KR20190103631A - Fluorescence compounds, complex nanoparticles comprising the same, and preparation method thereof - Google Patents

Abstract

The present invention relates to a fluorescent compound, a composite nanoparticle comprising the same, and a production method thereof. The fluorescent compound according to the present invention is not accumulated when administrating the fluorescent compound into the body, has excellent fluorescence intensity, is easy to dye and image in the body even in a small amount of use compared to conventional dyes, and thus is economically available. The fluorescent compound-bile acid-chitosan nanoparticles according to the present invention show excellent cancer targeting ability.

Description

Fluorescent compounds, complex nanoparticles comprising the same, and a method for preparing the same {Fluorescence compounds, complex nanoparticles comprising the same, and preparation method etc}

The present invention relates to a fluorescent compound, a composite nanoparticle comprising the same, and a preparation method thereof.

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. Although it varies slightly depending on the solvent and solubility characteristics, it has many advantages such as high molar extinction coefficient and is widely used for biological applications.

In addition, the dye compound having a cyanine chromophore may be usefully used in the use of an optical filter for an image display device or a resin composition for laser welding. Compounds having a high intensity absorption to a specific light include optical filters for image display devices such as liquid crystal displays, plasma display panels, electroluminescent displays, cathode ray tube displays, fluorescent display tubes, and optical elements of optical recording media such as DVD.R. It is widely used as. Optical filters require a function of selectively absorbing light of unnecessary wavelengths, and at the same time, wavelength light absorption of 480 to 500 nm and 540 to 560 nm is required to prevent reflection or glare of external light such as fluorescent lamps. In order to increase, the function which selectively absorbs the wavelength of a near infrared ray is calculated | required.

As described above, in order to apply industrially usefully, there is a continuous demand for the development of a novel dye having excellent optical and pH stability, but having a narrow absorption / luminescence wavelength range in a specific wavelength range and showing a high molar absorption coefficient.

1. Korean Patent Publication No. 10-2010-0094034 2. Korean Patent Publication No. 10-2013-0005381 3. Korean Patent Publication No. 10-2011-0136367 4. Korean Patent Publication No. 10-2011-0122314

The present invention is to provide a fluorescent compound, a labeling method using the same, etc. which can be used as a contrast agent composition in terms of fluorescence intensity, relative quantum efficiency, labeling rate.

In addition, the present invention is to provide a fluorescent compound-bile acid-chitosan nanoparticles having excellent cancer target ability and a method for preparing the same.

One aspect of the present invention is a fluorescent compound represented by the following formula (1).

[Formula 1]

Another aspect of the invention relates to a contrast agent composition comprising a fluorescent compound according to various embodiments of the present invention as an active ingredient.

Another aspect of the present invention relates to a contrast agent injection comprising a fluorescent compound according to various embodiments of the present invention as an active ingredient.

Another aspect of the invention is a fluorescent compound-bile acid-chitosan nano comprising a fluorescent compound according to various embodiments of the present invention, (a) bile acid-chitosan complex nanoparticles, (b) encapsulated in a bile acid-chitosan complex nanoparticles It's about the mouth.

Another aspect of the invention relates to a contrast agent composition comprising a fluorescent compound-bile acid-chitosan nanoparticles according to various embodiments of the present invention.

Another aspect of the invention relates to a contrast agent injection comprising fluorescent compound-bile acid-chitosan nanoparticles according to various embodiments of the invention.

Another aspect of the present invention is to prepare a bile acid solution by (A) mixing the first solvent, such as bile acid and methanol, NHS and the like, (B) a second solvent, such as glycol chitosan and methanol Preparing a chitosan solution by adding EDC to an aqueous solution, (C) adding the chitosan solution to the bile acid solution, dialysis and dispersing it, and then lyophilizing the bile acid-chitosan composite nanoparticle manufacturing method It is about.

Another aspect of the present invention is to prepare a bile acid-chitosan composite nanoparticle solution by (A) dissolving bile acid-chitosan composite nanoparticles in a first solvent such as a carbonate buffer solution, (B) in various embodiments of the present invention Preparing a fluorescent compound solution by dissolving the fluorescent compound in a second solvent such as DMSO, (C) the fluorescent compound solution into the bile acid-chitosan composite nanoparticle solution, and dialysis lyophilization. The present invention relates to a method for preparing a compound-bile acid-chitosan nanoparticle.

Another aspect of the present invention relates to a fluorescent compound labeling method comprising the step of binding a fluorescent compound according to various embodiments of the present invention with a labeling substance.

Another aspect of the present invention relates to a fluorescent compound labeling method comprising the step of binding a fluorescent compound-bile acid-chitosan nanoparticle with a labeling material according to various embodiments of the present invention.

