WO2022099658A1 - Cyanine compound, dye containing cyanine compound, and application of cyanine compound - Google Patents

Abstract

Disclosed in the present invention are a cyanine compound, a dye containing the cyanine compound, and an application of the cyanine compound. The cyanine compound has the structure of general formula I. X is selected from a group consisting of C(CH₃)₂, O, S, and Se. R₁ and R₂ are independently selected from a group consisting of H, C₁-C₁₈ alkyl groups, a phenyl group, OR₆, and a halogen, respectively; R₃ and R₄ are independently selected from a group consisting of C₁-C₁₈ alkyl groups, C₁-C₁₈ carboxyl groups, C₁-C₁₈ hydroxyl groups, C₁-C₁₈NR₅R₆, a benzyl group, and a substituted benzyl group, respectively, wherein a substituent of the substituted benzyl group is selected from a group consisting of C₁-C₁₈ alkyl groups, CN, COOH, NH₂, NO₂, OH, SH, C₁-C₆ alkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ acylamino groups, a halogen, and C₁-C₆ haloalkyl groups. R₅ and R₆ are independently selected from a group consisting of H and C₁-C₁₈ alkyl groups, respectively. Y^- is an anion. The cyanine compound according to the present invention has good permeability for living cells, and can enter a cell to dye a nucleic acid without destroying the cell membrane; moreover, the excitation light is blue-green light having a smaller wavelength.

Description

Cyanine compounds, dyes containing cyanine compounds and applications of cyanine compounds

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technical field

The present invention relates to the technical field of nucleic acid quantitative detection and biological dyeing, in particular to a cyanine compound, a dye containing the cyanine compound, and the application of the cyanine compound in the quantitative detection of nucleic acid and/or biological dyeing.

Background technique

DNA (DeoxyriboNucleic Acid, deoxyribonucleic acid) is a class of biological macromolecules with genetic information. Normal cells of the organism have relatively stable DNA diploid content, and abnormal changes will occur only when cancer or precancerous lesions with malignant potential occur. Therefore, the specific identification and precise measurement of DNA, especially in living cells, is of great significance in the early diagnosis of cancer.

Quantitative analysis of DNA by fluorescence technology has attracted the interest of researchers due to its advantages of high sensitivity, fast response, and convenient use of instruments. At present, the commercialized such dyes mainly include phenanthridines, acridines, imidazoles and cyanine families. However, each of these dyes has application limitations.

First, a large part of the dyes exhibit fluorescence quenching after binding to DNA, resulting in low practical value in visualization applications such as fluorescence imaging. Second, a considerable part of the application of dyes is limited to fixed cells, and it is necessary to increase the permeability of the cell membrane or similar methods to disintegrate the membrane to effectively fluorescently label biological samples. However, this fixation method often negatively affects the observation of the true morphology of cells and biological tissues, see [Kozubek S, Lukasova E, Amrichova J, Kozubek M, Liskova A, Slotova J. Anal Biochem 2000;282:29-38]. Third, most of the current dyes are highly toxic and carcinogenic, which further limits their application in living cells.

Among many kinds of fluorescent dyes, cyanine fluorescent dyes have the advantages of wide wavelength range, large molar extinction coefficient and moderate fluorescence quantum yield. Conversion materials, etc. have been widely used.

Some varieties of cyanine dyes have been commercialized, but most of these commercialized dyes have large molecules and complex structures. identification and detection. In addition, the excitation light (light emitted by a laser, or light absorbed by the dye) of most commercial cyanine dyes has a relatively large wavelength, such as orange light, which has a problem of poor recognition of tiny objects.

Therefore, a cyanine compound, a dye containing the cyanine compound and the application of the cyanine compound are required to at least partially solve the above problems.

SUMMARY OF THE INVENTION

A series of concepts in simplified form have been introduced in the Summary section, which are described in further detail in the Detailed Description section. The Summary of the Invention section of the present invention is not intended to attempt to limit the key features and essential technical features of the claimed technical solution, nor is it intended to attempt to determine the protection scope of the claimed technical solution.

In order to at least partially solve the above problems, a first aspect of the present invention provides a cyanine compound, which has the structure shown in the general formula I,

wherein X is selected from the group consisting of C(CH ₃ ) ₂ , O, S and Se;

R ₁ and R ₂ are each independently selected from the group consisting of H, C ₁ -C ₁₈ alkyl, phenyl, OR ₆ and halogen;

R ₃ and R ₄ are each independently selected from the group consisting of C ₁ -C ₁₈ alkyl, C ₁ -C ₁₈ carboxy, C ₁ -C ₁₈ hydroxy, C ₁ -C ₁₈ NR ₅ R ₆ , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C ₁ -C ₁₈ alkyl, CN, COOH, NH ₂ , NO ₂ , OH, SH, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkane the group consisting of amino, C ₁ -C ₆ amido, halogen and C ₁ -C ₆ haloalkyl;

R ₅ and R ₆ are each independently selected from the group consisting of H and C ₁ -C ₁₈ alkyl;

Y ^- is a negative ion.

The cyanine compound according to the present invention has good permeability of living cells, can enter cells to stain nucleic acid without destroying the cell membrane, has low toxicity and low carcinogenicity; and the excitation light of the cyanine compound of the present invention is The blue-green light with a smaller wavelength can identify tiny particles and improve the detection ability of small particles; the cyanine compound of the present invention can use ordinary green or blue semiconductor lasers as light sources, which greatly reduces the cost of use; in addition, the present invention The structure of the cyanine compound is simple, the raw materials for its preparation are readily available, the synthesis yield is high, and it is easy to realize industrialization.

Further, the X is selected from the group consisting of C(CH ₃ ) ₂ and S.

Further, the R ₁ and the R ₂ are each independently selected from the group consisting of H, C ₁ -C ₁₂ alkyl, phenyl, OR ₆ and halogen.

Further, each of said R ₁ and said R ₂ is independently selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, OR ₆ and halogen.

Further, the R ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl and halogen;

Further, the R ₂ is H.

Further, the R ₁ is selected from the group consisting of H, methyl, phenyl and Cl.

Further, the R ₃ and the R ₄ are each independently selected from C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ carboxyl, C ₁ -C ₁₂ hydroxyl, C ₁ -C ₁₂ NR ₅ R ₆ , benzyl The group consisting of substituted benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from C ₁ -C ₁₂ alkyl, CN, COOH, NH ₂ , NO ₂ , OH, SH, C ₁ -C ₆ alkoxy , the group consisting of C ₁ -C ₆ alkylamino, C ₁ -C ₆ amido, halogen and C ₁ -C ₆ haloalkyl.

Further, the R ₃ and the R ₄ are each independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ carboxyl, C ₁ -C ₆ hydroxyl, C ₁ -C ₆ NR ₅ R ₆ , benzyl the group consisting of substituted benzyl and substituted benzyl, wherein the substituent of said substituted benzyl is selected from C ₁ -C ₆ alkyl, CN, COOH, NH ₂ , NO ₂ , OH, SH, C ₁ -C ₆ alkoxy , the group consisting of C ₁ -C ₆ alkylamino, C ₁ -C ₆ amido, halogen and C ₁ -C ₆ haloalkyl.

Further, the R ₃ is selected from the group consisting of C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyl, C ₁ -C ₆ carboxyl, C ₁ -C ₆ NR ₅ R ₆ and benzyl;

Further, the R ₄ is selected from the group consisting of C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyl, C ₁ -C ₆ carboxyl, and benzyl.

Further, the R ₅ and the R ₆ are each independently selected from the group consisting of H and C ₁ -C ₁₂ alkyl.

Further, the R ₅ and the R ₆ are each independently selected from the group consisting of H and C ₁ -C ₆ alkyl.

Further, the R ₅ and the R ₆ are each independently C ₁ -C ₆ alkyl.

Further, the R ₅ and the R ₆ are ethyl.

Further, the R ₃ is selected from the group consisting of methyl, ethyl, benzyl, 4-(diethylamino)butyl, hydroxypropyl and hexylcarboxy.

Further, the R ₄ is selected from the group consisting of methyl, benzyl, carboxypentyl and hydroxypropyl.

Further, the Y ^- is selected from the group consisting of halogen anion, ClO ₄ ^- , PF ₆ ^- , BF ₄ ^- , CH ₃ COO ^- or OTs ^- .

Further, the cyanine compound comprises one of chemical formula I, chemical formula II, chemical formula III, chemical formula IV, chemical formula V, chemical formula VI, chemical formula VII, chemical formula VIII, chemical formula IX, chemical formula X, chemical formula XI, chemical formula XII and chemical formula XIII. the structure shown,

A second aspect of the present invention provides a dye comprising the cyanine compound described in the first aspect.

The dyes according to the present invention, including cyanine compounds, have good permeability to living cells, can enter cells to stain nucleic acids without destroying cell membranes, have low toxicity and low carcinogenicity; and, the cyanine compounds of the present invention have Both the excitation light and the emission light are blue-green light with a small wavelength, which can identify tiny particles and improve the detection ability of small particles; the cyanine compound of the present invention can use a common green semiconductor laser as a light source, which greatly reduces the use cost; In addition, the cyanine compound of the present invention has a simple structure, the raw materials for preparing the cyanine compound are readily available, the synthesis yield is high, and it is easy to realize industrialization.

The third aspect of the present invention relates to the application of the cyanine compound described in the first aspect above in the quantitative detection of nucleic acid and/or biological staining; or

The application of the fluorescent dye related to the second aspect above in the quantitative detection of nucleic acid and/or biological staining.

