WO2015064786A1 - Cyanine dye for labeling biomolecules and method for preparing same - Google Patents

Abstract

The present invention provides a cyanine dye capable of fluorescently labeling various biomolecules, since the cyanine dye has high fluorescent intensity at a near-infrared wavelength region, exhibits high stability under aqueous conditions, maintains stability during long periods of biomolecule staining, and shows excellent binding reactivity with biomolecules. A contrast medium composition including the cyanine dye according to the present invention as an active ingredient can be more effectively applied to contrast and obtain optical images of diseased sites by being administered into the body.

Description

Cyanine dye for biomolecular labeling and preparation method thereof

The present invention relates to a cyanine dye capable of fluorescently labeling various biomolecules and a method of preparing the same.

Since the biological material itself has low or no fluorescence in the visible and near-infrared regions, in the biological field, the biological material is used to observe biological phenomena at the cellular and subcellular level in or out of the living body or to project it in vivo to obtain optical images of contrast and diseased areas. Imaging data have been obtained through various techniques that utilize fluorescent dyes or specific biological materials that are pre-labeled with the optical device together with optical equipment.

Various optical anylsis instruments used in the biotechnologies use fluorescent dyes with excitation and emission wavelengths suitable for fluorescence observation based on built-in light sources and filters as base materials or reagents. Will be chosen.

Commonly used optical analysis equipment includes a fluoresce microscope, a confocal microscope, a flowcytometer, a microarray, a quantitative PCR system for cell observation. In addition to research equipment such as nucleic acid and protein separations, electrophoresis devices for analysis, and real-time in vivo imaging systems, the combination of immunoassay, PCR, and statistical techniques Nucleic acid and protein diagnostic kits (or biochips) based in vitro diagnostic equipment, as well as operating tables and endoscopic equipment for image-guided surgery, are known for their diagnosis and treatment. And equipment with higher levels of resolution and data processing capabilities are being developed.

In general, fluorescent dyes used for labeling biological molecules such as proteins or peptides are mostly anthranilate, 1-alkylthic isoindoles, pyrrolinones, and Bimanes, benzoxazole, benzimidazole, benzofuran, benzofurazan, naphthalenes, coumarins, cyanine, stilbenes, carbazoles ), Phenanthridine, anthracenes, bodipy, fluoresceins, eosins, rhodamines, pyrenes, chrysenes and acriri Structures such as acridines are included.

When screening fluorescent dye structures available in the biotechnologies among the many fluorophores exemplified above, fluorescence equipment and fluorescence equipment are generally used when most biomolecules are present in the medium in which they are present, ie, in aqueous and aqueous buffers. It is important to have an excitation and fluorescence wavelength that fits.

The dyes that can be applied mainly in the bio field should have low photobleaching and quenching in aqueous solutions or hydrophilic conditions, and have a large molecular extinction coefficient to absorb large amounts of light. It should be in the visible or near infrared region of 500 nm or more away from its fluorescence range and stable under various pH conditions, but the structure of the dye which can be used for biomolecular labeling which can satisfy the above limitations is limited.

Fluorescent chromogens that meet these requirements include cyanine, rhodamine, florcein, bodiphy, coumarin, acridine, and pyrene derivatives. The reactor is introduced to allow dyes to be combined with specific substituents in the biomolecular structure. Among them, xanthane-based florcene and rhodamine, and polymethine-based cyanine derivative dye compounds are mainly commercialized.

In particular, dye compounds with cyanine chromophores are generally excellent in optical and pH stability, have a narrow absorption and emission wavelength range, and have a fluorescence region of 500 to 800 nm, in addition to the advantages of being easy to synthesize compounds of various absorption / excitation wavelengths. Since it does not overlap with its own fluorescent region of the biomolecule, it is easy to analyze, and although there are some differences depending on the solvent and solubility characteristics, there are many advantages such as high molar absorption coefficient.

In general, when a fluorescent dye is labeled on a biomolecule, various functional groups present in the biomolecule, that is, amine, thiol, and hydroxy groups, are considered. In order to react, a fluorescent dye having a specific reactor is selected. For example, the substituents in the protein structure that can be combined with the reactor of the fluorescent dye, can be inferred from the chemical structure of the amino acid that is the basic unit structure, specifically, the amino group of lysine, cysteine Thiol group, histidine imidazole amine group, secondary aliphatic hydroxyl group of threonine, primary aliphatic hydroxyl group of serine, phenolic hydroxyl group of tyrosine, etc. It is also possible to bond with amino groups. Conversely, the C-terminus of an amino acid is reacted with an activation group such as succimidyl ester or maleimide, and then reacts with an amine or thiol. Covalent bonding with dyes is also widely used.

In addition, the reactor of the dye is biomolecules, sugars, glycoproteins, antibodies (antibody), biocompatible polymers having a monomer such as hyaluronic acid (hyaluronic acid), glycol chitosan (glycol chitosan), various drugs and It may also be combined with a substituent contained in the low molecular weight material. Reactors used in biomolecular labeling dyes or materials known to date can be classified according to substituents associated with biomolecules, and are also commonly used under respective common names.

Numerous reactors are known, but substituent selectivity, reaction rate, yield, reproducibility, stability, etc. have been proven for a long time by many researchers, and in recent years, only a few reactors are introduced into dyes for actual research or commercial purposes. For example, succinimidyl esters and isothiocyanates are most commonly used as reactors for the binding of amine groups to protein molecules, and are most commonly used for the thiol groups of protein molecules. It is maleimide, and dichlorotriazine is mainly chosen as a reactor for the purpose of bonding with the hydroxyl group of a protein molecule. However, in most cases, the reactors are difficult to maintain a long time reaction and storage stability are combined by a substitution reaction or in water-soluble conditions.

GE Healthcare U.S. Pat.Nos.5268486,6043025,6127134 and International Publication No.9633406 disclose the introduction of succinimidyl esters into various cyanine dye structures and staining for biomolecules that can be bound, such as antibodies and peptides. The method is also illustrated. However, this succinimidyl ester is not stable in an aqueous solution, and there is a problem in that N-hydroxy succinimide is produced as a by-product during the dyeing reaction.

The same applies to dyes having other color sources, but even in cyanine dyes, it is known that the color source is polymethine irrespective of whether or not a reactor is introduced, so that the fluorescence characteristics are almost the same, so that the absorption and emission wavelengths are hardly changed.

The most commonly used cyanine dyes include indole structures as hetero rings and succinimidyl ester groups as reactors, but in recent years maleimide, amine, hydrazide ( Many substituents and reactors such as hydrazide) are used alone or in combination.

Representative products having cyanine chromophores include Cy3 and Cy5, and indocyanine green is most known for the purpose of in vivo imaging. Indocyanine green is one of the few clinically approved dye compounds that has been approved by the US FDA. However, the fluorescence intensity of Cyanine green is low and generally known to accumulate well in the liver. Therefore, when looking at an image of a specific region, it is a obstacle in obtaining desired image data due to dye accumulated in the organ. Can act as In addition, the chemical structure of indocianin green itself does not include a reactor, so it is difficult to label biomolecules.