The fluorescent compound according to the present invention is excellent in fluorescence intensity, relative quantum efficiency and labeling rate, and thus can be more effectively used for labeling and dyeing target materials. In addition, it has excellent optical stability and shows stable fluorescence even for long time dyeing, and it does not accumulate when administered to the body, and has excellent fluorescence intensity, so that dyeing and imaging in the body can be used economically even with a small amount of use compared to conventional dyes. Do. In addition, the fluorescent compound-bile acid-chitosan nanoparticles according to the present invention show excellent cancer targeting ability.

1 and 2 are graphs showing the optical analysis results of Compound 1.
3 and 4 are graphs showing the results of optical analysis of compound 2.
5 is a fluorescence spectrum of Compound 1. FIG.
6 is a fluorescence spectrum of Compound 2. FIG.
7 is a result of in vivo imaging showing the distribution patterns of mouse 1 and compound 2 in the mouse.
8 is an ex vivo imaging result showing the distribution in the mouse body of Compound 1 and Compound 2.
9 is a graph showing fluorescence intensity values for each organ after ex vivo imaging.
10 is a result of in vivo imaging showing cancer targeting ability of Compound 1-HGC nanoparticles.
FIG. 11 is an ex vivo imaging result showing cancer targeting ability of Compound 1-HGC nanoparticles. FIG.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

One aspect of the present invention is a fluorescent compound represented by the following formula (1).

Is selected from, ^wherein _{X 1, X 2, X 3} , X 4 are the same or different and each independently represent hydrogen, SO ₃ H, SO ₃ together

X ₅ is SO ₃ H or SO ₃ ⁻ ,

(i) wherein Y ₁ has a structure of Formula 1a and Y ₂ is SO ₃ H or SO ₃ ⁻ , or (ii) Y ₁ is SO ₃ H or SO ₃ ⁻ and Y ₂ is Has the structure of

[Formula 1a]

N1 is a natural number of 2 to 7,

N3 is a natural number of 2 to 7,

N2 is a natural number of 1 to 5,

M1 is a natural number of 3 to 7,

M2 is a natural number of 2 to 5,

M3 is a natural number of 3 to 7,

Z is Cl.

It was confirmed that the fluorescent compound according to the present invention showed stronger fluorescence intensity than conventional commercial fluorescent dyes (cy 5.5, cy 7.5).

According to one embodiment, the _{X 1, X 2, X 3} , X 4 are each independently selected from SO ₃ H and SO _3, or both (i) hydrogen or (ii) ^- is selected from,

N1 is 3 or 4,

N3 is 3 or 4,

N2 is 2 or 3,

M1 is 5,

M2 is 3,

M3 is 5.

According to another embodiment, X ₁ , X ₂ , X ₃ , X ₄ are (i) all hydrogen or (ii) only one of them is SO ₃ ⁻ and the others are SO ₃ H.

According to another embodiment, the fluorescent compound has the structure of Formula 2a or Formula 2b.

[Formula 2a]

[Formula 2b]

As a result of observing the distribution patterns of Compound 1 and Compound 2, it was confirmed that Compound 1 and Compound 2 remained relatively large in kidney and liver, respectively.

Another aspect of the invention relates to a contrast agent composition comprising a fluorescent compound according to various embodiments of the present invention as an active ingredient.

Another aspect of the present invention relates to a contrast agent injection comprising a fluorescent compound according to various embodiments of the present invention as an active ingredient.

Another aspect of the invention is a fluorescent compound-bile acid-chitosan nano comprising a fluorescent compound according to various embodiments of the present invention, (a) bile acid-chitosan complex nanoparticles, (b) encapsulated in a bile acid-chitosan complex nanoparticles It is about particles.

It was confirmed that the fluorescent compound-bile acid-chitosan nanoparticles according to the present invention had excellent cancer targeting ability.

Another aspect of the invention relates to a contrast agent composition comprising a fluorescent compound-bile acid-chitosan nanoparticles according to various embodiments of the present invention.

Another aspect of the invention relates to a contrast agent injection comprising fluorescent compound-bile acid-chitosan nanoparticles according to various embodiments of the invention.

Another aspect of the present invention is to prepare a bile acid solution by (B) mixing the bile acid and methanol and NHS, (B) to prepare a chitosan solution by adding EDC to an aqueous solution containing glycol chitosan and methanol Step, (C) after the chitosan solution is added to the bile acid solution, dialysis and dispersion and lyophilized relates to a method for producing bile acid-chitosan composite nanoparticles.

Another aspect of the present invention is to prepare a bile acid-chitosan composite nanoparticles solution by dissolving bile acid-chitosan composite nanoparticles in a carbonate buffer solution, (B) a fluorescent compound according to various embodiments of the present invention DMSO Preparing a fluorescent compound solution by dissolving in (C) adding the fluorescent compound solution to the bile acid-chitosan composite nanoparticle solution, and lyophilizing the dialysis compound to the method of preparing a fluorescent compound-bile acid-chitosan nanoparticle. It is about.