Description of drawings

The following drawings of the present invention are incorporated herein as a part of the present invention for understanding of the present invention. The accompanying drawings illustrate embodiments of the present invention and their description, which serve to explain the principles of the present invention.

In the attached picture:

Fig. 1 shows the absorption spectrum of the compound B after DNA staining according to Example 2 of the present invention;

Fig. 2 shows the fluorescence spectrum after the compound B according to Example 2 of the present invention dyes DNA;

Figure 3 shows the absorption spectrum of compound B after RNA staining according to Example 2 of the present invention;

Fig. 4 shows the fluorescence spectrum after the compound B according to Example 2 of the present invention stains RNA;

Fig. 5 shows the variation curve of the fluorescence intensity after the compound B of Example 2 of the present invention stains DNA and RNA with nucleic acid concentration;

Figure 6 is a bright-field photomicrograph of live cells stained with Compound B according to Example 2 of the present invention;

Fig. 7 is the fluorescence micrograph after the compound B of Example 2 of the present invention stains the living cell;

Figure 8 is an overlay of the brightfield photomicrograph in Figure 6 and the fluorescence photomicrograph in Figure 7;

FIG. 9 shows the fluorescence spectrum after the compound C according to Example 3 of the present invention stains DNA;

Figure 10 shows the fluorescence spectrum after the compound C according to Example 3 of the present invention stains RNA;

Fig. 11 shows the variation curve of the fluorescence intensity after the compound C stained with DNA and RNA according to the embodiment 3 of the present invention as a function of nucleic acid concentration;

Figure 12 is a brightfield photomicrograph of live cells stained with Compound C according to Example 3 of the present invention; and

Figure 13 is a fluorescence micrograph of live cells stained with Compound C according to Example 3 of the present invention.

Detailed ways

In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without one or more of these details. In other instances, some technical features known in the art have not been described in order to avoid obscuring the present invention.

For a thorough understanding of the present invention, a detailed description will be set forth in the following description. Obviously, the implementation of the embodiments of the present invention is not limited to the specific details familiar to those skilled in the art. Preferred embodiments of the present invention are described in detail below, however, the present invention may have other embodiments in addition to these detailed descriptions.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments in accordance with the present invention. As used herein, the singular forms are also intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, it indicates the presence of the stated features, integers, steps, operations, elements and/or components, but does not exclude the presence of or in addition to one or more other features, integers, steps, operations, elements, components and/or combinations thereof.

In particular, as used throughout this application, the term alkyl may be understood in its broadest sense to mean any linear, branched or cyclic alkyl substituent. The term "C _1-18 alkyl" as used herein generally refers to a saturated hydrocarbon group having 1 to 18 carbon atoms in configuration, C _1-18 alkyl includes, but is not limited to, C _1-12 alkyl, C _{1 -6} alkyl, etc. Where not described as a "substituted alkyl", the term "alkyl" generally refers to an unsubstituted alkyl. Illustratively, the term alkyl includes the substituents methyl (Me), ethyl (Et), n-propyl (nPr), isopropyl (iPr), cyclopropyl, n-butyl (nBu), isobutyl (iBu), sec-butyl (sBu), tert-butyl (tBu), cyclobutyl, 2-methylbutyl, n-pentyl, sec-pentyl, tert-pentyl, 2-pentyl, neopentyl, Cyclopentyl, n-hexyl, sec-hexyl, tert-hexyl, 2-hexyl, 3-hexyl, neohexyl, cyclohexyl, 1-methylcyclopentyl, 2-methylpentyl, n-heptyl, 2-heptyl , 3-heptyl, 4-heptyl, cycloheptyl, 1-methylcyclohexyl, n-octyl, 2-ethylhexyl, cyclooctyl, 1-bicyclo[2,2,2]octyl, 2 -Bicyclo[2,2,2]-octyl, 2-(2,6-dimethyl)octyl, 3-(3,7-dimethyl)octyl, adamantyl, 2,2,2 -Trifluoroethyl, 1,1-dimethyl-n-hex-1-yl, 1,1-dimethyl-n-hept-1-yl, 1,1-dimethyl-n-oct-1-yl, 1,1-Dimethyl-n-decane-1-yl, 1,1-dimethyl-n-dodecane-1-yl, 1,1-dimethyl-n-tetradec-1-yl, 1 ,1-Dimethyl-n-hexadecan-1-yl, 1,1-dimethyl-n-octadec-1-yl, 1,1-diethyl-n-hex-1-yl, 1,1 -Diethyl-n-hept-1-yl, 1,1-diethyl-n-oct-1-yl, 1,1-diethyl-n-dec-1-yl, 1,1-diethyl- n-dodecan-1-yl, 1,1-diethyl-n-tetradec-1-yl, 1,1-diethyl-n-hexadecan-1-yl, 1,1-diethyl -n-Octadecan-1-yl, 1-(n-propyl)-cyclohex-1-yl, 1-(n-butyl)-cyclohex-1-yl, 1-(n-hexyl)-cyclohexyl- 1-yl, 1-(n-octyl)-cyclohex-1-yl and 1-(n-decyl)-cyclohex-1-yl, etc.

As used above and herein, the term carboxy includes any linear, branched or cyclic carboxy substituent. The term C _1-18 carboxyl as used herein generally refers to a carboxyl substituted group having 1 to 18 carbon atoms in configuration, and C _1-18 carboxyl includes, but is not limited to, C _1-12 carboxyl, C _1-6 carboxyl, etc. . In particular, the term carboxyl refers to a group having a carboxyl group attached to an alkyl group as previously defined. The term carboxyl exemplarily includes methylcarboxy, ethylcarboxy (carboxymethyl), propylcarboxy (carboxyethyl), butylcarboxy (carboxypropyl), pentylcarboxy (carboxybutyl), hexylcarboxy (carboxypentyl), heptylcarboxy Carboxyl (carboxyhexyl) and octylcarboxy (carboxyheptyl), and their isomers, etc.

As used above and herein, the term hydroxy includes any linear, branched or cyclic hydroxy substituent. The term C _1-18 hydroxy as used herein generally refers to a hydroxy-substituted group having 1 to 18 carbon atoms in configuration, C _1-18 hydroxy including but not limited to C _1-12 hydroxy, C _1-6 hydroxy, etc. . In particular, the term hydroxy refers to a group having a hydroxy group attached to an alkyl group as previously defined. The term hydroxy exemplarily includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, hydroxyheptyl, and hydroxyoctyl, isomers thereof, and the like.

As used above and herein, the term alkoxy includes any linear, branched or cyclic alkoxy substituent. In particular, the term alkoxy refers to an alkoxy group attached to an alkyl group as previously defined. The term alkoxy exemplarily includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and 2-methyl Butoxy, etc.

As used above and herein, the terms "halogen" and "halo" may be understood in the broadest sense to mean preferably fluorine, chlorine, bromine or iodine.

As used herein, unless not more specifically defined in a particular context, the color of emitted light is designated as follows: Blue-green wavelengths are approximately in the range of 420-560 nm.

A first aspect of the present invention provides a cyanine compound, which has the structure shown in general formula I,

wherein X is selected from the group consisting of C( _CH3 ) ₂ , O, S and Se. Preferably X is selected from the group consisting of C( _CH3 ) ₂ and S.

R ₁ and R ₂ are each independently selected from the group consisting of H, C ₁ -C ₁₈ alkyl, phenyl, OR ₆ and halogen. Preferably, R ₁ and R ₂ are each independently selected from the group consisting of H, phenyl, C ₁ -C ₁₈ alkyl and halogen. Preferably, R ₁ is selected from the group consisting of H, phenyl, C ₁ -C ₁₈ alkyl and halogen. Preferably, R ₁ is selected from the group consisting of H, phenyl, C ₁ -C ₁₈ alkyl and Cl. Preferably, R ₂ is selected from the group consisting of H, phenyl and halogen. Preferably, R ₂ is H.

R ₃ and R ₄ are each independently selected from the group consisting of C ₁ -C ₁₈ alkyl, C ₁ -C ₁₈ carboxy, C ₁ -C ₁₈ hydroxy, C ₁ -C ₁₈ NR ₅ R ₆ , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C ₁ -C ₁₈ alkyl, CN, COOH, NH ₂ , NO ₂ , OH, SH, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkane The group consisting of amino, C ₁ -C ₆ amido, halogen and C ₁ -C ₆ haloalkyl. Preferably, R ₃ and R ₄ are each independently selected from the group consisting of C ₁ -C ₁₈ alkyl, C ₁ -C ₁₈ carboxy, C ₁ -C ₁₈ hydroxy, C ₁ -C ₁₈ NR ₅ R ₆ and benzyl . Wherein, R ₃ is preferably a group consisting of C ₁ -C ₁₈ alkyl, C ₁ -C ₁₈ hydroxyl, C ₁ -C ₁₈ carboxyl, C ₁ -C ₁₈ NR ₅ R ₆ and benzyl; R ₄ is preferably C ₁ - the group consisting of C ₁₈ alkyl, C ₁ -C ₁₈ hydroxy, C ₁ -C ₁₈ carboxy and benzyl.

R ₃ and R ₄ are selected with larger polar groups, which can appropriately increase the molecular polarity, reduce the binding force to water-transporting substances such as membrane lipids and proteins in cells, and improve the specific binding to nucleic acids.

R ₅ and R ₆ are each independently selected from the group consisting of H and C ₁ -C ₁₈ alkyl. Preferably, R ₅ and R ₆ are each independently C ₁ -C ₁₈ alkyl.

Y ^- is a negative ion.

