cary Eclipse fluorescence spectrophotometer of Agilent corporation.

Cytotoxicity assays were performed using a Varioskan LUX Multimode Microplate Reader instrument from Thermofisher, USA.

EXAMPLE 1 preparation of Compound 1

The structural formula of compound 1:

example 1.1

2, 3-trimethyl-3H-pyrrolo [2,3-b ] pyridine (1g, 6.2mmol) and methyl iodide (1.70g, 12mmol) were added to a reaction flask containing 20mL of acetone and refluxed for 24H under a nitrogen atmosphere. After cooling to room temperature, the mixture was washed with 50ml of ethyl acetate and dried to obtain example 1.1 (1.79g, 5.9mmol, Y = 95%) as a brown solid powder compound.

Example 1.2

Phosphorus oxychloride (15.6g, 101.8mmol) is added into a reaction bottle containing 15ml of DMF and 15ml of dichloromethane which are uniformly mixed, stirring is carried out for 30min under a nitrogen atmosphere and ice bath, cyclohexanone (5g, 50.9mmol) is added, reflux is carried out for 5h at the temperature of 80 ℃, after cooling to the room temperature, the reaction liquid is dropped into ice water, and suction filtration is carried out, thus obtaining a yellow solid compound, namely example 1.2 (8g, 46.5mmol, Y = 91.3%).

Example 1.3

Compound example 1.1 (1g, 3.3mmol) and compound example 1.2 (0.85g, 5.0mmol) were added to a reaction flask containing 10ml of n-butanol and 4ml of toluene, which were uniformly mixed, and reacted at 110 ℃ under reflux for 2h, the solvent was removed by rotary evaporation, and the mixture was purified by a silica gel column to give compound example 1.3 (0.2g, 0.6mmol, y = 18%).

Production of Compound 1

Compound example 1.3 (0.2g, 0.6 mmol) and malononitrile (0.4g, 6 mmol) were added to a 10ml methanol reaction flask, followed by addition of 0.1ml piperidine, stirring overnight at room temperature, and suction filtration to give compound 1 (0.113g, 0.3mmol, y = 50%).

Example 2 preparation of Compound 2

The structural formula of compound 2:

compound example 1.3 (0.15g, 0.4mmol) and tert-butyl cyanoacetate (0.56g, 4mmol) were charged into a reaction flask of 8ml methanol, followed by addition of 0.1ml piperidine, stirring overnight at room temperature, and suction filtration to give compound 2 (0.08g, 0.18mmol, y = 43%).

Example 3 production of Compound 3

The structural formula of compound 3: used as a comparative example

Example 3.1

2,3,3-trimethyl-3H-indole (1g, 6.3mmol) and iodomethane (1.70g, 12mmol) were added to a reaction flask containing 20mL of acetone and refluxed for 24H under a nitrogen atmosphere. After cooling to room temperature, the mixture was washed with 50ml of ethyl acetate and dried to obtain compound example 3.1 (1.85g, 6.2mmol, y = 98%) as a pale pink solid powder.

Example 3.2

Compound example 3.1 (1g, 3.3mmol) and compound example 1.2 (0.85g, 5.0 mmol) were added to a reaction flask containing 10ml n-butanol and 4ml toluene, mixed well, reacted at 110 ℃ under reflux for 2h, the solvent was removed by rotary evaporation, and purified by silica gel column to give compound example 3.2 (0.26g, 0.79mmol, y = 24%).

Preparation of Compound 3

Compound example 3.2 (0.2g, 0.6 mmol) and malononitrile (0.4g, 6 mmol) were added to a 10ml methanol reaction flask, followed by addition of 0.1ml piperidine, stirring overnight at room temperature, and suction filtration to give compound 3 (0.15g, 0.4mmol, y = 66%).

Example 4 measurement of ultraviolet-visible absorption Spectrum and fluorescence Spectroscopy for

Compounds

1, 2 and 3

Accurately weighing the dye after vacuum drying by a ten-thousandth balance, preparing 2mmol/L DMF dye mother liquor into a brown sample bottle, and storing the brown sample bottle in a refrigerator at 4 ℃ for later use.