Another aspect of the present invention relates to a fluorescent compound labeling method comprising the step of binding a fluorescent compound according to various embodiments of the present invention with a labeling substance.

Another aspect of the present invention relates to a fluorescent compound labeling method comprising the step of binding a fluorescent compound-bile acid-chitosan nanoparticle with a labeling material according to various embodiments of the present invention.

In this case, the labeling material is at least one selected from fibers, biomolecules, nanoparticles and organic compounds, the labeling material comprises at least one functional group selected from amine groups, hydroxyl groups and thiol groups, Fluorescent compounds may bind.

In addition, the biomolecule may be selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins and siRNAs.

Hereinafter, the present invention will be described in more detail with reference to examples and the like, but the scope and contents of the present invention are not limited or interpreted by the following examples. In addition, if it is based on the disclosure of the present invention including the following examples, it will be apparent that those skilled in the art can easily carry out the present invention, the results of which are not specifically presented experimental results, these modifications and modifications are attached to the patent It goes without saying that it belongs to the claims.

In addition, the experimental results presented below are only representative of the experimental results of the Examples and Comparative Examples, and the effects of each of the various embodiments of the present invention not explicitly set forth below will be described in detail in the corresponding sections.

Example

Preparation Example 1 Preparation of Compound A

(1) Synthesis of Compound a

6-amino-1,3-naphthalenedisulfonic acid monosodium salt hydrate) (TCI) (15 g) was added to 200 mL of distilled water and dried under reduced pressure after ion exchange using an ion exchange resin for 12 hours (15 g, 100 %).

To tin (II) chloride, 40 mL of distilled water and 8 mL of hydrochloric acid (HCl) were mixed and cooled. 120 mL of distilled water and 25 mL of hydrochloric acid (HCl) were added to the solid material (15 g, 49.5 mmol, 1eq), and the reaction was carried out at 0 ° C. for 5 minutes. 50 mL of distilled water was added to sodium nitrite (3.4 g, 49.5 mmol, 1 eq), and the reaction product was added drop wise to the solvent for 30 minutes. After the cooled solution was added dropwise to the mixed solution and reacted at room temperature for 12 hours, the solvent was dried with a rotary evaporator and the particles were caught with diethyl ether, washed two or three times, and dried under reduced pressure (18 g, 114%).

(2) Synthesis of Compound b

Compound a (6 g, 18.8 mmol, 1 eq,) and 3-methyl-2-butanone (6.05 mL, 56.4 mmol, 3.02 eq, TCI), potassium acetate (3.7 g, 37.6 mmol, 2 eq, Duksan) After addition to 50 mL of acetic acid, the mixture was reacted with heating under reflux at 140 ° C. for 12 hours. After cooling to room temperature, the solvent was dried under reduced pressure, washed with ethyl acetate two or three times, and dried under reduced pressure (7.4 g, 107%).

R _f = 0.57 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

(3) Synthesis of Compound c

Compound b (7.4 g, 20.3 mmol, 1 eq) and 1,3-propanesultone (7.9 mL, 90.5 mmol, 4.46 eq, TCI), sodium acetate (2 g, 40.6 mmol, 2 eq) After addition to 20 mL of nitrile, the reaction was heated to reflux at 120 ° C. for 12 hours. Cooled to room temperature and dried under reduced pressure (15 g, 153%).

(4) Synthesis of Compound d

Compound c (15 g, 30.4 mmol, 1 eq) and malonaldehyde dianilide hydrochloride (7.8 g, 30.4 mmol, 1 eq, TCI), triethylamine (4.2 mL, 30.4 mmol, 1 eq, TCI) were added to acetic acid 100 After addition to the mL and reacted by heating to reflux at 140 ℃ for 4 hours. After cooling to room temperature, the mixture was washed two or three times with ethyl acetate and dried under reduced pressure. Thereafter, the mixture was purified by silica gel 60 normal chromatography with a developing solution of isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v (12 g, 60%).

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

(5) Synthesis of Compound e

Ethyl 2-methylacetoacetate (24.2 mL, 0.16 mol, 1 eq, TCI) and ethyl-6-bromohexanoate (34 mL, 0.17 mol, 1.1 eq, TCI), sodium ethoxide (64 mL, 0.77 mol , 4.8 eq, TCI) was added 200 mL of ethanol and reacted by heating under reflux at 120 ° C. for 12 hours. After cooling to room temperature, a small amount of 1 M hydrochloric acid was added dropwise to make the pH neutral (pH = 7). After extraction with chloroform and distilled water, the solvent was dried under reduced pressure. Thereafter, the nucleic acid and ethyl acetate were purified by silica gel 60 normal chromatography with a developing solution (48 g, 106.6%).