Specifically, the above-mentioned C ₁ -C ₁₈ alkyl group may further be a C ₁ -C ₁₂ alkyl group. Preferably, the C ₁ -C ₁₂ alkyl group may further be a C ₁ -C ₆ alkyl group. More preferably, the _C1 - _C6 alkyl group may be the group consisting of methyl, ethyl, propyl and butyl. In particular, R ₅ and R ₆ are each independently ethyl. _In particular, _R3 and R4 may each independently be methyl, and _R3 may also be ethyl. Exemplarily, the above-mentioned C ₁ -C ₆ alkyl group may be a straight-chain alkyl group or a branched-chain alkyl group that is an isomer of the straight-chain alkyl group.

The above-mentioned C ₁ -C ₁₈ carboxyl group may further be a C ₁ -C ₁₂ carboxyl group. Preferably, the C ₁ -C ₁₂ carboxyl group may further be a C ₁ -C ₆ carboxyl group. More preferably, the C ₁ -C ₆ carboxyl group may be the group consisting of methylcarboxy, ethylcarboxy, propylcarboxy, butylcarboxy, pentylcarboxy and hexylcarboxy. In particular, R ₃ and R ₄ may each independently be hexylcarboxy (carboxypentyl). Exemplarily, the above-mentioned C ₁ -C ₆ carboxyl group may be a straight-chain carboxyl group, or may be a branched-chain carboxyl group that is an isomer of a straight-chain carboxyl group.

The above-mentioned C ₁ -C ₁₈ hydroxyl group may further be a C ₁ -C ₁₂ hydroxyl group. Preferably, the C ₁ -C ₁₂ hydroxyl group may further be a C ₁ -C ₆ hydroxyl group. More preferably, the C ₁ -C ₆ hydroxyl group may be the group consisting of hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl. _In particular, R4 may be hydroxypropyl. Exemplarily, the above-mentioned C ₁ -C ₆ hydroxyl groups may be straight-chain hydroxyl groups or branched-chain hydroxyl groups that are isomers of straight-chain hydroxyl groups.

The above-mentioned C ₁ -C ₁₈ NR ₅ R ₆ may further be C ₁ -C ₁₂ NR ₅ R ₆ . Preferably, C ₁ -C ₁₂ NR ₅ R ₆ may further be C ₁ -C ₆ NR ₅ R ₆ . More preferably, C ₁ -C ₆ NR ₅ R ₆ may be composed of -CH ₂ NR ₅ R ₆ , -(CH ₂ ) ₂ NR ₅ R ₆ , -(CH ₂ ) ₃ NR ₅ R ₆ , -(CH ₂ ) ₄ NR ₅ R ₆ and the group consisting of -(CH ₂ ) ₅ NR ₅ R ₆ . Further preferably, R ₃ and R ₄ are each independently -(CH ₂ ) ₄ NR ₅ R ₆ . In particular, _R3 may be 4-(diethylamino)butyl.

The cyanine compound according to the present invention has good permeability of living cells, can enter cells to stain nucleic acid without destroying the cell membrane, and has low toxicity and low carcinogenicity.

In addition, the excitation light of the cyanine compound of the present invention is blue-green light with a small wavelength, which can identify fine particles and improve the detection capability of small particles.

The cyanine compound of the present invention can use a common green semiconductor laser as a light source, which greatly reduces the use cost.

In addition, the cyanine compound of the present invention according to the present invention has a simple structure, readily available raw materials for its preparation, high synthesis yield, and is easy to realize industrialization.

More specifically, the cyanine compound of the present invention can be a compound comprising chemical formula I, chemical formula II, chemical formula III, chemical formula IV, chemical formula V, chemical formula VI, chemical formula VII, chemical formula VIII, chemical formula IX, chemical formula X, chemical formula XI, chemical formula XII and chemical formula One of the structures shown in XIII,

Now, the present invention will be described in more detail with reference to Examples. These exemplary embodiments may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. It should be understood that these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of these exemplary embodiments to those skilled in the art.

Example 1

To prepare compound A having the structure shown in chemical formula I,

In this embodiment, X is S, R ₁ is H, R ₂ is H, R ₃ is benzyl, and R ₄ is benzyl.

The specific preparation method of compound A is as follows:

The first step, prepares 2-methylthiobenzothiazole (right side of reaction 1) according to following reaction formula I,

Measure 20 mL of DMF into a container, and add 10 mmol of 2-mercaptobenzothiazole (the left side of reaction formula I), 11 mmol of methane and 12 mmol of sodium carbonate into the container. The reaction was stirred for 8 hours under nitrogen protection and a temperature of 110°C, and then the reaction was stopped.

The resulting mixture was cooled to room temperature, after which it was poured into a large amount of water. Extract 3 times with an appropriate amount of ethyl acetate, and combine the extracted organic phases. The organic phase was washed twice with distilled water, and then dried over anhydrous magnesium sulfate overnight.

Then, the material after drying overnight was purified by a chromatographic column to obtain about 8.5 mmol of orange-yellow solid powder, which was 2-methylthiobenzothiazole. The yield of Scheme I is about 85%.

In the second step, prepare 1-benzyl-4-methylpyridine quaternary ammonium salt (right side of reaction formula II) according to following reaction formula II,

Measure 50 mL of toluene in a container, and add 10 mmol of 4-picoline (the left side of reaction formula II) and 12 mmol of benzyl bromide into the container. The reaction was stopped under reflux and stirring for 8 hours under nitrogen protection.

The reacted mixture was subjected to suction filtration, and then the filter cake was washed three times with 50 mL of toluene to obtain a crude product.

The above crude product was purified by a chromatographic column to obtain a white solid powder of about 7.3 mmol, and the white solid powder was 1-benzyl-4-methylpyridine quaternary ammonium salt. The yield of this reaction formula II is about 73%.

The 3rd step, prepares 3-benzyl-2-thione benzothiazole (the right side of reaction formula III) according to following reaction formula III,

Measure 30 mL of toluene into a container, and add 3 mmol of 2-methylthiobenzothiazole (the left side of reaction formula III) obtained in the first step of the reaction, 4 mmol of benzyl bromide and 5 mmol of potassium carbonate into the container. The reaction was stirred for 8 hours under nitrogen protection and a temperature of 110°C.

The reacted mixture was suction filtered, and then the filter cake was washed three times with 50 mL of toluene to obtain a crude product.

The above crude product is purified by a chromatographic column to obtain about 2.1 mmol of white solid powder, which is 3-benzyl-2-thionebenzothiazole, and the yield of reaction formula III is about 70%.

The 4th step, prepares 3-benzyl-2-ethylsulfanyl benzothiazole (reaction formula IV right side) according to following reaction formula IV,

In a container, 30 mL of dichloromethane was weighed, and 2 mmol of 3-benzyl-2-thione benzothiazole obtained in the third step reaction and 2 mmol of sulfonium salt were added to the container. The mixture was stirred at reflux for 24 hours under nitrogen protection.

The product was purified by a chromatographic column to obtain a white solid powder of about 1.1 mmol, and the white solid powder was 3-benzyl-2-ethylthiobenzothiazole. The yield of Scheme IV is about 55%.

The 5th step, prepares compound A (reaction formula V right side) according to following reaction formula V,

Measure 30 mL of ethanol in the container, weigh 1 mmol of the 1-benzyl-4-methylpyridine quaternary ammonium salt obtained in the second step reaction, and weigh the 3-benzyl-2-ethyl sulfide obtained in the fourth step reaction 1 mmol of benzothiazole was added to the vessel.

The mixture was stirred at reflux for 24 hours under a nitrogen blanket. After the product was purified by chromatographic column, about 0.3 mmol of orange-yellow solid powder was obtained, and the orange-yellow solid powder was compound A. The yield of Reaction V is about 30%.

The NMR test of compound A was carried out, and the results were as follows.

¹ H-NMR (500MHz, DMSO, TMS): δ4.00(s, 3H), 5.59(s, 2H), 6.39(s, 1H), 7.26-7.31(m, 4H), 7.35-7.38(t, 2H), 7.40-7.42(d, 2H), 7.45-7.48(t, 1H), 7.52-7.54(d, 1H), 7.94-7.96(d, 1H), 8.33-8.35(d, 2H).

After verification, the test result conforms to the structure of chemical formula I.

Example 2

To prepare compound B having the structure shown in chemical formula II,

In this embodiment, X is S, R ₁ is H, R ₂ is H, R ₃ is methyl, and R ₄ is methyl.

The specific preparation method of compound B is as follows:

In the first step, 5 mL of dichloromethane was weighed into a round-bottomed flask with a capacity of 25 mL, and 10 mmol of 4-picoline and 10 mmol of methyl iodide were added to the round-bottomed flask. The reaction was stopped after stirring for 1 h under nitrogen protection and room temperature.

The resulting mixture was precipitated and filtered, and the filter cake was washed with dichloromethane. The filtrate was dried to give a white granular solid which was 1,4-lutidine quaternary ammonium salt. The crude yield of the above reaction was about 97%.

In the second step, measure 10 mL of anhydrous tetrahydrofuran into a round-bottomed flask with a capacity of 50 mL, and add 11.84 mmol of 2-mercaptobenzothiazole to the round-bottomed flask. Under stirring conditions, 14.21 mmol of methyl iodide was slowly added dropwise to the round-bottomed flask, and the reaction was carried out overnight.

A small amount of petroleum ether was added to the obtained mixture, and the mixture was left for precipitation, filtered, and the filter cake was washed with a large amount of petroleum ether. The filtrate was dried to give a white solid which was 2-methylthiobenzothiazole. The crude yield of the second step reaction was about 98%.