An object of the present invention can be widely used in observing biomolecules in the fields of proteomics, optical molecular imaging, etc., and a novel near-infrared fluorescent cyanine compound which is easy to label biomolecules and a method of preparing the same. To provide.

In order to achieve the above object, the present invention provides a cyanine compound represented by the following [Formula 1] and a preparation method thereof.

[Formula 1]

In [Formula 1],

Y is selected from oxygen, sulfur and CR ₆ R ₇ ,

Z is oxygen or sulfur,

R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups,

R ₃ is a hydroxy group, hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NHNH-4 -R ₈ -6-R ₉ -1,3,5-triazine and NH-A-SO ₂ CH = CH ₂ ,

A is selected from (CH ₂ ) _n , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ),

R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group,

R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups,

m and m 'are the same as or different from each other, and each independently an integer of 1 to 23,

1, n, p and q are the same as or different from each other, and each independently an integer of 1 to 10.

The present invention also provides a near-infrared fluorescent contrast agent composition comprising the cyanine compound represented by Formula 1 as an active ingredient.

The cyanine compound according to the present invention has high fluorescence intensity in the near-infrared wavelength region, has high stability in water-soluble conditions, and is easy to store for a long time, and is stable even for long-term dyeing of biomolecules, and its binding reactivity with biological molecules Excellent but no reaction by-products can be produced can be used more effectively for the labeling and staining of biomolecules. In addition, the cyanine compound according to the present invention does not accumulate in the body and is completely released, exhibiting strong fluorescence without residual toxicity, and the contrast agent composition comprising the same as an active ingredient is more effectively used to obtain optical images of contrast and diseased areas by being administered in vivo. Can be.

1 is an image showing the results of labeling and electrophoresis on proteins using cyanine-based compounds according to one embodiment and comparative example of the present invention.

Figure 2 is the result of measuring the near infrared fluorescence intensity according to the preparation and comparative example of the present invention.

3 is an imaging image of measuring the distribution tendency and residual amount in the body after administration of a cyanine compound according to one embodiment of the present invention and a comparative example.

The present invention, while maintaining the excellent performance of the cyanine chromophore, in particular to produce a compound having a high fluorescence intensity in the near-infrared wavelength region, while showing a high stability in water-soluble buffer conditions, while being reactive with biomolecules, nanoparticles or organic compounds This excellent cyanine compound was prepared to complete the present invention. Most of the cyanine compounds according to the present invention are designed to target water as a medium and to be stable to heat when applied to biological molecules.

Hereinafter, the present invention will be described in more detail.

The present invention provides a cyanine compound represented by the following [Formula 1].

[Formula 1]

In [Formula 1],

Y is selected from oxygen, sulfur and CR ₆ R ₇ ,

Z is oxygen or sulfur,

R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups,

R ₃ is a hydroxy group, hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NHNH-4 -R ₈ -6-R ₉ -1,3,5-triazine and NH-A-SO ₂ CH = CH ₂ ,

A is selected from (CH ₂ ) _n , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ),

R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group,

R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups,

m and m 'are the same as or different from each other, and each independently an integer of 1 to 23,

1, n, p and q are the same as or different from each other, and each independently an integer of 1 to 10.

를 의미한다.In the _{R 3 2-NHNH-4-} R 8 -6-R 9 -1,3,5-triazine is

Means.

According to the present invention, [Formula 1] may be a compound represented by the following [Formula 2] or [Formula 3].

[Formula 2]

[Formula 3]

In [Formula 2] or [Formula 3],

Y, Z, R ₁ , R ₁ ', R ₂ , R ₂ ', l, m and m 'are as defined for [Formula 1],

R ₄ is hydrogen or succinimide,

R ₅ is an amine, (CH ₂ ) _p NH ₂ , (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NH-4-R ₈ -6-R ₉ -1,3,5-triazine And A-SO ₂ CH = CH ₂ ,

A is selected from (CH ₂ ) _r , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ),

R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups,

n, p and q are the same as or different from each other, and each independently an integer of 1 to 10.

[Formula 1] according to the present invention may be selected from the group consisting of [Formula 4] to [Formula 7] more specifically.

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

In [Formula 4] to [Formula 7]

Y, R ₁ , R ₁ ', R ₂ , R ₂ ', l, m and m 'are as defined for the [Formula 1],

R ₄ and R ₅ are as defined for the above [Formula 2] or [Formula 3].

According to the present invention, concretely exemplifying the compound represented by [Formula 1] may be any one or more selected from the group consisting of the following [Formula 8] to [Formula 35], but is not limited thereto.

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

[Formula 18]

[Formula 19]

[Formula 20]

[Formula 21]

[Formula 22]

[Formula 23]

[Formula 24]

[Formula 25]

[Formula 26]

[Formula 27]

[Formula 28]

[Formula 29]

[Formula 30]

[Formula 31]

[Formula 32]

[Formula 33]

[Formula 34]

[Formula 35]

The cyanine compound represented by [Formula 1] according to the present invention may exhibit fluorescence at a wavelength of 500 to 800 nm, which is a near infrared fluorescent region.

The cyanine compound represented by [Formula 1] according to the present invention may be labeled on a biomolecule, a nanoparticle or an organic compound including an amine group, a hydroxyl group or a thiol group, and the biomolecule is a protein, a peptide, a carbohydrate, a sugar, At least one selected from the group consisting of fats, antibodies, proteoglycans, glycoproteins, and siRNAs.

For example, the cyanine compound represented by the above [Formula 1] according to the present invention can be easily dyed to the protein by the reaction of the protein with the compound of Formula 1 as in the case of the conventional cyanine dye.

Method for labeling the cyanine compound represented by the above [Formula 1] is a solvent selected from the group consisting of phosphate buffer, carbonate buffer and tris buffer as a solvent, dimethyl sulfoxide, dimethylformamide, methanol, ethanol and acetonitrile By using an organic solvent selected from the group, or water, the reaction of the compound of [Formula 1] and the biological molecules, nanoparticles or organic compounds at pH 5 to 12. The reaction is sufficient for 30 minutes to 48 hours at a temperature of 20 to 80 ℃.

In the case of biomolecules, most of them are dissolved in a predetermined buffer from the packaging unit, and in order to secure the stability of the biomolecules, a separate buffer or pH is often required, and thus it is not easy to adjust the parameters. The compound of formula 1 according to the present invention is easily reacted with the protein in various buffers, reaction temperature, pH conditions, etc. to express fluorescence, and thus is suitable for use for biomolecular labeling.

It provides a method for producing a cyanine compound represented by the above [Formula 1] according to the present invention.

The cyanine compound represented by the above [Formula 1] according to the present invention may be prepared by reacting the compound of the following [Formula 36] with the compound of the following [Formula 37].

[Formula 36]

[Formula 37]

In [Formula 36] or [Formula 37]

X is a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine,

Y, Z, R ₁ , R ₁ ′, R ₂ , R ₂ ′, R ₃ , l and m are the same as defined for Formula 1 above.

According to the present invention, the step of preparing the compound of [Formula 1] by reacting the compound of [Formula 36] with the compound of [Formula 37] may proceed in one step, but is not limited thereto. It may proceed to the above.