When testing the ultraviolet visible absorption spectrum and the fluorescence spectrum, a liquid-transferring gun is used for measuring 6 mu L of dye mother liquor, the dye mother liquor is dissolved in a quartz cuvette containing 3mL of solvent to be tested, the mixture is uniformly mixed, the concentration of the obtained dye is 4.0 mu mol/L, and the dye is used for testing the absorption spectrum and the fluorescence emission spectrum. All tests were done at 25 ℃.

As shown in FIG. 4, the absorption spectrum of compound 1 was measured in ethanol to show that the absorption spectrum reached 711nm, which is near infrared (650-900 nm), and that the absorption spectrum was red-shifted up to 81nm compared to 630nm, which is the absorption spectrum of compound 3, which is a comparative molecule.

As shown in FIG. 5, the maximum emission wavelength of compound 1 was 733nm, which reached the near infrared region (650-900 nm), and the emission spectrum was red-shifted by as much as 40nm, compared to 693nm, which is the maximum emission wavelength of comparative compound 3, as determined by the results of the emission spectrum in ethanol.

Meanwhile, as shown in fig. 4 and 5, the maximum absorption and emission waves of the molecule 2 are 620nm and 734nm respectively, and the Stokes shift of the molecule is up to 114nm while the molecule has longer absorption and emission wavelengths, while the Stokes shift of the common dye is generally less than 30nm. The large Stokes shift can greatly reduce the signal-to-noise ratio and fluorescence self-quenching when the dye is used for fluorescence imaging, and can be well used in organisms.

Example 5 photostability tests were performed on

Compounds

1 and 2

Using the mother solution obtained in example 4, 6 μ L of the dye mother solution was measured using a pipette, and dissolved in a quartz cuvette containing 3mL of the solvent to be tested, and mixed uniformly to obtain a dye concentration of 4.0 μmol/L for testing the light stability. All tests were done at 25 ℃.

As shown in FIG. 6, the absorption intensity of the

compounds

1 and 2 is not obviously reduced within 2h under the irradiation of a xenon lamp light source, which indicates that the compounds have good light stability. Compared with most conventional cyanine dyes, such as commercialized indocyanine green (ICG), the absorption intensity of the cyanine dye is obviously reduced within 2h of illumination. The dye can be well applied to the fields of biological identification imaging, tumor photodynamic and photothermal therapy and the like.

EXAMPLE 6 cytotoxicity assays of

Compounds

1 and 2

The toxicity of the dye molecules to the cells was assessed by MTT assay. The principle is as follows: succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, but dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value of the formazan is measured at 490nm wavelength by using an enzyme-linked immunosorbent assay instrument, so that the number of living cells can be indirectly reflected.

MCF-7 cells are inoculated in a 96-well plate, after a period of culture, compounds 1 and 2 with certain concentrations are respectively added into different wells, the concentrations of the compounds are respectively 0-32 mu mol/L, and the cell activity is detected through an MTT experiment after the cells are continuously incubated for 24h. The experimental data are shown in figure 7, and even after MCF-7 cells are cultured for 24 hours by using the

compounds

1 and 2 with the maximum concentration of 32 mu mol/L, the cells still show good survival rate, which indicates that the azaindole hemicyanine dye has very good biocompatibility, does not generate toxic and side effects on the cells within the working concentration range, and can be applied to the fields of biology and medicine.

The foregoing is a further description of the invention in connection with specific preferred embodiments thereof and is not intended to limit the invention to the particular forms disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the scope of the invention as defined in the appended claims.