In the third step, measure 10 mL of toluene and put it into a round-bottomed flask with a capacity of 50 mL, and add 10 mmol of the 2-methylthiobenzothiazole obtained in the second step into it. Under nitrogen protection, 12 mmol of methyl iodide was slowly added dropwise to the round-bottomed flask, and the reaction was refluxed at a temperature of 110 °C for 24 h.

The resulting mixture was cooled to room temperature and precipitated and filtered, and the filter cake was washed with dichloromethane. The filtrate was dried to obtain a yellowish solid powder, which was 3-methyl-2-methylthiobenzothiazole quaternary ammonium salt. The crude yield of the third step reaction is about 30%.

In the fourth step, 5 mL of anhydrous ethanol was weighed and put into a double-necked round-bottomed flask with a capacity of 50 mL, and 3 mmol of the 3-methyl-2-methylthiobenzothiazole obtained by the reaction in the third step was added into it.

Under nitrogen protection, 10 mmol of triethylamine was added dropwise to the double-necked round-bottomed flask while stirring. 3.5 mmol of the 1,4-lutidine quaternary ammonium salt obtained in the first step was dissolved in 5 mL of anhydrous ethanol, and the obtained solution was added dropwise into the above two-necked round-bottomed flask. The reaction was refluxed for 7 h at a temperature of 60 °C. After that, the reaction solution was evaporated to dryness, and it was separated by a neutral silica gel chromatography column. In the separation process, a mixed solvent of dichloromethane and methanol was used as the eluent. The separated yellow components were collected and evaporated to dryness to obtain a yellow solid powder, which was compound B, and the crude yield was about 20%.

The NMR test of compound B was carried out, and the results were as follows.

¹ H-NMR (500MHz, DMSO, TMS): δ 3.73(s, 3H), 3.99(s, 3H), 6.25(s, 1H), 7.29-7.32(t, 1H), 7.40-7.41(d, 2H), 7.51-7.54 (t, 1H), 7.58-7.60 (d, 1H), 7.90-7.92 (d, 1H), 8.29-8.30 (d, 2H).

It was verified that the test result conformed to the structure of chemical formula II.

Example 3

To prepare compound C having the structure shown in chemical formula III,

In this embodiment, X is S, R ₁ is H, R ₂ is H, R ₃ is benzyl, and R ₄ is methyl.

The specific preparation method of compound C is as follows:

In the first step, 10 mL of toluene was weighed into a round-bottomed flask with a capacity of 50 mL, and 10 mmol of 2-methylthiobenzothiazole was added to it. Under nitrogen protection, 12 mmol of benzyl bromide was slowly added dropwise to the round-bottomed flask, and the reaction was continued at 110° C. for 24 h under reflux.

The reaction mixture was cooled to room temperature, after which the mixture was precipitated and filtered, and the filter cake was washed with dichloromethane. The filtrate was dried to obtain a brownish-yellow solid powder, which was 3-benzyl-2-thionebenzothiazole, and the crude yield was about 25%.

In the second step, measure 5mL of dichloromethane and put it into a round-bottomed flask with a capacity of 25mL, then add 0.4mmol of 3-benzyl-2-thione benzothiazole into it, and cool it to -20°C in liquid nitrogen. Under stirring, 0.5 mmol triethyloxonium tetrafluoroboric acid was slowly added dropwise, and the reaction was stopped after continuous stirring for 6 h. Excess petroleum ether was added to the mixture, and the mixture was allowed to settle for precipitation and filtration, and the filter cake was washed with petroleum ether. After drying, a white solid powder, 3-benzyl-2-ethylthiobenzothiazole quaternary ammonium salt, was obtained with a crude yield of 30%.

In the third step, 5 mL of absolute ethanol was added to a double-necked round-bottomed flask with a capacity of 50 mL, and 3 mmol of 3-benzyl-2-ethylthiobenzothiazole obtained in the second step was weighed and added to the above-mentioned double-necked flask. in a round bottom flask.

Under nitrogen protection and stirring, 10 mmol of triethylamine was added dropwise to a two-necked round-bottomed flask. 3.5 mmol of 1,4-lutidine was dissolved in 5 mL of absolute ethanol, and then the solution was added dropwise to the above two-necked round-bottomed flask, and the reaction was refluxed at 60° C. for 7 h. After that, the reaction solution was evaporated to dryness, and separated by a neutral silica gel chromatography column. In the separation process, a mixed solvent of dichloromethane and methanol was used as an eluent. The separated yellow components were collected and evaporated to dryness to obtain a yellow solid powder, namely Compound C, with a crude yield of about 22%.

The NMR test of compound C was carried out, and the results were as follows.

¹ H-NMR (500MHz, DMSO, TMS): δ4.00(s, 3H), 5.59(s, 2H), 6.39(s, 1H), 7.26-7.31(m, 4H), 7.35-7.38(t, 2H), 7.40-7.42(d, 2H), 7.45-7.48(t, 1H), 7.52-7.54(d, 1H), 7.94-7.96(d, 1H), 8.33-8.35(d, 2H).

It was verified that the test result conformed to the structure of chemical formula III.

Example 4

To prepare compound D having the structure shown in formula IV,

In this embodiment, X is S, R ₁ is H, R ₂ is H, R ₃ is 4-(diethylamino)butyl, and R ₄ is methyl.

The specific preparation method of compound D is as follows:

In the first step, 13.4mmol (2g, 1eq) of 2-methylbenzothiazole and 67.0mmol (14.52g, 5eq) of 1,4-dibromobutane were added to a microwave tube with a capacity of 20mL, and it was microwaved Heated to 140°C and reacted for 3 hours.

After cooling the microwave tube, a large amount of solid was precipitated. The reaction solution was filtered, the obtained filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The filtrate was dried in vacuo to give 2.5 g of a white solid as 3-(4-bromobutyl)-2-methylbenzothiazole quaternary ammonium salt.

In the second step, 10 mmol (2.05 g, 1 eq) of 4-iodopyridine and 50 mmol (7.1 g, 5 eq) of iodomethane were added to a microwave tube with a capacity of 20 mL, and 10 mL of dichloromethane was added to the microwave tube.

The microwave tube was heated to 60°C by microwave and the reaction was maintained for 2 hours. After it was cooled down, a large amount of solid was precipitated. The reaction solution was filtered, the obtained filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The filtrate was dried in vacuo to give 2.0 g of a brown solid as 1-methyl-4-iodopyridine quaternary ammonium salt.

In the third step, 10 mL of methanol was added to a microwave tube with a capacity of 20 mL, and 2.75 mmol (1 g, 1 eq) of 3-(4-bromobutyl)-2-methylbenzothiazole quaternary amine obtained in the first reaction was weighed salt and dissolve it in methanol in a microwave tube.

Weigh 2.75mmol (955mg, 1eq) of the 1-methyl-4-iodopyridine quaternary ammonium salt obtained in the second step reaction at room temperature, weigh 5.5mmol (462mg, 2.5eq) of sodium bicarbonate, put the two were added to the reaction flask (microwave tube).

The reaction vial was microwaved to 60°C for 2 hours. After that, the reaction solution was cooled and filtered, and the filtrate was concentrated, and then separated through a silica gel column. In the separation process, a mixture of dichloromethane and methanol was used as an eluent. The isolated product was collected to obtain 400 mg of yellow solid powder, namely 3-(4-bromobutyl)-2-((1-methylpyridine-4(1H)-methylene)methyl)benzothiazole, which was The yield is about 32%.

The fourth step, weigh 0.88mmol (400mg, 1eq) of the 3-(4-bromobutyl)-2-((1-methylpyridine-4(1H)-methylene)methan obtained in the third step base) benzothiazole, weigh 4.4 mmol (321 mg, 5 eq) of diethylamine and add both to a 20 mL capacity microwave tube.

The above microwave tube was microwave heated to 60°C and maintained for 2 hours. After that, the reaction solution was cooled and filtered, and the filtrate was concentrated, and then separated through a silica gel column. In the separation process, a mixture of dichloromethane and methanol was used as an eluent. The isolated product was collected to obtain 150 mg of yellow solid powder, namely compound D, whose name was 3-(4-(diethylamino)butyl)-2-((1-methylpyridine-4(1H)-methylene) ) methyl) benzothiazole quaternary ammonium salt. The yield of compound D was about 38%.

Compound D was tested by liquid chromatography-mass spectrometry, and the results were as follows.

LCMS: Calculated Exact Mass=368.22, Found[M+H]+(ESI+)=368.22.

The results show that the calculated value of the molecular weight of Compound D corresponds to the measured value, which proves that Compound D has the structure of Chemical Formula IV.

The NMR test of compound D was carried out, and the results were as follows.

¹ H-NMR (400 MHz, DMSO): δ 8.33 (d, J=7.1 Hz, 2H), 7.93 (d, J=7.7 Hz, 1H), 7.64 (s, 1H), 7.54 (s, 1H), 7.46(d, J=7.2Hz, 2H), 7.32(s, 1H), 6.36(s, 1H), 4.00(s, 3H), 3.12(d, J=5.8Hz, 6H), 1.78(s, 4H) ), 1.20 (d, J=15.5, 8.3 Hz, 8H).

It was verified that the test result conformed to the structure of chemical formula IV.

Example 5

To prepare compound E having the structure shown in formula V,

In this embodiment, X is S, R ₁ is Cl, R ₂ is H, R ₃ is methyl, and R ₄ is methyl.

The specific preparation method of compound E is as follows:

In a two-necked round-bottomed flask with a capacity of 50 mL, 5 mL of absolute ethanol was added, and then 3 mmol of 3-methyl-2-methylthio-6-chlorobenzothiazole was added. Under nitrogen protection and stirring, 10 mmol of triethylamine was added dropwise to a two-necked round-bottomed flask.