For example, in Formula 1, R ₃ may be a hydroxy group, but N-hydroxysuccinimide group, hydrazinyl group, NH (CH ₂ ) _p NH ₂ , NH- (CH ₂ ) _q -N ( CO) ₂ C ₂ H ₂ , 2-NHNH-4-R ₈ -6-R ₉ -1,3,5-triazine or NH-A-SO ₂ CH = CH ₂ ,

R ₃ is a hydrazinyl group, NH (CH ₂ ) _p NH ₂ , NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NHNH-4-R ₈ -6-R ₉ -1 If any one selected from, 3,5-triazine and NH-A-SO ₂ CH = CH ₂ ,

The compound of [Formula 36] is reacted with a compound of [Formula 37], wherein R ₃ is a hydroxy group to prepare the following [Formula 2a], followed by hydrazine, NH ₂ (CH ₂ ) _p NH ₂ , and NH ₂ (CH ₂ ). _q- N (CO) ₂ C ₂ H ₂ , 2-NH ₂ NH-4-R ₈ -6-R ₉ -1,3,5-triazine or NH ₂ -A-SO ₂ CH = CH ₂ The compound of Formula 3 may also be prepared.

[Formula 2a]

In Formula [2a], Y, Z, R ₁ , R ₁ ′, R ₂ , R ₂ ′, l, and m are the same as defined for Formula 1 above.

In addition, the step of preparing the compound of [Formula 3] from the compound of [Formula 2a] may proceed to 1-step, but is not limited to this may be proceeded to 2-step or more.

For example, in order to improve the reactivity, the compound of [Formula 2a] is reacted with N, N′-disuccinimidyl carbonate (N, N′-Disuccinimidyl carbonate) to prepare a compound represented by the following [Formula 2b] This was followed by hydrazine, NH ₂ (CH ₂ ) _p NH ₂ , NH ₂ (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NH ₂ NH-4-R ₈ -6-R _9- By reacting with an amine compound selected from 1,3,5-triazine and NH ₂ -A-SO ₂ CH = CH ₂ , the compound of Chemical Formula 3 may be prepared, wherein the amine compound is protected with a protecting group. It may be an amine compound, the protecting group may be, but is not limited to, di-tert-butyl dicarbonate.

[Formula 2b]

In [Formula 2a]

Y, Z, R ₁ , R ₁ ′, R ₂ , R ₂ ′, l, and m are as defined for Formula 1 above.

In addition, in the amine compound, the cyanine compound of [Chemical Formula 3] having 4,6-dichloro-1,3,5-triazine as a functional group is a compound of [Chemical Formula 2a] or [Chemical Formula 2b] and 4,6 It may also be prepared by reacting dichloro-2-hydrazinyl-1,3,5-triazine, but the compound of [Formula 2a] or [Formula 2b] is reacted with tert-butyl carbazate to introduce a hydrazine group It may then be further prepared by reacting with cyanuric chloride.

The solvent used for the reaction is one selected from the group consisting of 1,2-dichloroethane, dichloromethane, chloroform, acetonitrile, dioxane, tetrahydrofuran, N, N-dimethylformamide, methanol, and ethanol. Can be more than

N, N-Diisopropylethylamine (DIPEA), triethylamine (TEA), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (1-ethyl- 3- (3-dimethyl-aminopropyl) -carbodiimide; EDC), 4- (dimethylamino) -pyridine (4- (Dimethylamino) -pyridine; DMAP), 1-ethyl O- (benzotriazol-1-yl)- N, N, N ', N'-tetramethyluronium hexafluorophosphate (O- (Benzotriazol-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate; HBTU), hydroxybenzotria It may be carried out by further comprising one or more selected from the group consisting of sol (Hydroxybenzotriazole (HOBT) and sodium bicarbonate.

According to the present invention, the compound of [Formula 36] may be prepared by reacting at least one compound selected from the compound of Formula 38 and the compound of Formula 38a with the compound of Formula 39.

[Formula 38]

[Formula 38a]

[Formula 39]

In [Formula 38] or [Formula 38a],

Y, R ₁ , R ₁ ', R ₂ , R ₂ ' and m are the same as defined in [Formula 1],

In Chemical Formula 39, X is any one selected from the group of halogens, and in general, the salt may be determined according to phosphorus oxychloride or phosphorus bromide, which is commonly used in synthesis.

A brief description of the preparation method of the cyanine compound represented by [Formula 1] according to the present invention is given by the following [Scheme 1].

Scheme 1

In [Scheme 1], Y, Z, R ₁ , R ₂ , R ₃ , l, and m are the same as defined in [Formula 1],

X is a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine,

[Scheme 1] shows a method for producing using the compound of [Chemical Formula 38], but is not limited thereto, the compound of [Chemical Formula 38a] may be used, and [Chemical Formula 38] and [Chemical Formula 38a] may be used together. It may be.

On the other hand, the compound of [Formula 38] or [Formula 38a] may be prepared by the same method as [Scheme 2].

Scheme 2

In [Scheme 2], Y, R ₁ , R ₂ and m are as defined in [Formula 1], wherein X is a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

The present invention provides a contrast agent composition comprising a cyanine compound represented by the above [Formula 1].

The cyanine compound represented by the above [Formula 1] according to the present invention maintains excellent performance of the cyanine chromophore, while the composition comprising the same as an active ingredient has a high fluorescence intensity in the near-infrared wavelength region of 500 to 800 nm. Useful as imaging contrast agent.

The present invention provides a compound labeling method comprising the step of combining the cyanine compound represented by the above [Formula 1] and the compound to be labeled.

According to the present invention, the labeling compound may be at least one selected from biomolecules, nanoparticles or organic compounds, and the labeling compound may include at least one functional group selected from an amine group, a hydroxyl group, and a thiol group.

The label is a combination of the carboxyl group, succinimidyl ester group, vinylsulfone group, amine group or halogen group present in the compound of [Formula 1] and the amine group, hydroxyl group or thiol group present in the biomolecule, nanoparticle or organic compound. It is done by.

The biomolecule may be any one or more selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins, and siRNAs.

The compound of [Formula 1] according to the present invention can be easily dyed to the protein by the reaction of the protein with the compound of [Formula 1].

Thus, the label uses a solvent selected from the group consisting of phosphate buffer, carbonate buffer and tris buffer, an organic solvent selected from the group consisting of dimethyl sulfoxide, dimethylformamide, methanol, ethanol and acetonitrile, or water as a solvent. And reacting the compound of [Formula 1] with the biomolecule, nanoparticles, or organic compound at pH 5-12. The reaction is sufficient for 30 minutes to 48 hours at a temperature of 20 to 80 ℃.

On the other hand, in the case of biomolecules are most often dissolved in a predetermined buffer solution from the packaging unit, in order to ensure the stability of the biomolecules in many cases it requires a separate buffer or pH is not easy to adjust to the variable. The compound of [Formula 1] according to the present invention has a high stability in water-soluble conditions, not only easy to store for a long time, but also easily reacts with the protein in various buffers, reaction temperature, pH conditions, etc. to express fluorescence, for biomolecular labeling Suitable for use as

Hereinafter, the present invention will be described in more detail through examples and comparative experiments. However, the examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example

Experimental apparatus, analytical equipment, and reagents used in Examples and Comparative Experiments will be described.