R _f = 0.4 (normal, silica gel, nucleic acid: ethyl acetate = 9: 1 v / v)

(6) Synthesis of Compound f

To compound e (48 g, 0.16 mol, 1 eq) and sodium hydroxide (21.4 g, 0.51 mol, 3.2 eq, Deoksan) in methanol (165 mL, 3.9 mol, 24.4 eq) and distilled water (54.6 mL, 2.89 mol, 18.1 eq ) Was added and reacted with heating at 50 ° C. for 12 hours. After drying under reduced pressure, 200 mL of 1M hydrochloric acid was added dropwise to make the pH acidic (pH = 1). After extraction with ethyl acetate and distilled water, the solvent was dried under reduced pressure (30 g, 95.2%).

R _f = 0.0 (normal, silica gel, nucleic acid: ethyl acetate = 9: 1 v / v)

(7) Synthesis of Compound g

87 mL of acetic acid was added to compound f (15.8 g, 84.8 mmol, 1.5 eq) and compound 5-1 (18 g, 56.5 mmol, 1 eq), followed by reaction under heating and reflux at 120 ° C. for 5 hours. It was cooled to room temperature and filtered to remove the solid formed, and the filtrate was dried under reduced pressure. Thereafter, the mixture was purified by silica gel 60 normal chromatography using isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v (30 g, 115.3%).

R _f = 0.45 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

(8) Synthesis of Compound h

Compound g (30 g, 63.9 mmol, 1 eq) and 1,3-propanesultone (37.5 mL, 428.1 mmol, 6.7 eq, TCI), sodium acetate (7.2 g, 105.4 mmol, 1,65 eq) were acetonitrile After addition to 45 mL, the reaction was heated at reflux at 120 ° C. for 5 hours. Cooled to room temperature and dried under reduced pressure. The aqueous acetonitrile solution was then purified by RP-C18 reverse phase chromatography as a developing solution (52 g, 140%).

(9) Synthesis of Compound A

Compound h (15 g, 25.9 mmol, 1.5 eq) and compound d (11.5 g, 17.3 mmol, 1 eq) were completed in dimethylformamide. Triethylamine (20.5 mL, 147 mmol, 8.5 eq, TCI) was added to the solution, followed by addition of acetic anhydride (9 mL, 95.1 mmol, 5.5 eq, Duksan), followed by reaction at room temperature for 1 hour. After washing 2-3 times with ethyl acetate, the mixture was dried under reduced pressure. Thereafter, the mixture was purified by silica gel 60 normal chromatography with a developing solution of isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v (12 g, 60%).

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

Example 1 Preparation of Compound 1

(1) Synthesis of Compound A-1

Compound A (500 mg, 0.5 mmol, 1 eq) and N, N-diisuccinimidyl carbonate (DSC, 384 mg, 1.5 mmol, 3 eq, TCI), N, N-diisopropylethylamine (DIPEA) (870 μL, 5 mmol, 10 eq, TCI) was added to 50 mL of dimethylformamide and then reacted with heating at 40 ° C. for 1 hour. After cooling to room temperature, the particles were caught using diethyl ether and the resulting solid particles were dried under reduced pressure (500 mg, 91%).

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

LC / MS, calculated 1215.14, found 1216.2

(2) Synthesis of Compound A-2

1,3-diaminopropane (9.72 μL, 0.1165 mmol, 1 eq) was added to Compound A-1 (500 mg, 0.035 mmol, 1 eq) in 10 mL of DMF, followed by reaction at room temperature for 30 minutes. After the reaction, the resulting solid particles were filtered off. After washing two or three times with ethyl acetate, the mixture was dried under reduced pressure. Acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography with a developing solution to obtain pure Compound A-2 (400 mg, 75.6%).

R _f = 0.33 (normal, silica gel, acetonitrile: distilled water = 4: 1 v / v)

LC / MS, calculated 1174.2, found 1175.3

(3) Synthesis of Compound A-3

Cyanuric chloride (383 mg, 2.08 mmol, 5.5 eq) was added to 35 mL of acetonitrile and 35 mL of ice, and the temperature was mixed at 0 to 5 ° C. for 0.5 hour. Compound A-2 (400 mg, 0.378 mmol, 1eq) and 191 mg of sodium bicarbonate were added thereto, and the temperature was reacted at 0-5 ° C. for 2 hours. After drying under reduced pressure, an acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography with a developing solution to give pure Compound A-3 (373 mg, 81.9%).