3.5 mmol of 1,4-lutidine was dissolved in 5 mL of absolute ethanol, the obtained solution was added dropwise to the above double-necked round-bottomed flask, and the reaction was refluxed at 60° C. for 7 h.

The reaction solution was evaporated to dryness and separated through a neutral silica gel column. In the separation process, a mixed solvent of dichloromethane and methanol was used as the eluent. The separated yellow components were collected and evaporated to dryness to finally obtain a yellow solid powder, namely Compound E, with a crude yield of about 20%.

The NMR test of compound E was carried out, and the results were as follows.

¹ H-NMR (500MHz, DMSO, TMS): δ 3.73(s, 3H), 3.99(s, 3H), 6.25(s, 1H), 7.29-7.32(t, 1H), 7.40-7.41(d, 2H), 7.51-7.54 (t, 1H), 7.58-7.60 (s, 1H), 8.29-8.30 (d, 2H).

It was verified that the test results conformed to the structure of chemical formula V.

Example 6

To prepare compound F having the structure shown in chemical formula VI,

In this embodiment, X is C(CH ₃ ) ₂ , R ₁ is H, R ₂ is H, R ₃ is ethyl, and R ₄ is methyl.

The specific preparation method of compound F is as follows:

In the first step, 25mmol (4g, 1eq) of 2,3,3-trimethyl-3H-indole and 37.7mmol (5.89g, 1.5mmol) of iodoethane were added to a 20mL capacity microwave tube, and Thereto was added 5 mL of toluene.

The microwave tube was microwaved to 140°C, and the reaction was carried out for 3 hours. After the reaction was completed, the temperature of the microwave tube was cooled, and a large amount of solid was precipitated. The reaction solution was filtered, the filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The finally obtained solid was dried in vacuo to finally obtain 7.34 g of a red solid, which was 1-ethyl-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt.

In the second step, 10 mmol (2.05 g, 1 eq) of 4-iodopyridine and 100 mmol (14.2 g, 10 eq) of methyl iodide were added to a single-neck flask with a capacity of 100 mL. The reaction was stirred for 3 hours under nitrogen protection and an oil bath at 60°C. After the reaction was completed, the single-neck flask was cooled down, and a large amount of solid was precipitated. The reaction solution was filtered, the filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The solid obtained by the second filtration was vacuum-dried to finally obtain 2.0 g of a brown solid, which was 1-methyl-4-iodopyridine quaternary ammonium salt.

The third step, weigh 3.17mmol (1g, 1eq) of the 1-ethyl-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt obtained in the first step, and dissolve it in the reaction flask in 10 mL of acetonitrile.

Weigh 3.17 mmol (1 g, 1 eq) of the 1-methyl-4-iodopyridine quaternary ammonium salt obtained from the second step reaction, weigh 7.94 mmol (665 mg, 2.5 eq) of sodium bicarbonate, mix the two at room temperature. were added to the reaction flask. The reaction vial was microwaved to 100°C and the reaction was maintained for 2 hours.

After the reaction, the reaction solution was extracted with dichloromethane, and the organic phase obtained by extraction was washed with water, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated. Then, the concentrated product was separated through a silica gel column, and a mixture of dichloromethane and methanol was used as an eluent during the separation process, and 500 mg of a yellow oily crude product was collected. The crude product was treated with Pre-HPLC to obtain 100 mg of the final product as a yellow oil, which was compound F.

Compound F was tested by liquid chromatography-mass spectrometry, and the results were as follows.

LCMS: Calculated Exact Mass: 279.19, Found[M+H]+(ESI+)=279.2.

The results show that the calculated value of the molecular weight of compound F corresponds to the test value, which proves that compound F has the structure of chemical formula VI.

The NMR test of compound F was carried out, and the results were as follows.

¹ H-NMR (400MHz, DMSO): δ8.33 (s, 2H), 7.70 (s, 1H), 7.42-7.40 (m, 1H), 7.29 (m, 1H), 7.06 (t, J=7.3Hz) , 2H), 5.76 (s, 1H), 4.04 (s, 3H), 3.92 (s, 2H), 1.73 (s, 3H), 1.54 (s, 3H), 1.32 (s, 3H).

It was verified that the test results conformed to the structure of chemical formula VI.

Example 7

To prepare compound G having the structure shown in chemical formula VII,

In this embodiment, X is C(CH ₃ ) ₂ , R ₁ is H, R ₂ is H, R ₃ is benzyl, and R ₄ is methyl.

The specific preparation method of compound G is as follows:

In the first step, 12 mmol (2 g, 1 eq) of 2,3,3-trimethyl-3H-indole and 60 mmol (10 g, 5 mol) of benzyl bromide were added to a microwave tube of 20 mL capacity. The microwave tube was microwave-heated and allowed to react at 140°C for 1 hour. After the reaction was completed, the temperature was lowered, and a large amount of solid was precipitated. The reaction solution was filtered, the filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The solid obtained by the second filtration was dried under vacuum to finally obtain 1.7 g of a white solid, which was 1-benzyl-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt.

In the second step, 10 mmol (2.05 g, 1 eq) of 4-iodopyridine and 100 mmol (14.2 g, 10 eq) of methyl iodide were added to a single-neck flask with a capacity of 100 mL, and the reaction was stirred under nitrogen protection and a 60 °C oil bath for reaction 3 Hour. After the reaction was completed, the single-neck flask was cooled, and a large amount of solid was precipitated. The reaction solution was filtered, the filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The solid obtained by the second filtration was vacuum-dried to finally obtain 2.0 g of a brown solid, which was 1-methyl-4-iodopyridine quaternary ammonium salt.

The 3rd step, in the reactor, add 10 milliliters of methylene chloride, take by weighing the 1-benzyl-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt 3mmol ( 1 g, 1 eq), which was dissolved in dichloromethane. Then add 1 mL of methanol to the reactor

At room temperature, weigh 3 mmol (1.1 g, 1 eq) of the 1-methyl-4-iodopyridine quaternary ammonium salt obtained from the second step reaction, weigh 7 mmol (630 mg, 2.3 eq) of sodium bicarbonate, and mix the two Each was added to the reactor, after which the reaction was stirred at room temperature for 3 hours. After the reaction, the reaction solution was filtered and concentrated, and the concentrated solution was separated through a silica gel column, and a mixture of dichloromethane and methanol was used as an eluent during the separation process. The isolated product was collected to obtain 366 mg of yellow solid powder, which was compound G, and the yield was about 35.2%.

Compound G was tested by liquid chromatography-mass spectrometry, and the results were as follows.

LCMS: Calculated Exact Mass=341.2, Found[M+H]+(ESI+)=341.2.

The results show that the calculated value of the molecular weight of compound G corresponds to the test value, which proves that compound G has the structure of chemical formula VII.

The NMR test of compound G was carried out, and the results were as follows.

¹ H-NMR (400MHz, MeOD): δ 8.16(s, 2H), 7.61(s, 1H), 7.41-7.18(m, 7H), 7.09(s, 2H), 6.96(s, 1H), 5.72 (d, J=26.5 Hz, 1H), 5.10 (d, J=59.2 Hz, 2H), 4.07 (s, 3H), 1.82 (s, 3H), 1.50 (s, 3H).

After verification, the test result conforms to the structure of chemical formula VII.

Example 8

To prepare compound H having the structure shown in chemical formula VIII,

In this embodiment, X is S, R ₁ is H, R ₂ is H, R ₃ is methyl, and R ₄ is hydroxypropyl.

The specific preparation method of compound H is as follows:

In a 50-mL double-necked round-bottomed flask, 5 mL of absolute ethanol was added, and 2.55 mmol of 3-methyl-2-methylthiobenzothiazole was added therein. Under nitrogen protection and stirring conditions, 10 mmol of triethylamine was added dropwise to a two-necked round-bottomed flask.

3.04 mmol of 1-(3-hydroxypropyl)-4-methylpyridine was dissolved in 5 mL of absolute ethanol, and the obtained solution was added dropwise to the above two-necked round-bottomed flask, and the reaction was heated under reflux at 60 °C. 1.5h.

After the reaction, the reaction solution was evaporated to dryness and separated through a neutral silica gel column, and a mixed solvent of dichloromethane and methanol was used as the eluent in the separation process. The separated yellow components were collected, evaporated to dryness, and finally a yellow solid powder was obtained, which was compound H, and the crude yield was about 20%.

The NMR test of compound H was carried out, and the results were as follows.

¹ H-NMR (500MHz, DMSO, TMS): δ1.95-2.00(m, 2H), 3.44(s, 2H), 3.74(s, 3H), 4.28-4.31(t, 2H), 4.74(s, 1H), 6.27(s, 1H), 7.30-7.33(t, 1H), 7.40-7.42(d, 2H), 7.51-7.54(t, 1H), 7.60-7.61(d, 1H), 7.92-7.93( d, 1H), 8.35-8.37 (d, 2H).

It is verified that the test result conforms to the structure of chemical formula VIII.

Example 9

To prepare compound I having the structure shown in formula IX,

In this embodiment, X is C(CH ₃ ) ₂ , R ₁ is H, R ₂ is H, R ₃ is hexylcarboxy, and R ₄ is methyl.

The specific preparation method of compound I is as follows:

In the first step, 25mmol (4g, 1eq) of 2,3,3-trimethyl-3H-indole and 37.7mmol (7.3g, 1.5eq) of 6-bromohexanoic acid were added to a 20mL capacity microwave tube , and 5 mL of toluene was added to it. After that, microwave heating was performed on the microwave tube, and the reaction was carried out at 140° C. for 3 hours.