Bruker Avance 500 was used as an instrument for FT-NMR spectroscopy, and LC / MS was measured by electrospray ionization (ESI) using LC / MSD SL of Agilent Technology.

Absorption values at the absorption wavelength and the maximum wavelength of the synthesized dye were measured by Lambda 45 from Perkin Elmer, and emission values at the emission wavelength and the maximum emission wavelength were obtained using LS 55 from Perkin Elmer.

Column chromatography for separation and purification of organic compounds used Merck kieselgel 60 (230-400 mesh) as a silica gel in the normal phase, silica gel 60 GF254 for thin layer chromatography (TLC) (0.25 mm, Merck) was applied, and the compound identification on TLC used ultraviolet rays of 254 nm and 365 nm. In the reverse phase, TLC used a glass plate coated with silica gel 60 RP-18 F254S (0.25 mm, Merck), and column chromatography used Fraction Collector, a medium pressure liquid chromatography (MPLC) device from Buchi. Reverse phase column Lichroprep RP-18 (40-63 μm, available from Merck) was used in conjunction with R-660.

An electrophoresis device for gel electrophoresis experiments is to use BIO-RAD's PowerPac Basic Power Supply (Catalog No. 164-5050) to Amershame Biosciences' SE260, a mini-vertical gel electrophoresis unit. It was. Lonza's PAGEr Gold Precast Gels (Polyacrylamide gels for protein electrophoresis, 10-20% Tris-Glycine gels, Catalog No. 59506) were used.

Reagents were mainly used by Aldrich and TCI. Solvents requiring purification were used in accordance with known methods and all reactions were carried out under a nitrogen stream unless otherwise indicated. NMR solvents were DMSO- d ₆ or D ₂ O from Aldrich and Cambridge Isotope Laboratories Inc. The relative positions of the signals were based on tetramethylsilane (TMS) in the solvent or on NMR solvents. Chemical shifts are expressed in ppm from the standard, and data are available for chemical shift multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), intergration, and coupling constants (Hz). ) In order.

Preparation Example 1 Compound of Preparation Example 1

Stage 1

10 g (53 mmol, 1 eq) of p-hydrazinobenzenesulfonic acid and 17.18 mL (160 mmol, 3.02 eq) of 3-methyl-2-butanone After addition to 30 mL of acetic acid, the reaction was heated under reflux for 4 hours. After cooling to room temperature, the resulting solid particles were filtered. After washing 2-3 times with ethyl acetate, the mixture was dried under reduced pressure. (11.34 g, 89%)

R _f = 0.68 (RP-C18, acetonitrile / water 1: 4 v / v)

1.427 g (25.4 mmol, 1.2 eq) of potassium hydroxide was dissolved in 35 mL of propanol, 5.073 g (21.2 mmol, 1 eq) of the solid material obtained above was dissolved in 35 mL of methanol, added dropwise, and stirred at room temperature for 24 hours, Filtration gave yellow solid particles. (5.35 g, 90%)

R _f = 0.68 (RP-C18, acetonitrile: distilled water = 1: 4 v / v)

2 steps

6.0 g (21.6 mmol, 1 eq) of the compound prepared in step 1, 6.4 mL (68.9 mmol, 3.1 eq) of 1,4-butanesultone, and 2.1 g (25.9 mmol) of sodium acetate , 1.2 eq) was added to 10 mL of acetonitrile and heated to reflux for 12 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, the solvent was removed, ethyl acetate was added, the filtrate was dried under reduced pressure, and a pink solid was obtained. (6.3 g, 100%)

Rf = 0.33 (silicagel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

3 steps

60.8 mL (2 mol, 5 eq) of N, N-dimethylformamide (DMF) and 40 mL of dichloromethane were mixed and the temperature was cooled to 0 ° C. 45.2 mL (1.5 mol, 3.7 eq) of phosphorus oxychloride was dissolved in 40 mL of dichloromethane, and then added dropwise to the cooled solution for 10 minutes. To the mixture was added dropwise a solution of 11.7 mL (0.4 mol, 1 eq) of cyclohexanone in 60 mL of dichloromethane for 10 minutes. After refluxing for 3 hours and cooling to room temperature, 31.0 mL (1.0 mol, 2.7 eq) of aniline was added thereto, and the mixture was further stirred at room temperature for 1 hour. After completion of the reaction, 50 mL of distilled water was added to the mixed solution and stored in a cold place. (38 g, 26%)

Rf = 0.99 (silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

4 steps

5 g (13.3 mmol, 2 eq) of 2 steps of compound, 2.4 g (6.6 mmol, 1 eq) of 3 steps of compound and 1.4 g (17.1 mmol, 2.5 eq) of sodium acetate were mixed in 6 mL of ethanol and then Stir for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, crystals were formed using 80 mL of ethyl acetate, filtered, and dried under reduced pressure. (3.9 g, 66%)

Rf = 0.5 (silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

5 steps

After dissolving 200 mg (0.22 mmol, 1 eq) of 4 steps of compound in 1 mL of DMF, 62.1 mg (0.45 mmol, 2 eq) and 3- (4-hydroxyphenyl) propionic acid (6) of potassium carbonate 148.7 mg (0.9 mmol, 4eq) was added to 3- (3-hydroxyphenyl) propionic acid) and stirred at 40 ° C for 12 hours. After completion of the reaction, 40 mL of ethyl acetate was added to obtain crystals, filtered and dried under reduced pressure. A 10% acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography with a developing solution to obtain the desired compound of Preparation Example 1. (16.8 mg, 7%)

Rf = 0.67 (RP-C18, acetonitrile: distilled water = 3: 7 v / v)

¹ H NMR (500 MHz, DMSO-d ₆ ): δ 8.28 (m, 1H), 8.21 (m, 1H), 7.76-7.71 (m, 3H), 7.64-7.58 (m, 4H), 7.41 (m, 1H), 7.37 (d, 1H, J = 8 Hz), 7.19 (d, 1H, J = 8 Hz), 6.32 (d, 1H, J = 13.5 Hz), 6.17 (d, 1H, J = 14 Hz) , 4.17 (m, 4H), 2.73-2.71 (m, 2H), 2.71-2.67 (m, 2H), 2.37 (m, 4H), 1.90-1.61 (m, 10H), 1.50 (m, 2H), 1.33 (s, 12H)

LC / MS: 1017 (Note, Theoretical C ₄₇ H ₅₇ N ₂ O ₁₅ S ₄ ⁺ 1017.26)

λ _abs (MeOH): 787 nm (ε = 1.46 x 10 ⁵ M ^-1 cm ^-1 ), λ _fl (MeOH): 813 nm

Example 1 Compound of Formula 8

40 mg (0.0393 mmol, 1 eq) of Preparation Example 1 was completely dissolved in 0.5 ml of DMF, followed by 20.5 uL (N, N-Diisopropylethylamine) of N, N-Diisopropylethylamine (0.118 mmol, 3 eq) And 30 mg (0.118 mmol, 3 eq) of N, N-disuccinimidyl carbonate were added and stirred at room temperature for 1 hour. After confirming the reaction, ether was added to form crystals, filtered and dried to form a precursor.