R _f = 0.47 (isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)

LC / MS, calculated 1321.14, found 1322.8

(4) Synthesis of Compound A-4

Compound A-3 (373 mg, 0.309 mmol, 1eq), 6-aminohexanoic acid (284 mg, 2.16 mmol, 7 eq), N, N-diisopropylethylamine (DIPEA) (538 μL, 3.09 mmol, 10 eq, TCI) was added to 30 mL of distilled water and reacted at room temperature for 1 hour. Then purified by reverse phase chromatography using an aqueous solution of acetonitrile as a developing solution to give the pure compound A-4 (235 mg, 58.5%).

R _f = 0.33 (isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)

LC / MS, calculated 1416.26, found 1416.9

(5) Synthesis of Compound 1

Compound A-4 (235 mg, 0.18 mmol, 1 eq) and N, N-diisuccinimidyl carbonate (DSC) (139 mg, 0.542 mmol, 3 eq, TCI), N, N-diisopropylethylamine (DIPEA, 314 μL, 1.8 mmol, 10 eq, TCI) was added to 20 mL of dimethylformamide, followed by reaction at room temperature for 1 hour. The resulting solid particles were dried under reduced pressure after catching the particles with diethyl ether. Then purified by reverse phase chromatography using an aqueous solution of acetonitrile as a developing solution to give the pure compound 1 (39 mg, 15.5%).

R _f = 0.65 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

LC / MS, 1513.28, found 1514.3

Preparation Example 2 Preparation of Compound B

(1) Synthesis of Compound i

1,1,2-trimethylbenz indole (2 g, 9.56 mmol, 1 eq) and 1,4-butanesultone (2.9 mL, 28.7 mmol, 3.01 eq, TCI), sodium acetate (941 mg, 11.5 mmol, 1, 2 eq) was added to 10 mL of acetonitrile and then reacted with heating under reflux at 100 ° C. for 12 hours. After cooling to room temperature, the resulting solid particles were filtered after removing the solvent. After washing 2-3 times with ethyl acetate and dried under reduced pressure (1.56 g, 47.3%).

R _f = 0.37 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

(2) Synthesis of Compound j

1,1,2-trimethylbenz indole (2 g, 9.56 mmol, 1 eq) and 6-bromohexanoic acid (5.6 g, 28.7 mmol, 3.01 eq, TCI), sodium acetate (941 mg, 11.5 mmol, 1 , 2 eq) was added to 10 mL of acetonitrile and then reacted with heating under reflux at 100 ° C. for 12 hours. After cooling to room temperature, the resulting solid particles were filtered after removing the solvent. After washing 2-3 times with ethyl acetate and dried under reduced pressure (1.01 g, 33.7%).

R _f = 0.37 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

(3) Synthesis of Compound k

Compound i (15 g, 30.4 mmol, 1 eq) and glutacondianilide hydrochloride (7.5 g, 30.4 mmol, 1 eq, TCI), triethylamine (4.2 mL, 30.4 mmol, 1 eq, TCI) was added to 100 mL of acetic acid and then reacted by heating to reflux at 140 ° C. for 4 hours. After cooling to room temperature, the mixture was washed two or three times with ethyl acetate and dried under reduced pressure. Then, the mixture was purified by Silica gel 60 normal chromatography with isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v (16 g, 96.9%).

R _f = 0.5 normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

(4) Synthesis of Compound B

Compound j (15 g, 46.3 mmol, 1.5 eq) and compound k (16.7 g, 30.9 mmol, 1 eq) were completed in dimethylformamy. Triethylamine (20.5 mL, 147 mmol, 8.5 eq, TCI) was added to the solution, followed by addition of acetic anhydride (9 mL, 95.1 mmol, 5.5 eq, Duksan), followed by reaction at room temperature for 1 hour. After washing 2-3 times with ethyl acetate, the mixture was dried under reduced pressure. Thereafter, the mixture was purified by silica gel 60 normal chromatography with isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v (5.7 g, 24.9%).

R _f = 0.5 normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

Example 2: Preparation of Compound 2

(1) Synthesis of Compound B-1

Compound B (500 mg, 0.67 mmol, 1 eq) and N, N-diisuccinimidyl carbonate (DSC) (518 mg, 2.0 mmol, 3 eq, TCI), N, N-diisopropylethylamine (DIPEA ) (1.1 mL, 6.7 mmol, 10 eq, TCI) was added to 50 mL of dimethylformamide, followed by reaction at 40 ° C. for 1 hour. After cooling to room temperature, the particles were caught using diethyl ether and the resulting solid particles were dried under reduced pressure (500 mg, 89%).