After the reaction was completed, the microwave tube was cooled, and a large amount of solid was precipitated. The reaction solution was filtered, the filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The solid obtained by the second filtration was dried under vacuum to finally obtain 7.34 g of a red solid, which was 1-hexylcarboxy-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt.

In the second step, 10mmol (2.05g, 1eq) of 4-iodopyridine and 100mmol (14.2g, 10eq) of methyl iodide were added to a 100mL single-neck flask, and the reaction was stirred under nitrogen protection and in an oil bath at 60°C 3 hours.

After the reaction was completed, the single-neck flask was cooled, and a large amount of solid was precipitated. The reaction solution was filtered, the filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The solid obtained by the second filtration was vacuum-dried to finally obtain 2.0 g of a brown solid, which was 1-methyl-4-iodopyridine quaternary ammonium salt.

In the third step, take 3.17mmol (1g, 1eq) of 1-hexanecarboxylic acid-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt obtained by the first step reaction, and dissolve it in 10 mL of acetonitrile in the reaction vial.

Weigh 3.17 mmol (1 g, 1 eq) of the 1-methyl-4-iodopyridine quaternary ammonium salt obtained from the second step reaction, weigh 7.94 mmol (665 mg, 2.5 eq) of sodium bicarbonate, mix the two at room temperature. were added to the reaction flask. The reaction vial was microwaved to 100°C and the reaction was maintained for 2 hours.

After the reaction, the reaction solution was extracted with dichloromethane, and the organic phase obtained by extraction was washed with water, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated. Then, the concentrated product was separated through a silica gel column, and a mixture of dichloromethane and methanol was used as an eluent during the separation process, and 500 mg of a yellow oily crude product was collected. The crude product was treated with Pre-HPLC to obtain 100 mg of the final product as a yellow oil, which was compound I.

The NMR test of compound I was carried out, and the results were as follows.

¹ H-NMR (400MHz, DMSO): δ8.33 (s, 2H), 7.70 (s, 1H), 7.42-7.40 (m, 1H), 7.29 (m, 1H), 7.06 (t, J=7.3Hz) , 2H), 5.76 (s, 1H), 4.04 (s, 3H), 3.92 (s, 2H), 1.73 (s, 3H), 1.54 (s, 3H), 1.32 (s, 3H).

It was verified that the test results conformed to the structure of chemical formula IX.

Example 10

To prepare compound J having the structure shown in formula X,

In this embodiment, X is S, R ₁ is H, R ₂ is H, R ₃ is carboxypentyl, and R ₄ is methyl.

The specific preparation method of compound J is as follows:

In the first step, 10 mL of ethyl acetate was placed in a round-bottomed flask with a capacity of 50 mL, and 5.37 mmol of 4-picoline and 10.7 mmol of 6-bromohexanoic acid were added to the above round-bottomed flask. Under reflux reaction for 8h. The obtained mixture was recrystallized with ethyl acetate and suction filtered, and ethyl acetate was used for thorough washing during suction filtration. The obtained filtrate was dissolved in methanol and evaporated to dryness. After final drying, a yellow oily liquid was obtained, which was 1-(5-carboxypentyl)-4-methylpyridine, and the crude yield was about 91.07%.

In the second step, 10 mL of anhydrous tetrahydrofuran was placed in a round-bottomed flask with a capacity of 50 mL, and 11.84 mmol of 2-mercaptobenzothiazole was added to the round-bottomed flask, and then slowly added dropwise to the round-bottomed flask while stirring. 14.21 mmol of iodomethane, reacted overnight. After that, a small amount of petroleum ether was added to the mixture obtained by the reaction and allowed to stand for precipitation. It was then filtered and the filtrate was washed with copious amounts of petroleum ether. Finally, the filtrate was dried to obtain a white solid, which was 2-methylthiobenzothiazole, and the crude yield was about 98%.

In the third step, 10 mL of toluene was placed in a round-bottomed flask with a capacity of 50 mL, and 10 mmol of 2-methylthiobenzothiazole was added to the above-mentioned round-bottomed flask. Then, under nitrogen protection, 12 mmol of methyl iodide was slowly added dropwise to the round-bottomed flask, and the reaction was continued at 110° C. and reflux for 24 h. The reaction mixture was cooled to room temperature, after which the mixture was filtered, and the filtrate was washed with dichloromethane. Finally, the filtrate was dried to obtain a yellowish solid powder, which was 3-methyl-2-methylthiobenzothiazole, and the crude yield was about 30%.

In the fourth step, 5 mL of dichloromethane was added to a 25 mL capacity round bottom flask. 0.500 mmol of 3-methyl-2-methylthiobenzothiazole and 0.509 mmol of 1-(5-carboxypentyl)-4-methylpyridine were added to the above round bottom flask. After stirring for 10 min at room temperature and in the dark, 756 μL of triethylamine was slowly added dropwise thereto, and then the reaction was carried out at room temperature and in the dark for about 21 h. After that, the mixture obtained by the reaction was evaporated to dryness, and the residue was separated through a neutral silica gel column using a mixed solvent of dichloromethane and methanol as an eluent. The separated yellow components were collected and evaporated to dryness to finally obtain a brownish-yellow viscous substance, namely Compound J, with a crude yield of 50%.

The NMR test of compound J was carried out, and the results were as follows.

¹ H-NMR (400 MHz, DMSO): δ 8.36 (s, 1H), 7.90 (d, J=7.6 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.51 (t, J=7.4 Hz, 1H), 7.40(s, 1H), 7.30(t, J=7.1Hz, 1H), 6.25(s, 1H), 4.21(s, 1H), 3.72(s, 3H), 2.10(s, 1H) ), 1.82 (s, 1H), 1.53 (s, 1H), 1.28 (s, 1H).

It was verified that the test results conformed to the structure of chemical formula X.

Example 11

To prepare compound K having the structure shown in chemical formula XI,

In this embodiment, X is S, R ₁ is H, R ₂ is H, R ₃ is hydroxypropyl, and R ₄ is methyl.

The specific preparation method of compound K is as follows:

The first step: 4.88 mmol of 4-iodopyridine and 9.76 mmol of methyl iodide were added to a round-bottomed flask with a capacity of 25 mL and contained 5 mL of tetrahydrofuran, and the reaction was refluxed at a temperature of 75° C. for 3 h. The mixture obtained by the reaction was thoroughly washed with ethyl acetate, and dried to obtain a brown-yellow solid powder, which was 4-iodo-1-methylpyridine quaternary ammonium salt, and the crude yield was about 93%.

The second step: 670.19 mmol of 2-methylbenzothiazole was added to a double-necked round-bottomed flask with a capacity of 25 mL, and 804.23 mmol of 3-bromo-1-propanol was slowly added dropwise to it. The reaction was continued for about 8 h, and the reactor was cooled to room temperature after completion. The resulting mixture was added to diethyl ether for standing precipitation and filtered, after which the filter cake was washed with a large amount of diethyl ether. A green solid was obtained after drying, which was 3-(3-hydroxypropyl)-2-methylbenzothiazole in about 54% crude yield.

The third step: 4mL anhydrous methanol is charged into the double-necked round-bottomed flask of 25mL capacity, then 480.08mmol of the obtained 3-(3-hydroxypropyl)- 2-methylbenzothiazole, then add 576.10 mmol of NaHCO ₃ (dissolved in 2 mL of water), stir at room temperature for 0.5 h, and then add 576.10 mmol of 4-iodo-1-methyl obtained in the first step above The pyridine was added to a double-necked round-bottomed flask, and the reaction was carried out overnight at 110° C. under reflux conditions. The obtained reaction solution was washed with water, extracted with dichloromethane, dried and concentrated, and finally crystallized by adding ethyl acetate and filtered. The crude product obtained by filtration was separated through a neutral silica gel column (the eluent was a mixed solvent of dichloromethane and methanol), and the separated yellow components were collected and evaporated to dryness. Finally, a yellow solid powder is obtained, which is compound K, and its crude yield is about 1.12%.

The NMR test of compound K was carried out, and the results were as follows.

¹ H-NMR (400MHz, DMSO): δ 8.31 (d, J=6.7Hz, 1H), 7.92 (d, J=7.8Hz, 1H), 7.60-7.48 (m, 1H), 7.41 (d, J =6.6Hz, 1H), 7.31(t, J=7.5Hz, 1H), 6.30(s, 1H), 4.81(t, J=4.8Hz, 1H), 4.31(t, J=7.2Hz, 1H), 3.99 (s, 1H), 3.64-3.45 (m, 1H), 2.00-1.73 (m, 1H).

It was verified that the test result conformed to the structure of chemical formula XI.

Example 12

To prepare compound L having the structure shown in chemical formula XII,

In this embodiment, X is S, R ₁ is methyl, R ₂ is H, R ₃ is methyl, and R ₄ is methyl.

The specific preparation method of compound L is as follows:

The first step: put 5mL of tetrahydrofuran into a 25mL capacity round-bottomed flask, then add 4.88mmol of 4-iodopyridine and 9.76mmol of iodomethane, and react at 75°C and reflux for 3h. The mixture obtained by the reaction was thoroughly washed with ethyl acetate, and dried to obtain a brownish-yellow solid powder, which was 4-iodo-1-picoline quaternary ammonium salt, and the crude yield was about 93%.

Step 2: Add 612.60 mmol of 2,6-dimethylbenzothiazole to a double-necked round-bottomed flask with a capacity of 10 mL, slowly add 1.23 mmol of methyl iodide dropwise while stirring, and then at a temperature of 70 °C Reflux reaction for 1.5h. After the reaction was completed, the reactor was cooled to room temperature, and ethyl acetate was added to the mixture obtained by the reaction for precipitation and filtration, and then the filter cake was washed with a large amount of ethyl acetate. The filtrate was dried to obtain a white solid, which was 2,3,6-trimethylbenzothiazole, in a crude yield of about 98%.