The resulting precursor was completely dissolved in 0.5 ml of DMF and then 20.5 uL (0.118 mmol, 3 eq) of N, N-diaisopropyledylamine was added thereto. 8 mg (0.0393 mmol, 1 eq) of 2- (2'-chloroethylsulfonyl) -ethylamine hydrochloride) was dissolved in DMF dropwise and stirred for 24 hours at room temperature. At the end of the reaction, ethyl acetate was added to form crystals, which were then filtered and concentrated and separated using RP-C18 reverse phase chromatography. The separated material was dried under a reduced pressure to obtain a compound of [Formula 8]. (15 mg, 33.5%)

Rf = 0.4 (RP-C18, acetonitrile: distilled water = 1: 2 v / v)

Example 2 Compounds of Formula 9

200 mg (0.197 mmol, 1 eq) of the compound of Preparation Example 1 were dissolved in 5 ml of DMF, and 137 μL (0.788 mmol, 4 eq) of N, N-diaisopropyledylamine and O- (benzotriazole-1 -Yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (O- (Benzotriazol-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate) 149 mg (0.394) mmol, 2 eq) was added and stirred at room temperature for 1 hour. To the mixed solution was added 26 mg (0.197 mmol, 1 eq) of tert-Butyl carbazate and further stirred at room temperature for 1 hour. After the reaction was completed, ethyl acetate was added to form crystals, followed by filtration and drying under reduced pressure, followed by purification by silica gel normal chromatography.

The obtained compound was added to 6 mL of chloroform and 3 mL of distilled water, and completely dissolved. 12 mL of trifluoroacetic acid was added thereto, followed by stirring at room temperature for 1 hour. The reacted compound was dried under reduced pressure and purified by silica gel normal chromatography to obtain the compound of [Formula 9]. (58 mg, 28.5%)

 Rf = 0.6 (RP-C18, acetonitrile: distilled water = 3: 7 v / v)

Example 3 Compounds of Formula 10

After dissolving 200 mg (0.197 mmol, 1 eq) of the compound of Preparation Example 1 in 5 ml of DMF, 137 μL (0.788 mmol, 4 eq) of N, N-diaisopropyledylamine and O- (benzotriazole- 1-yl) -N, N, N ', N'-tetramethyluroni hexafluorophosphate 149 mg (0.394 mmol, 2 eq) was added thereto and stirred at room temperature for 1 hour. 118 mg (1.97 mmol, 10 eq) of ethylene diamine was added to the mixed solution and reacted at room temperature for 4 hours. After completion of the reaction, ethyl acetate was added to form a crystal, filtered, dried under reduced pressure, and purified by silica gel normal chromatography to obtain a compound of [Formula 10]. (74.7 mg, 35.8%)

 Rf = 0.55 (RP-C18, acetonitrile: distilled water = 3: 7 v / v)

Example 4: Compound of Formula 11

After dissolving 200 mg (0.197 mmol, 1 eq) of the compound of Preparation Example 1 in 5 ml of DMF, 137 μL (0.788 mmol, 4 eq) of N, N-diaisopropyledylamine and O- (benzotriazole- 1-yl) -N, N, N ', N'-tetramethyluroni hexafluorophosphate 149 mg (0.394 mmol, 2 eq) was added thereto and stirred at room temperature for 1 hour. 75 mg (0.296 mmol, 1.5 eq) of N- (2-aminoethyl) maleimide was added to the mixed solution and reacted at room temperature for 4 hours. After the reaction was completed, ethyl acetate was added to form crystals, followed by filtration and pressure drying, and purification by silica gel normal chromatography to obtain a compound of [Formula 11]. (85.7 mg, 38.2%)

 Rf = 0.50 (RP-C18, acetonitrile: distilled water = 3: 7 v / v)

Example 5 Compounds of Formula 12

17 mg (0.093 mmol, 1 eq) of cyanuric chloride was dissolved in 10 ml of distilled water, cooled to 0 ° C., and stirred for 30 minutes. 110 mg (0.093 mmol, 1 eq) of the compound of [Formula 9] prepared in Example 2 were added while maintaining the temperature, followed by stirring for 10 minutes. 15 mg of sodium bicarbonate was added to the reaction solution, followed by reaction for 2 hours while maintaining the temperature. 15% aqueous acetonitrile solution was purified by RP-C18 reverse phase chromatography with a developing solution to obtain a compound of [Formula 12]. (47 mg, 42.9%)

Preparation Example 2 Compound of Preparation Example 2

Stage 1

2,3,3-trimethylindolenine 1.0 g (6.2 mmol, 1 eq), 1,4-butanesultone 1.8 mL (20.0 mmol, 3.1 eq) And 0.6 g (7.5 mmol, 1.2 eq) of sodium acetate were added to 5 mL of acetonitrile and then heated to reflux for 12 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, ethyl acetate was added to form crystals, filtered and dried under reduced pressure to obtain a pink solid. (2.3 g, 100%)

Rf = 0.26 (silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

2 steps

0.5 g (1.6 mmol, 2 eq) of the first step compound of Preparation Example 2, 0.3 g (0.8 mmol, 1 eq) of the compound prepared in step 3 of Preparation Example 1, and 0.17 g (2.1 mmol, 2.5 eq) of sodium acetate were prepared. It was dissolved in 20 mL of ethanol and reacted at 50 ° C. for 1 hour. After completion of the reaction, the mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, crystals were formed with 80 mL of ethyl acetate, filtered, and dried under reduced pressure. (1.1 g, 92%)

Rf = 0.5 (silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

3 steps

200 mg (0.27 mmol, 1eq) of the second step compound of Preparation Example 2 was dissolved in 1 mL of DMF, followed by 75.7 mg (0.54 mmol, 2 eq) of potassium carbonate and 3-1.8- (4-hydroxyphenyl) propionic acid. mg (0.54 mmol, 2 eq) was added thereto and stirred at 40 ° C. for 12 hours. After completion of the reaction, 40 mL of ethyl acetate was added to form crystals, filtered and dried under reduced pressure. Purification by silica gel normal chromatography using a chloroform: methyl alcohol = 4: 3 solvent gave the desired compound. (53.1 mg, 23%)

Rf = 0.66 (silica gel, chloroform: methyl alcohol = 4: 3 v / v)

¹ H NMR (500 MHz, DMSO-d ₆ ): δ 8.12 (m, 1H), 8.01 (m, 2H), 7.94 (m, 2H), 7.77 (m, 2H), 7.72 (m, 1H), 7.58 (m, 1H), 7.45 (m, 1H), 7.37 (m, 1H), 7.29 (m, 1H), 7.22 (d, 1H, J = 8.5 Hz), 7.16-7.12 (m, 2H), 6.39 ( d, 1H, J = 14.5 Hz), 4.41 (t, 2H, J = 7.0 Hz), 3.98 (dd, 2H, J = 26.7 Hz, 2 Hz), 3.72 (t, 2H, J = 8.8 Hz), 2.77 -2.73 (m, 4H), 2.59 (t, 2H, J = 6.5 Hz), 2.33 (t, 2H, J = 7.5 Hz), 2.05-2.02 (m, 2H), 1.96-1.90 (m, 2H), 1.90-1.87 (m, 2H), 1.87-1.84 (m, 4H), 1.58 (s, 12H)