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

LC / MS, calculated 827.36, found 828.1

(2) Synthesis of Compound B-2

To compound B-1 (500 mg, 0.6 mmol, 1 eq) was added 1,3-diaminopropane) (49.8 μL, 0.6 mmol, 1 eq) to 10 mL of DMF, followed by 30 minutes at room temperature. Reacted. Solid particles produced after the reaction were filtered. After washing 2-3 times with ethyl acetate, the mixture was dried under reduced pressure. Acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography with a developing solution to obtain pure Compound B-2 (327 mg, 68.4%).

R _f = 0.33 (normal, silica gel, acetonitrile: distilled water = 4: 1 v / v)

LC / MS, calculated 786.42, found 785.9

(3) Synthesis of Compound B-3

Cyanuric chloride (416 mg, 2.26 mmol, 5.5 eq) was added to 35 mL of acetonitrile and 35 mL of ice, and the temperature was mixed at 0 to 5 ° C. for 0.5 hour. Compound B-2 (327 mg, 0.41 mmol, 1eq) and 200 mg of sodium bicarbonate were added thereto, and the temperature was reacted at 0˜5 ° C. for 2 hours. After drying under reduced pressure, an acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography with a developing solution to obtain pure Compound B-3 (157 mg, 40.6%).

R _f = 0.47 (isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)

LC / MS, calculated 933.36 found 932.6

(4) Synthesis of Compound B-4

Compound B-3 (157 mg, 0.166 mmol, 1eq), 6-aminohexanoic acid (153 mg, 1.16 mmol, 7 eq), N, N-diisopropylethylamine (DIPEA) (279 μL, 1.6 mmol, 10 eq, TCI) was added to 30 mL of distilled water and reacted at room temperature for 1 hour. Then purified by reverse phase chromatography using an aqueous solution of acetonitrile as a developing solution to give pure Compound B-4 (112 mg, 65.5%).

R _f = 0.33 (isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)

LC / MS, calculated 1028.47, measured 1027.8

(5) Synthesis of Compound 2

Compound B-4 (112 mg, 0.11 mmol, 1 eq) and N, N-diisuccinimidyl carbonate (DSC) (83.7 mg, 0.33 mmol, 3 eq, TCI), N, N-diisopropylethylamine (DIPEA) (192 μL, 1.1 mmol, 10 eq, TCI) was added to 20 mL of dimethylformamide, followed by reaction at room temperature for 1 hour. The resulting solid particles were dried under reduced pressure after catching the particles with diethyl ether. Then purified by reverse phase chromatography using an aqueous solution of acetonitrile as a developing solution to give pure Compound 2 (57 mg, 46%).

R _f = 0.65 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)

LC / MS, calculated 1124.5, measured 1125.1

Preparation Example 3 Preparation of Bile Acid-Chitosan Nanoparticles

(1) Purification of Chitosan

1 g of glycol chitosan (GC) (100 kDa) was placed in a 250 mL Erlenmeyer flask, dissolved in 80 mL of ultrapure water, and dialyzed several times using a dialysis membrane (MWCO 100 kDa). After completion of dialysis, the mixture was cooled at -80 ° C for 12 hours and then lyophilized to obtain purified GC (0.571 g, 57.1%),

(2) Synthesis of Bile Acid-Chitosan (HGC) Nanoparticles

500 mg of purified GC was completed in 60 mL of ultrapure water, and then 60 mL of methanol was further added thereto, followed by sufficient stirring. 150 mg of bile acid (5β-cholanic acid) was added to 120 mL of methanol and completed. 1000 mL of methanol was added to 150 mg N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), followed by stirring into a GC solution. 72 mg of N-hydroxysuccinimide (NHS) was added to 1 mL of methanol and stirred, and then added to a bile acid solution and stirred. The bile acid mixed solution was added to the GC mixed pressure solution, and the mixture was stirred at room temperature overnight. The reaction solution was filtered using a syringe filter (pore size 80 μm), and then placed in a dialysis membrane (MWCO 12-14 kDa), followed by dialysis for 4 days. After completion of dialysis, the dispersion was dispersed in an ultrasonic disperser (Waterbath sonicator) for 30 seconds and then lyophilized to obtain HGC nanoparticles (0.492 g, 75.7%).

Day 1 ~ 2 Dialysis Solution: Methanol / Ultra Pure Water 3: 1 v / v

Day 3 dialysis solution: methanol / ultra pure water 1: 1 v / v

Day 4 dialysis solution: ultrapure water

Example 3: Preparation of Compound 1 -HGC

(1) Synthesis of Compound 1-HGC Nanoparticles

Bile acid-chitosan (HGC) nanoparticles (20 mg, 0.21 μmol, 1 eq) were added to 8 mL carbonate buffer solution (pH = 9.0) and completed. Compound 1 (1.29mg, 0.85 μmol) was dissolved in DMSO at a concentration of 10 mg / mL, and then added dropwise to HGC solution to react with room temperature overnight. The reaction solution was dialyzed for 5 days using a dialysis membrane (MWCO 12-14 kDa). The dialysis-compounded compound was lyophilized to obtain compound 1-HGC nanoparticles (15 mg, 75%).