The third step: put 4mL of anhydrous methanol into a double-necked round-bottomed flask with a capacity of 25mL, and then add 560.94mmol of 2,3,6-trimethylbenzothiazole obtained in the second step above and 1.12mmol of NaHCO. ₃ (dissolved in 1 mL of water), stirred at room temperature for 0.5 h. After dissolving 673.13 mmol of 4-iodo-1-methylpyridine obtained in the first step above in 5 mL of anhydrous methanol, it was added dropwise into a round-bottomed flask, and the reaction was carried out at 110° C. and reflux for 8 h. The obtained reaction solution was washed with water, extracted with dichloromethane, dried and concentrated, and finally crystallized by adding ethyl acetate and filtered. The crude product obtained by filtration is separated by a neutral silica gel column (the mixed solvent of dichloromethane and methanol is used as the eluent), the yellow component obtained by the separation is collected and evaporated to dryness, and finally a yellow solid powder is obtained, which is compound L. The yield is about 4.2%.

The NMR test of compound L was carried out, and the results were as follows.

¹ H-NMR (400 MHz, DMSO): δ 8.26 (d, J=7.1 Hz, 1H), 7.72 (s, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.36 (t, J=9.0 Hz, 1H), 6.21 (s, 1H), 3.97 (s, 2H), 3.71 (s, 2H), 2.39 (s, 1H).

It was verified that the test results conformed to the structure of chemical formula XII.

Example 13

To prepare compound M having the structure shown in chemical formula XIII,

In this embodiment, X is S, R ₁ is phenyl, R ₂ is H, R ₃ is methyl, and R ₄ is methyl.

The specific preparation method of compound M is as follows:

The first step: put 5mL of tetrahydrofuran into a 25mL capacity round-bottomed flask, then add 4.88mmol of 4-iodopyridine and 9.76mmol of iodomethane, and react at 75°C and reflux for 3h. The mixture obtained by the reaction was thoroughly washed with ethyl acetate, and dried to obtain a brownish-yellow solid powder, which was 4-iodo-1-picoline quaternary ammonium salt, and the crude yield was about 93%.

The second step: 501.83 mmol of 2-methylnaphthalenethiazole was added to a double-necked round-bottomed flask with a capacity of 25 mL, then 4 mL of chloroform was added, and 1.00 mmol of methyl iodide was slowly added dropwise while stirring. The reaction was carried out under reflux for about 8h. After the reaction was completed, the reactor was cooled to room temperature, and diethyl ether was added to the obtained mixture for precipitation and filtration, and the filter cake was washed with a large amount of diethyl ether. The filtrate was dried to obtain a yellow solid, which was 2,3-dimethylnaphthalenethiazole, and the crude yield was about 56.35%.

The 3rd step: 4mL anhydrous methanol was charged into the double-necked round bottom flask of 25mL, then 466.62mmol of the 2,3-dimethylnaphthalene thiazole obtained in the second step was added, and 559.95mmol of NaHCO (dissolved ₎ was added. in 2 mL of water) and stirred at room temperature for 0.5 h. Then, 559.95 mmol of 4-iodo-1-methylpyridine obtained in the first step was added into the reactor, and the reaction was carried out under reflux at a temperature of 110° C. overnight. The resulting reaction solution was washed with water, extracted with dichloromethane, dried and concentrated, and finally crystallized by adding ethyl acetate and filtered. The crude product obtained by filtration is separated by a neutral silica gel column (the mixed solvent of dichloromethane and methanol is used as the eluent), the separated yellow components are collected, evaporated to dryness, and finally a yellow solid powder is obtained, which is compound M, The crude yield was about 1.40%.

The NMR test of compound M was carried out, and the results were as follows.

¹ H-NMR (400 MHz, DMSO): δ 8.30 (d, J=6.9 Hz, 1H), 8.13 (dd, J=11.7, 8.6 Hz, 1H), 7.99 (d, J=7.9 Hz, 1H), 7.90 (d, J=9.1Hz, 1H), 7.73 (t, J=7.7Hz, 1H), 7.59 (t, J=7.5=Hz, 1H), 7.49 (d, J=7.2Hz, 1H), 6.34 (s, 1H), 4.00 (s, 1H), 3.86 (s, 1H).

It was verified that the test result conformed to the structure of chemical formula XIII.

Calf thymus DNA and RNA were stained with the compounds synthesized in Examples 1-13, and absorption (excitation) and fluorescence (emission) spectra were measured using a UV-Vis spectrophotometer and a fluorescence spectrophotometer, respectively. The result is that the excitation light wavelengths of the compounds synthesized in Examples 1-13 are in the blue-green range.

Live HeLa cells were stained with the compounds synthesized in Examples 1-13 and observed using a confocal laser scanning microscope. The results show that the compounds synthesized in Examples 1-13 can stain HeLa cells without destroying the cell membrane, and the images are clear.

The following will take Compound B synthesized in Example 2 and Compound C synthesized in Example 3 as examples to describe the calf thymus nucleic acid staining experiment and the HeLa live cell staining experiment in detail.

Example 14

The staining experiment of compound B on calf thymus DNA:

The configuration concentrations are 10μg/mL, 20μg/mL, 30μg/mL, 40μg/mL, 50μg/mL, 60μg/mL, 70μg/mL, 80μg/mL, 90μg/mL, 100μg/mL, 110μg/mL, 120μg/mL mL, 130 μg/mL, 140 μg/mL, 150 μg/mL, 200 μg/mL of calf thymus DNA in water.

A certain amount of compound B was dissolved in DMSO (dimethyl sulfoxide), and a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH of 7.4 and a concentration of 10 mmol/L was added thereto to prepare a certain amount of compound B. concentration of Compound B in buffer solution.

A certain amount of compound B buffer solution was mixed with a certain amount of the above-mentioned aqueous solutions of calf thymus DNA with different concentrations, placed in a cuvette and left at 37°C for 3 minutes, and then the absorption spectrum was measured. And a certain amount of compound B buffer solution and a certain amount of water were used as a comparative test. The instrument used was an ultraviolet-visible spectrophotometer, model Hp8453.

The absorption spectrum of compound B as a function of DNA concentration is shown in Figure 1. In the figure, taking 440 nm as an example, the nucleic acid concentrations represented by the different curves from top to bottom along the ordinate increase sequentially. Among them, the highest curve represents 0 μg/mL, that is, no DNA; the lowest curve represents the nucleic acid concentration of 200 μg/mL. It can be seen from the figure that compound B has the strongest absorption of light with a wavelength of about 440 nm, and the light near this wavelength is blue-green light.

Take the wavelength at the maximum absorption peak (such as around 440 nm) of the absorption curve of the calf thymus DNA solution of each concentration, and use the light of this wavelength as the excitation light to mix the above-mentioned calf thymus DNA solutions of different concentrations with compound B. The solution was subjected to fluorescence excitation experiment, and the fluorescence spectrum was measured. The instrument used was a fluorescence spectrophotometer, model FP-6500.

The fluorescence spectrum of compound B as a function of DNA concentration is shown in Figure 2. In the figure, taking 475 nm as an example, the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease in turn. Among them, the curve at the highest position represents a DNA concentration of 200 μg/mL; the curve at the lowest position (almost overlapping the horizontal axis) represents 0 μg/mL, that is, no DNA. It can be seen from the figure that the fluorescence emitted by the excited compound B has the highest intensity near 485 nm, indicating that the compound B can successfully emit fluorescence and can be used as a fluorescent dye.

The staining experiment of compound B on calf thymus RNA is similar to the above-mentioned DNA staining experiment process, and will not be repeated here. The absorption spectrum of compound B with the change of calf thymus RNA concentration is shown in FIG. 3 , and the fluorescence spectrum of compound B with the change of calf thymus RNA concentration is shown in FIG. 4 .

In Fig. 3, taking 440 nm as an example, the nucleic acid concentrations represented by different curves from top to bottom along the ordinate increase sequentially. Among them, the highest curve represents 0 μg/mL, that is, no RNA; the lowest curve represents the RNA concentration of 200 μg/mL. It can be seen from the figure that compound B has the strongest absorption of light with a wavelength of about 440 nm, and the light near this wavelength is blue-green light.

In Fig. 4, taking 475 nm as an example, the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease sequentially. Among them, the curve at the highest position represents the RNA concentration of 200 μg/mL; the curve at the lowest position (almost overlapping the horizontal axis) represents 0 μg/mL, that is, no RNA. It can be seen from the figure that the fluorescence emitted by the excited compound B has the highest intensity near 485 nm, indicating that the compound B can successfully emit fluorescence and can be used as a fluorescent dye.

It can be seen that compound B has good absorption of blue-green laser with short wavelength, and the short wavelength is conducive to the identification of small particle objects. Common semiconductor green laser or blue laser can be used as the light source, and the cost is low.

Figure 5 shows a graph of the fluorescence intensity of compound B stained for DNA and RNA as a function of nucleic acid concentration. In the figure, it is more clearly shown that as the nucleic acid concentration increases, the fluorescence intensity of compound B is higher. In the absence of nucleic acid, compound B is excited and hardly emits light, which is beneficial to reduce the background interference of background fluorescence and can improve the sensitivity.

Example 15

The staining test of compound B on live HeLa cells. The instrument used was a confocal laser scanning microscope, model FV1000IX81, Japan.