LC / MS: 895 (Reference, theoretical C ₄₇ H ₅₆ KN ₂ O ₉ S ₂ ⁺ 895.31)

λ _abs (MeOH): 805 nm (ε = 3.26 x 104 M ^-1 cm ^-1 ), λ _fl (MeOH): 825 nm

Example 6: Compounds of Formula 13

After 40 mg (0.046 mmol, 1 eq) of the compound of Preparation Example 2 was completely dissolved in 0.5 ml of DMF, 23.4 uL (0.14 mmol, 3 eq) of N, N-diaisopropylethylamine and N, N-disuccinimi 35.8 mg (0.14 mmol, 3 eq) of dicarbonate was added thereto, and the resultant was stirred at room temperature for 1 hour. After completion of the reaction, ether was added to form crystals, filtered and dried. After complete drying, 23.4 uL (0.14 mmol, 3 eq) of N, N-diaisopropylethylamine was added after completely dissolving in 0.5 ml of DMF. Further, 9.6 mg (0.046 mmol, 1 eq) of 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride was added dropwise in DMF, followed by stirring at room temperature for at least 24 hours. After the reaction was completed, ethyl acetate was added to form a crystal, followed by filtration and drying under reduced pressure. Using acetonitrile 50% solution as a mobile phase was separated by reverse phase chromatography, and the separated material was dried under reduced pressure to obtain the desired compound of formula (13).

Rf = 0.4 (RP-C18, acetonitrile: distilled water = 1: 1 v / v)

LC / MS = 973.13 (reference, theoretical value C ₅₁ H ₆₃ N ₃ O ₁₀ S ₃ 973.37)

λ _abs (MeOH): 805 nm (ε = 1.24 x 10 ⁵ M ^-1 cm ^-1 ), λ _fl (MeOH): 825 nm

Preparation Example 3

Stage 1

2,3,3-trimethylindolenine 10.0 mL (62.8 mmol, 1 eq) and 15.2 mL (1-iodopropane) 15.2 mL (157.2 mmol, 2.5 eq) for 5 hours Heated to reflux. After the reaction was completed, the mixture was cooled to room temperature, ethyl acetate was added thereto to form crystals, and then filtered and dried under reduced pressure to obtain a solid. (12.7 g, 100%)

Rf = 0.5 (silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

2 steps

5.0 g (24.7 mmol, 2 eq) of the compound prepared in Step 1 of Preparation Example 3, 4.4 g (12.3 mmol, 1 eq) of the compound prepared in Step 3 of Preparation Example 1, and 2.6 g (31.7 mmol, 2.5 of Sodium Acetate) eq) was dissolved in 20 mL of ethyl alcohol and reacted at 50 ° C. for 1 hour. After the reaction was completed, the mixture was cooled to room temperature, concentrated under reduced pressure, crystals were formed with ethyl acetate, filtered, and dried under reduced pressure. (5.3 g, 79%)

Rf = 0.83 (silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

3 steps

500 mg (0.92 mmol, 1eq) of the compound prepared in Step 2 of Preparation Example 3 was dissolved in 2.5 mL of DMF, followed by 255.6 mg (1.85 mmol, 2 eq) of potassium carbonate and 3- (4-hydroxyphenyl) propionic 307.4 mg (1.85 mmol, 2 eq) of acid was added thereto and stirred at 40 ° C. for 5 hours. After completion of the reaction, 120 mL of ethyl acetate was added to form crystals, filtered and dried under reduced pressure. Purification by silica gel normal chromatography with chloroform: methyl alcohol (6: 1) gave pure compound 7-3. (62.0 mg, 10%)

Rf = 0.30 (silica gel, chloroform: methyl alcohol = 4: 1 v / v)

¹ H NMR (500 MHz, DMSO-d ₆ ): δ 7.82 (m, 2H), 7.49 (m, 2H), 7.24 (m, 2H), 7.21-7.18 (m, 2H), 7.04 (m, 2H) , 6.98 (m, 2H), 6.65 (d, 2H, J = 8.5 Hz), 6.18 (d, 2H, J = 14.5 Hz), 4.10 (d, 4H, J = 7 Hz), 2.75-2.67 (m, 8H), 2.43-2.37 (m, 4H), 1.93 (m, 4H), 1.76-1.67 (m, 4H), 1.27 (s, 12H)

LC / MS = 669.4 (Reference, theoretical value C ₄₅ H ₅₃ N ₂ O ₃ 669.41)

λ _abs (MeOH): 766 nm (ε = 1.87 x 10 ⁵ M ^-1 cm ^-1 ), λ _fl (MeOH): 798 nm

Example 7 Compounds of Formula 18

Except for using the compound of Preparation Example 3 instead of the compound of Preparation Example 1 the desired compound of [Formula 18] was prepared by the method of Example 1.

Rf = 0.5 (silica gel, chloroform: methanol = 4: 3 v / v)

LC / MS = 786.49 (Note, Theoretical C ₄₉ H ₆₀ N ₃ O ₄ S ⁺ 786.43)

λ _abs (MeOH): 766 nm (ε = 1.51 x 10 ⁵ M ^-1 cm ^-1 ), λ _fl (MeOH): 793 nm

Preparation Example 4

Using the method of Preparation Example 3, except that 4-mercaptohydrocinnamic acid was used instead of 3- (4-hydroxyphenyl) propionic acid used in Step 3 of Preparation Example 3 The desired compound was synthesized (710 mg, 18%).

Rf = 0.31 (silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

¹ H NMR (500 MHz, DMSO-d ₆ ): δ 8.50 (m, 1H), 8.18 (m, 1H), 8.01 (m, 1H), 7.85 (m, 1H), 7.66 (m, 2H), 7.33 (m, 1H), 7.19 (d, 1H, J = 8.1 Hz), 6.99 (dd, 1H, J = 9.95 Hz, 5.50 Hz), 6.26 (dd, 2H, J = 4 Hz, 2.1 Hz), 6.17 ( d, 1H, J = 13.15 Hz), 6.23 (m, 1H), 3.64-3.58 (m, 4H), 3.16-3.11 (m, 2H), 2.05 (t, 2H, J = 7.3 Hz), 1.27-1.19 (m, 18 H)

LC / MS = 685.56 (reference, theoretical value C ₄₅ H ₅₃ N ₂ O ₂ S 685.98)

Example 8 Compounds of Formula 23

Except for using the compound of Preparation Example 4 instead of the compound of Preparation Example 1 to the desired compound of the formula [23] by the method of Example 1.

Example 9 Compounds of Formula 24

Except for using the compound of Preparation Example 4 instead of the compound of Preparation Example 1 to the desired compound of the formula [Formula 24] by the method of Example 2.

Example 10 Compounds of Formula 25

Except for using the compound of Preparation Example 4 instead of the compound of Preparation Example 1 to prepare a compound of the formula [25] by the method of Example 3.

Example 11 Compound of Formula 26

Except for using the compound of Preparation Example 4 instead of the compound of Preparation Example 1 the desired compound of the formula [26] was prepared by the method of Example 4.

Example 12 Compounds of Formula 27

Except for using the compound of Preparation Example 4 instead of the compound of Preparation Example 1 the desired compound of the formula [26] was prepared by the method of Example 5.