Day 1-2 dialysis solution: 8.4 g / 1 L of purified salt / ultra pure water

-Day 3 ~ 5 Dialysis Solution: Ultrapure Water

Example 4: Preparation of Compound 2-HGC

(2) Synthesis of Compound 2-HGC Nanoparticles

Bile acid-chitosan (HGC) nanoparticles (20 mg, 0.21 μmol, 1 eq) were added to 8 mL carbonate buffer solution (carbonate buffer, pH = 9.0) and completed. Compound 2 (0.96mg, 0.85 μmol) was dissolved in DMSO at a concentration of 10 mg / mL, and then added dropwise to HGC solution to react at room temperature overnight. The reaction solution was dialyzed for 5 days using a dialysis membrane (MWCO 12-14 kDa). The dialysis completed compound was lyophilized to obtain HGC-FSD ICG nanoparticles (17 mg, 85%).

Day 1-2 dialysis solution: 8.4 g / 1 L of purified salt / ultra pure water

-Day 3 ~ 5 Dialysis Solution: Ultrapure Water

Test Example 1 Optical Analysis of a Compound

(1) Absorption, fluorescence wavelength analysis of compound 1

The absorption and fluorescence wavelength of Compound 1 were analyzed. Stock solution was prepared by adding DMF to Compound 1. Stock solutions were prepared at a concentration of 10 mg / mL and diluted to a concentration of 6.6 μM using pH 7.4 10 mM Phosphate buffered saline (hereinafter, referred to as 1 × PBS). Absorption values at the maximum wavelength and absorption wavelength of the synthesized material were measured using an Agilent Cary 8454 instrument, and the maximum absorption value at 678 nm was confirmed (FIG. 1).

Fluorescence values at the fluorescence wavelength and the maximum fluorescence wavelength were measured using LS-55 manufactured by Perkin Elmer. Excitation was set to 678 nm, the measured maximum absorption wavelength, and fluorescence was measured using a reagent diluted to 66 nM (Fig. 2).

(2) Absorption, fluorescence wavelength analysis of compound 2

The absorption and fluorescence wavelength of Compound 2 were analyzed. DMF was added to compound 2 to prepare a stock solution. Stock solutions were prepared at a concentration of 10 mg / mL and diluted to a concentration of 6.6 μM using Methanol. Absorption values at the maximum wavelength and absorption wavelength of the synthesized material were measured using an Agilent Cary 8454 instrument, and the maximum absorption value at 786 nm was confirmed (FIG. 3).

Fluorescence values at the fluorescence wavelength and the maximum fluorescence wavelength were measured using LS-55 manufactured by Perkin Elmer, and the fluorescence was measured with a reagent diluted to 0.22 μM after setting Excitation to the measured maximum absorption wavelength of 786 nm. 4).

(3) Comparison of control phosphor and fluorescence intensity

The fluorescence intensities of Compound 1, Compound 2 and control fluorescent dyes (Cy 5.5, Cy 7.5) were compared. DMF was added to four fluorescent dyes to prepare a stock solution. The concentration was made equal to 10 mg / mL. Compound 1 and Cy 5.5 were then diluted to a concentration of 1 nM using pH 7.4 10 mM Phosphate buffered saline (hereinafter referred to as 1x PBS) and measured for fluorescence under the setting of Excitation 678 nm, and Compound 2 and Cy 7.5 using Methanol. After dilution to a concentration of 10 nM it was measured under Excitation 768 nm setting. The measurement was performed using PerkinElmer's LS 55 Fluorescence spectrometer.

5 and 6 show fluorescence spectra of Compounds 1 and 2, and the fluorescence intensities of Compounds 1 and 2 were relatively stronger than those of all control dyes (Cy 5.5 and Cy 7.5). It was.

Test Example 2: Animal Experimental Analysis of Compound 1 and Compound 2

(1) Measurement of distribution patterns in the mouse

Compound 1 and compound 2 were dissolved in DMSO at 10 mg / mL to make a stock solution, diluted with 1X PBS, and injected through the tail vein of the mouse at 5 μg / 200 μL. Mice were male 6-week-old male Balb / c-nu (20 g) mice. Imaging was performed in vivo 1 hour, 2 hours, 4 hours, 8 hours, 24 hours after injection of the compound using the IVIS spectrum (PerkinElmer), followed by Liver, Lung, Spleen, Kidney , Heart was extracted and ex vivo imaging was performed. In this case, Compound 1 was performed at 710 nm / 760 nm (Excitation / Emission) Compound 2 was set to 745 nm / 820 nm (Excitation / Emission).