Claims

1. An azaindole hemicyanine fluorescent compound, which is characterized in that: the dye has a structure of a general formula I:

in the general formula I, the compound has the following structure,

R ₁ and R ₃ One selected from hydrogen, halogen, alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, aryl, alkylsulfonate, arylsulfonate, alkylsulfonate, or arylsulfonate;

R ₂ one selected from the group consisting of hydrogen, alkyl having 1 to 18 carbons, carboxyalkyl having 1 to 18 carbons, alkylsulfonate, aryl, alkylsulfonate, arylsulfonate, alkylsulfonate, or arylsulfonate having 1 to 18 carbons;

R ₄ one selected from hydrogen, cyano, carboxyl and esters having 1 to 8 carbons;

R ₅ one selected from halogen, alkoxy sulfonate with 1-18 carbons, aryloxy sulfonate, alkoxy sulfonate, aryloxy sulfonate, alkyl imino with 1-18 carbons, and aryl imino.

2. A method of synthesizing a compound according to claim 1, wherein: the method comprises the following steps:

(1) In an organic solvent at 60-90 deg.C, containing R ₁ Substituted 2, 3-trimethyl-3H-pyrrolo [2,3-b ] s]Pyridine Y-1 with N-alkylating agent R ₂ Substituted halogenated alkane reacts for 3 to 24 hours and is converted into the compound containing N-R ₂ Y-2 of a substituted side chain; the molar ratio of the compound Y-1 to the N-alkylating agent is 1-10;

(2) Slowly and dropwise adding the corresponding phosphorus trihalide into a mixed solution of DMF and dichloromethane at the temperature of 0-30 ℃, stirring for 0.5-3h at the volume ratio of the DMF to the dichloromethane of 1 ₃ Heating up the substituted cyclohexanone for reflux reaction, and then performing neutralization reaction to obtain an intermediate product S-1;

(3) Adding the intermediate product Y-2 and the intermediate product S-1 into an organic solvent, heating to 80-130 ℃, performing reflux reaction for 1-3h, removing the solvent by rotary evaporation, and purifying by a silica gel column to obtain an intermediate product Y-3; the molar ratio of the intermediate product Y-2 to the intermediate product S-1 is 1-3;

(4) Dissolving the intermediate product Y-3 and a cyanogen compound in methanol, adding a small amount of piperidine serving as a catalyst, stirring at room temperature for reaction for 5 hours, and filtering to obtain an intermediate product Y-4; the molar ratio of the intermediate product Y-3 to the cyanogen compound is 1;

dissolving the intermediate product Y-4 in an organic solvent, adding the corresponding R ₅ The side chain reacts with sodium carbonate at 25-80 ℃ to obtain the azaindole hemicyanine fluorescent compound I.

3. The method of synthesis according to claim 2, characterized in that: the molar ratio of the compound Y-1 to the N-alkylating agent is 1.

4. The method of synthesis according to claim 2, characterized in that: in the step (1), the organic solvent is at least one selected from ethanol, benzene, toluene and o-dichlorobenzene; the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, ether, acetone and propanol.

5. The method of synthesis according to claim 2, characterized in that: in the step (3), the organic solvent is selected from any one or a mixture of several of benzene, toluene, o-dichlorobenzene, methanol, ethanol, propanol and butanol.

6. The method of synthesis according to claim 2, characterized in that: in the step (4), the cyanide compound is selected from any one of acetonitrile, malononitrile, cyanoacetic acid, or cyanoacetate having 1 to 8 carbons.

7. The method of synthesis according to claim 2, characterized in that: in the step (4), the organic solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile and ethyl acetate.

8. The method of synthesis according to claim 2, characterized in that: in the step (5), the organic solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile and dichloromethane.

9. The method of synthesis according to claim 2, characterized in that: in the above step (5), if the group R ₅ And selecting halogen to obtain the azaindole hemicyanine fluorescent compound I.

10. The use of an azaindole hemicyanine fluorescent compound as claimed in claim 1, wherein: the application is that the hemicyanine fluorescent compound is used for cell imaging, protein labeling, specific recognition of antibody, nucleic acid labeling and DNA sequencing and is used for preparing preparations for tumor photodynamic and photothermal therapy; when the hemicyanine fluorescent compound is used for detection, the fluorescence imaging emission wavelength is 600-950nm.