Test Example 1: Protein Labeling Experiment

Test Example 1.1 Protein Labeling Experiment of Compound of Example 6

NIR-797 isothiocyanate (excitation wavelength 795 nm, fluorescence wavelength 817 nm, Aldrich), a commercial cyanine dye, and FNI-774 (excitation wavelength 777 nm) for comparison with cyanine dyes exhibiting near infrared fluorescence. , Fluorescence wavelength 802 nm, BioActs) and 1 mg of Example 6 (excitation wavelength 805 nm, fluorescence wavelength 825 nm) were prepared by dissolving each in 100 μL of DMF.

30 μg of albumin (Albumin from bovine serum, lyophilized powder, Aldrich) protein was dissolved in 30 μL of 0.1 M bicarbonate / carbonate buffer (pH 9.0), and then aliquoted into three, and the three fluorescent dye solutions were added to each protein solution. μL was added and mixed vigorously and reacted at room temperature for 4 hours.

The entire amount of the reaction solution was loaded on the gel, and SDS-PAGE gel electrophoresis was performed at 125 V for 2 hours, and the results are shown in FIG. 1.

In Figure 1, the leftmost marker is a protein marker for checking the size of the protein, 1 is NIR-797 isothiocyanate, 2 is FNI-774, 3 is the formula of [Formula 13] according to Example 6 of the present invention Compound.

As can be seen in Figure 1, the compound (3) of Example 6 according to the present invention was mostly labeled with a protein. On the other hand, Comparative Examples NIR-797 isothiocyanate (1) and FNI-774 (3) can be confirmed that the amount of dye remaining unlabeled in the protein.

Test Example 1.2 Protein Labeling Experiment of Compound of Example 8

The protein labeling experiment was carried out in the same manner as in Test Example 1.1, except that the compound of Example 8 was used instead of the compound of Example 6.

Comparative Examples NIR-797 isothiocyanate (1) and FNI-774 (3) were not labeled on the protein, but a significant amount of dye remained, but the compound of Example 8 (compound 23) according to the present invention was mostly present in the protein. Labeled and compared to the compound of Example 6 of Test Example 1.1, it was found that the amount of dye remaining unlabeled in the protein was less.

Test Example 2 Measurement of Near-Infrared Fluorescence Intensity

Test Example 2.1 Measurement of Near-Infrared Fluorescence Intensity of Preparation Example 3

Fluorescence intensity evaluation was performed through direct administration in vivo. Considering that the compound of Example 6 is a compound capable of directly reacting with a biomolecule, a comparative evaluation with indocyanine green was performed using Preparation Example 3, which is a preliminary compound, for indirect fluorescence intensity comparison.

As a comparative example, Diagnogreen (Daiichi), a clinical indocyanine green product, was used. Preparation Example 3 and Comparative Example were prepared by repeating the method of preparing 0.4 mL of 0.5 mg / mL each with PBS as a solvent, diluting with PBS at half the concentration, and preparing 0.2 mL and 9 samples, respectively, as a control. A total of 10 samples were prepared by adding 0.2 mL of PBS. Ten tubes were placed side by side in an fluorescence imaging device for animals (IVIS) and photographed using an ICG wavelength detection filter, which is shown in FIG. 2.

In FIG. 2, the magnitude of fluorescence intensity represents fluorescence with high red color and low blue color. As can be seen in the photograph of FIG. 2, Preparation Example 3 showed a higher fluorescence intensity than the Comparative Example, and was observed to have low self-quenching phenomenon even at high concentrations.

Test Example 2.2 Near-infrared fluorescence intensity measurement of Preparation Example 4

For the same reasons as in Test Example 2.1, except for using Preparation Example 4 instead of Preparation Example 3, the near infrared fluorescence intensity was measured by the method of Test Example 2.1.

Compound according to Preparation Example 4 exhibited a higher fluorescence intensity than Comparative Example, was confirmed to be higher than Preparation Example 3, as in Preparation Example 3 was observed that almost no self-quenching phenomenon at high concentrations for biomolecular labeling It was expected to be available as a phosphor.

Experimental Example 3 Experiment of Confirmation of Distribution Trend in Animals after Animal Administration

Test Example 3.1 Animal Experiment of Preparation Example 3

(1) experimental animal preparation

Three 5-week-old Balb / c nude mice (female, Jungang Lab Animal, Inc) were purchased and had a one-week acclimatization period. Compliant.

(2) Injecting Fluorescent Dye Solution into Experimental Animal

For the same reason as in Experiment 2, animal experiments were performed using Preparation Example 3, a preliminary compound of Example 6. 0.5 mL was prepared at a concentration of 0.00781 mg / mL and 0.0313 mg / mL, respectively, for preparation of the injection solution of Comparative Example and Preparation Example 3, and 0.5 mL of sterile 1XPBS was prepared for the control.

Three mice were respiratory anesthesia using 2% isoflurane and oxygen. Three mice were photographed using an IVIS equipped with a respiratory anesthesia device in order to take an image before injection injection under anesthesia (exposure time 1 second, using an ICG filter). 0.2 mL of the prepared PBS, Comparative Example, Preparation Example 3 injections were inhaled with a sterile syringe (BD Ultra-FineTM IIm Short Needle, 1 mL, 0.25 mm (31 G) X 8 mm) and tail vein injection.

Fluorescence images were taken after 10 minutes, 15 minutes, 20 minutes, 40 minutes and 24 hours after injection, and repeated anesthesia was performed 30 seconds before the point of time without exposure to the anesthetic until the next time point after each shot. It is shown in Figure 3.

As shown in FIG. 3, Preparation Example 3 showed similar distribution and migration in the body as in Comparative Example (Clinical ICG), and after 24 hours, no fluorescence was detected in the body, and thus all were released. In particular, it exhibited about 3.5 times stronger fluorescence intensity than the comparative example, and showed a tendency consistent with the result of the fluorescence intensity measured in Test Example 2.

Experimental Example 3.2 Animal Experiment of Preparation Example 4

Except for using the compound of Preparation Example 4 instead of the compound of Preparation Example 3 the animal experiment was carried out by the method of Test Example 3.1. Fluorescence images were taken after 10 minutes, 15 minutes, 20 minutes, 40 minutes and 24 hours after injection, and repeated anesthesia was performed 30 seconds before the point of time without exposure to the anesthetic until the next time point after each shot. The distribution distribution in the body was confirmed.

After 10 minutes of drug administration, strong fluorescence was observed in the liver, and gradually shifted to the bile after 20 minutes of drug administration. After 1 hour, the amount of the compound of Comparative Example was small, but the compound of Preparation Example 4 showed fluorescence in liver and bile, and was not detected in the body after 24 hours. Based on the above results, it is determined that the compound according to the present invention can be usefully used for long time imaging.

Considering the results in Test Examples 1 to 3 above, in the case of using the cyanine dye of formula 1 according to the present invention labeled indocyanine green or a near-infrared dye having a similar wavelength as the comparative example It is expected to show a relatively high fluorescence intensity, and thus may be usefully used.