Compound B was added to PBS buffer to prepare a compound B buffer solution with a concentration of 1 mmol/L. HeLa cells were cultured in a six-well plate, and 10 μL of the above compound B buffer solution was added thereto. It was then incubated for 30 min in a cell culture incubator at 37°C and 5% CO ₂ .

After the incubation, the cells were washed three times with PBS, and then the cell culture medium was added, and the cell morphology was observed using a confocal laser scanning microscope. The observation results are shown in the bright field micrograph of the representative area shown in Fig. 6, from which the living cells with complete morphology can be clearly seen.

The above representative regions were excited using the 488 nm channel and observed with a 100x oil lens, and repeated three times. Results Fluorescence micrographs of HeLa live cells stained by Compound B are shown in FIG. 7 . From the figure, it can be clearly seen that each cell can emit fluorescence.

FIG. 8 is an overlay of the brightfield micrograph of FIG. 6 and the fluorescence micrograph of FIG. 7 . In the figure, it can be seen more clearly that the nuclei are stained and the cell structure is intact.

It can be seen from Figures 6 to 8 that Compound B can effectively penetrate the living cell membrane to stain nucleic acids without destroying the cell structure, so that it can be stained and observed in the state of cell survival, which is more conducive to the morphology and type of cells. to identify.

Example 16

In the staining experiment of compound C on calf thymus DNA and RNA, the experimental process was similar to that in Example 10, except that the nucleic acid solution with a concentration of 200 μg/mL was not prepared. The specific process is not repeated here.

Figure 9 shows the fluorescence spectrum of compound C as a function of calf thymus DNA concentration. In the figure, taking 475 nm as an example, the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease in turn. Among them, the curve at the highest position represents a DNA concentration of 200 μg/mL; the curve at the lowest position (almost overlapping the horizontal axis) represents 0 μg/mL, that is, no DNA. It can be seen from the figure that the fluorescence emitted by the excited compound C has the highest intensity near 485 nm, indicating that the compound C can successfully emit fluorescence and can be used as a fluorescent dye.

Figure 10 shows the fluorescence spectrum of Compound C as a function of calf thymus RNA concentration. In the figure, taking 475 nm as an example, the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease sequentially. Among them, the curve at the highest position represents the RNA concentration of 200 μg/mL; the curve at the lowest position (almost overlapping the horizontal axis) represents 0 μg/mL, that is, no RNA. It can be seen from the figure that the fluorescence emitted by the excited compound C has the highest intensity near 485 nm, indicating that the compound C can successfully emit fluorescence and can be used as a fluorescent dye.

Figure 11 shows a graph of the fluorescence intensity of compound C after staining for DNA and RNA as a function of nucleic acid concentration. In the figure, it is more clearly shown that as the nucleic acid concentration increases, the fluorescence intensity of compound C is higher.

Example 17

The staining test of compound C on live HeLa cells. The instrument used was a confocal laser scanning microscope, model FV1000IX81, Japan. The experimental process is similar to that of Example 15, and is not repeated here.

Figure 12 shows a bright-field micrograph of a representative area of HeLa live cells stained by compound C, from which the morphologically intact live cells can be clearly seen, and the cell structure is intact and not damaged.

Figure 13 shows fluorescence micrographs of representative regions of compound C staining of live HeLa cells. From the figure, it can be clearly seen that each cell can emit fluorescence, which proves that HeLa has been successfully stained with high identification.

To sum up, the compounds of Examples 1 to 9 of the present invention can penetrate the living cell membrane without destroying the original cell structure, and can effectively stain nucleic acids; and the wavelength range of the absorbed light is located in the blue-green light region , can use blue or green laser, low cost.

Table 1

Numbering	compound	Absorbed light color	Can penetrate living cell membranes	Whether to destroy the cell structure
Example 1	Compound A	blue-green light	can	no
Example 2	Compound B	blue-green light	can	no
Example 3	Compound C	blue-green light	can	no
Example 4	Compound D	blue-green light	can	no
Example 5	Compound E	blue-green light	can	no
Example 6	Compound F	blue-green light	can	no
Example 7	Compound G	blue-green light	can	no
Example 8	Compound H	blue-green light	can	no
Example 9	Compound I	blue-green light	can	no

Example 10	Compound J	blue-green light	can	no
Example 11	Compound K	blue-green light	can	no
Example 12	Compound L	blue-green light	can	no
Example 13	Compound M	blue-green light	can	no

Therefore, the cyanine compounds of the present invention can be used as dyes, especially as fluorescent dyes, as referred to in the second aspect of the present invention.

In addition, the above-mentioned fluorescent dye containing the cyanine compound of the present invention can be used for quantitative detection of nucleic acid and/or biological staining. In particular, it can be used in live cell staining.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of this invention. The terminology used herein is for the purpose of describing a particular implementation only and is not intended to limit the present invention. A feature described herein in one embodiment may be used in another embodiment alone or in combination with other features, unless the feature is not applicable in the other embodiment or stated otherwise.

The present invention has been described by the above-mentioned embodiments, but it should be understood that the above-mentioned embodiments are only for the purpose of illustration and description, and are not intended to limit the present invention to the scope of the described embodiments. In addition, those skilled in the art can understand that the present invention is not limited to the above-mentioned embodiments, and more variations and modifications can also be made according to the teachings of the present invention, and these variations and modifications all fall within the protection claimed in the present invention. within the range. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (17)

1. A kind of cyanine compound, it is characterized in that, has the structure shown in general formula I,

   

   wherein X is selected from the group consisting of C(CH ₃ ) ₂ , O, S and Se;

   R ₁ and R ₂ are each independently selected from the group consisting of H, C ₁ -C ₁₈ alkyl, phenyl, OR ₆ and halogen;

   R ₃ and R ₄ are each independently selected from the group consisting of C ₁ -C ₁₈ alkyl, C ₁ -C ₁₈ carboxy, C ₁ -C ₁₈ hydroxy, C ₁ -C ₁₈ NR ₅ R ₆ , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C ₁ -C ₁₈ alkyl, CN, COOH, NH ₂ , NO ₂ , OH, SH, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkane the group consisting of amino, C ₁ -C ₆ amido, halogen and C ₁ -C ₆ haloalkyl;

   R ₅ and R ₆ are each independently selected from the group consisting of H and C ₁ -C ₁₈ alkyl;

   Y ^- is a negative ion.
2. The cyanine compound of claim 1, wherein the X is selected from the group consisting of C(CH ₃ ) ₂ and S.
3. The cyanine compound of claim 1, wherein the R ₁ and the R ₂ are each independently selected from the group consisting of H, C ₁ -C ₁₂ alkyl, phenyl, OR ₆ and halogen.
4. The cyanine compound of claim 3, wherein the R ₁ and the R ₂ are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl, OR ₆ and halogen.
5. The cyanine compound according to claim 4, wherein the R ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, phenyl and halogen.
6. The cyanine compound according to claim 4, wherein the R ₂ is H.
7. The cyanine compound according to claim 1, wherein said R ₃ and said R ₄ are each independently selected from C ₁ -C ₁₂ alkyl group, C ₁ -C ₁₂ carboxyl group, C ₁ -C ₁₂ hydroxyl group , C ₁ -C ₁₂ NR ₅ R ₆ , benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from the group consisting of C ₁ -C ₁₂ alkyl, CN, COOH, NH ₂ , NO ₂ , The group consisting of OH, SH, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylamino, C ₁ -C ₆ amido, halogen and C ₁ -C ₆ haloalkyl.
8. The cyanine compound according to claim 7, wherein said R ₃ and said R ₄ are each independently selected from C ₁ -C ₆ alkyl group, C ₁ -C ₆ carboxyl group, C ₁ -C ₆ hydroxyl group , C ₁ -C ₆ NR ₅ R ₆ , benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from the group consisting of C ₁ -C ₆ alkyl, CN, COOH, NH ₂ , NO ₂ , The group consisting of OH, SH, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylamino, C ₁ -C ₆ amido, halogen and C ₁ -C ₆ haloalkyl.
9. The cyanine compound according to claim 8, wherein the R ₃ is selected from the group consisting of C ₁ -C ₆ alkyl group, C ₁ -C ₆ hydroxyl group, C ₁ -C ₆ carboxyl group, C ₁ -C ₆ NR ₅ The group consisting of R ₆ and benzyl.
10. The cyanine compound of claim 8, wherein the R ₄ is selected from the group consisting of C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxyl, C ₁ -C ₆ carboxyl, and benzyl.
11. The cyanine compound of claim 1, wherein the R ₅ and the R ₆ are each independently selected from the group consisting of H and C ₁ -C ₁₂ alkyl.
12. The cyanine compound according to claim 11, wherein the R ₅ and the R ₆ are each independently selected from the group consisting of H and C ₁ -C ₆ alkyl.
13. The cyanine compound according to claim 12, wherein the R ₅ and the R ₆ are each independently a C ₁ -C ₆ alkyl group.
14. The cyanine compound according to claim 1, wherein the Y- is selected from the group consisting of halogen anion, ClO ₄ ^- , PF ₆ ^- , BF ₄ ^- , CH ₃ COO ^- or OTs ^{- .}
15. The cyanine compound according to claim 1, wherein the cyanine compound comprises chemical formula I, chemical formula II, chemical formula III, chemical formula IV, chemical formula V, chemical formula VI, chemical formula VII, chemical formula VIII, chemical formula IX, chemical formula X , a structure shown in one of chemical formula XI, chemical formula XII and chemical formula XIII,

    

    
16. A dye, characterized by comprising the cyanine compound described in any one of claims 1-15.
17. The application of the cyanine compound of any one of claims 1-15 in the quantitative detection of nucleic acid, biological staining, and/or blood cell analysis.

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