Since the biomolecules have weak or no fluorescence in the visible and near-infrared regions, fluorescence may be labeled on the biological material in order to observe biological phenomena or obtain optical images in vivo. However, biomolecules often require a separate buffer or pH condition to ensure stability, and it is important to have high stability and fluorescence intensity in a medium in which most biomolecules exist, that is, in water solubility.

The cyanine compound according to the present invention has high fluorescence intensity in the near-infrared wavelength region, high stability in water-soluble conditions, stable even in long-term dyeing of biomolecules, and excellent binding reactivity with biological molecules. In addition, the cyanine compound according to the present invention does not accumulate in the body and is completely released, exhibiting strong fluorescence without residual toxicity, and the contrast agent composition comprising the same as an active ingredient is more effectively used to obtain optical images of contrast and diseased areas by being administered in vivo. Can be.

Claims (14)

Cyanine compound represented by the following [Formula 1]: [Formula 1]

In [Formula 1], Y is selected from oxygen, sulfur and CR ₆ R ₇ , Z is oxygen or sulfur, R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups, R ₃ is a hydroxy group, hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NHNH-4 -R ₈ -6-R ₉ -1,3,5-triazine and NH-A-SO ₂ CH = CH ₂ , A is selected from (CH ₂ ) _n , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ), R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group, R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups, m and m 'are the same as or different from each other, and each independently an integer of 1 to 23, 1, n, p and q are the same as or different from each other, and each independently an integer of 1 to 10. The cyanine compound according to claim 1, wherein [Chemical Formula 1] is [Chemical Formula 2] or [Chemical Formula 3]: [Formula 2]

[Formula 3]

In [Formula 2] or [Formula 3], Y is selected from oxygen, sulfur and CR ₆ R ₇ , Z is oxygen or sulfur, R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups, R ₄ is hydrogen or succinimide, R ₅ is an amine, (CH ₂ ) _p NH ₂ , (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NH-4-R ₈ -6-R ₉ -1,3,5-triazine And A-SO ₂ CH = CH ₂ , A is selected from (CH ₂ ) _r , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ), R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group, R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups, m and m 'are the same as or different from each other, and each independently an integer of 1 to 23, 1, n, p and q are the same as or different from each other, and each independently an integer of 1 to 10. The cyanine compound according to claim 1, wherein the compound of [Formula 1] is selected from the group consisting of [Formula 4] to [Formula 7]: [Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

In [Formula 4] to [Formula 7] Y is selected from oxygen, sulfur and CR ₆ R ₇ R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups, R ₄ is hydrogen or succinimide, R ₅ is an amine, (CH ₂ ) _p NH ₂ , (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NH-4-R ₈ -6-R ₉ -1,3,5-triazine And A-SO ₂ CH = CH ₂ , A is selected from (CH ₂ ) _r , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ), R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group, R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups, m and m 'are the same as or different from each other, and each independently an integer of 1 to 23, 1, n, p and q are the same as or different from each other, and each independently an integer of 1 to 10. According to claim 1, wherein [Chemical Formula 1] is a cyanine compound, characterized in that any one or more selected from the group consisting of [Formula 8] to [Formula 35]: [Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

[Formula 18]

[Formula 19]

[Formula 20]

[Formula 21]

[Formula 22]

[Formula 23]

[Formula 24]

[Formula 25]

[Formula 26]

[Formula 27]

[Formula 28]

[Formula 29]

[Formula 30]

[Formula 31]

[Formula 32]

[Formula 33]

[Formula 34]

[Formula 35]

The cyanine compound according to claim 1, wherein the cyanine compound exhibits fluorescence at a wavelength of 500 to 800 nm. The cyanine compound according to claim 1, wherein the cyanine compound is labeled with a biomolecule, a nanoparticle or an organic compound including an amine group, a hydroxyl group or a thiol group. The cyanine compound according to claim 6, wherein the biomolecule is at least one selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins, and siRNAs. A process for preparing a cyanine compound, characterized in that prepared by reacting the compound of formula 36 with the compound of formula 37: [Formula 36]

[Formula 37]

In [Formula 36] or [Formula 37], X is halogen, Y is selected from oxygen, sulfur and CR ₆ R ₇ , Z is oxygen or sulfur, R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups, R ₃ is a hydroxy group, hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NHNH-4 -R ₈ -6-R ₉ -1,3,5-triazine and NH-A-SO ₂ CH = CH ₂ , A is selected from (CH ₂ ) _n , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ), R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group, R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups, m and m 'are the same as or different from each other, and each independently an integer of 1 to 23, 1, n, p and q are the same as or different from each other, and each independently an integer of 1 to 10. The method of claim 8, wherein the compound of Formula 36 is prepared by reacting at least one compound selected from the compound of Formula 38 and the compound of Formula 38a with the compound of Formula 39: Method for preparing the cyanine compound: [Formula 38]

[Formula 38a]

[Formula 39]

In [Formula 38], [Formula 38a] or [Formula 39], X is halogen, Y is selected from oxygen, sulfur and CR ₆ R ₇ , R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups, R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group, m and m 'are the same as or different from each other, and each independently an integer of 1 to 23. Cyanine represented by [Formula 3] comprising the step of reacting a compound of formula [2] with a compound in which an amine group of NH ₂ R ₅ or NH ₂ R ₅ is protected with a protecting group to obtain a compound of [Formula 3] Preparation of Compounds: [Formula 2]

[Formula 3]

In [Formula 2] or [Formula 3], Y is selected from oxygen, sulfur and CR ₆ R ₇ , Z is oxygen or sulfur, R ₁ , R ₁ ′, R _2, and R ₂ ′ are the same as or different from each other, and are each independently hydrogen, an alkyl group of 1 to 6 carbon atoms, an alkyl group of 7 to 10 carbon atoms, an alkoxy group of 1 to 6 carbon atoms, a sulfonic acid group, and Selected from sulfonate groups, R ₄ is hydrogen or succinimide, R ₅ is an amine, (CH ₂ ) _p NH ₂ , (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 2-NH-4-R ₈ -6-R ₉ -1,3,5-triazine And A-SO ₂ CH = CH ₂ , A is selected from (CH ₂ ) _r , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ ), R ₆ and R ₇ are the same as or different from each other, and are each independently selected from a C 1-6 alkyl group and a C 7-10 alkyl group, R ₈ and R ₉ are the same as or different from each other, and are each independently selected from halogen, NH ₂ and hydrazine groups, l is an integer from 1 to 9, m and m 'are the same as or different from each other, and each independently an integer of 1 to 23, n, p and q are the same as or different from each other, and each independently an integer of 1 to 10, The protecting group is Di-tert-butyl dicarbonate. A contrast agent composition comprising the cyanine compound of claim 1 as an active ingredient. 12. The contrast agent composition according to claim 11, wherein the contrast agent exhibits fluorescence at a wavelength of 500 to 800 nm. A compound labeling method comprising the step of combining the cyanine compound of claim 1 with a compound to be labeled, The labeling target compound is at least one selected from biomolecules, nanoparticles, and organic compounds, The compound to be labeled includes at least one functional group selected from an amine group, a hydroxyl group and a thiol group, Compound labeling method characterized in that the cyanine compound of claim 1 is bonded to the functional group. The method of claim 13, wherein the biomolecule is selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins, and siRNAs.

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