As a result of the in vivo imaging of FIG. 7, Compound 1 had dye spread over each organ of the mouse at 1 hour after injection of the compound, and almost all at 24 hours after gradually passing through the urine over 2 hours. It was confirmed that the compound escaped. Compound 1 was confirmed that a lot of accumulation in the Kidney of the mouse when 1 hour after the injection of the compound, it was confirmed that the passage through the urine over time.

As a result of the ex vivo imaging of FIG. 8, Compound 1 was found to have many residues in Kidney and Compound 2 in Liver. After ex vivo imaging, fluorescence intensity values for each organ are shown in FIG. 9.

(2) Confirmation of Cancer Target Capability by Using Xenograft Mouse Model of Compound 1-HGC Nanoparticles

To determine the cancer targetability of Compound 1-HGC, 6-week-old male Balb / c-nu (20 g) was injected with subcutaneous injection of SCC7 cells (1x106 / 0.1mL), a Squamous cell carcinoma cell line, on the left thigh of the mouse. A model was produced. Compound 1-HGC was dissolved in distilled water (hereinafter referred to as DW) at a concentration of 1 mg / mL to prepare a stock solution.When the volume of the mouse tumor became 60 to 80 mm ³ , Compound 1-HGC was injected through 120 μg tail vein. Injection. In vivo imaging was performed under the setting of 710 nm / 760 nm (Excitation / Emission) using the IVIS spectrum (PerkinElmer). After 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours of nanoparticle injection, ex vivo imaging was performed after 24 hours of imaging, followed by extraction of Liver, Lung, Spleen, Kidney, Heart, Tumor, The analysis was performed using software from the IVIS spectrum.

As a result of the in vivo imaging analysis of FIG. 10, it was confirmed that the cells were distributed throughout the body until 8 hours after injection and gradually accumulated in tumors and exited through metabolism except for nanoparticles accumulated in tumors at 24 hours.

In FIG. 11, organs were extracted to confirm accumulation in organs other than the tumor, and ex vivo imaging was performed to compare the fluorescence intensity of organs. As a result, it was confirmed that the compound 1-HGC was accumulated in Kidney. It seems to be because In vivo and ex vivo imaging confirmed the cancer targeting ability of Compound 1-HGC, and confirmed that Compound 1 can be used in various animal experiments.

Claims (15)

Fluorescent compound represented by the following formula (1):
[Formula 1]

Is selected from, ^wherein _{X 1, X 2, X 3} , X 4 are the same or different and each independently represent hydrogen, SO ₃ H, SO ₃ together
X ₅ is SO ₃ H or SO ₃ ⁻ ,
(i) wherein Y ₁ has a structure of Formula 1a and Y ₂ is SO ₃ H or SO ₃ ⁻ , or (ii) Y ₁ is SO ₃ H or SO ₃ ⁻ and Y ₂ is Has the structure of
[Formula 1a]

N1 is a natural number of 2 to 7,
N3 is a natural number of 2 to 7,
N2 is a natural number of 1 to 5,
M1 is a natural number of 3 to 7,
M2 is a natural number of 2 to 5,
M3 is a natural number of 3 to 7,
Z is Cl. The compound of claim 1, wherein X ₁ , X ₂ , X ₃ , X ₄ are (i) all hydrogen or (ii) each independently selected from SO ₃ H and SO ₃ ⁻ ,
N1 is 3 or 4,
N3 is 3 or 4,
N2 is 2 or 3,
M1 is 5,
M2 is 3,
M3 is a fluorescent compound, characterized in that 5. The fluorescent compound according to claim 2, wherein X ₁ , X ₂ , X ₃ , X ₄ are (i) all hydrogen or (ii) only one of them is SO ₃ ⁻ and the other is SO ₃ H. The fluorescent compound of claim 3, wherein the fluorescent compound has a structure of Formula 2a or Formula 2b:
[Formula 2a]

[Formula 2b]

A contrast agent composition comprising the fluorescent compound according to any one of claims 1 to 4 as an active ingredient. A contrast agent injection comprising the fluorescent compound according to any one of claims 1 to 4 as an active ingredient. A fluorescent compound-bile acid-chitosan nanoparticle comprising (a) a bile acid-chitosan composite nanoparticle, and (b) a fluorescent compound encapsulated in a bile acid-chitosan composite nanoparticle,
The fluorescent compound is a fluorescent compound according to any one of claims 1 to 4, characterized in that the fluorescent compound-bile acid-chitosan nanoparticles. A contrast agent composition comprising the fluorescent compound-bile acid-chitosan nanoparticles according to claim 7. A contrast agent injection comprising the fluorescent compound-bile acid-chitosan nanoparticles according to claim 7. Bile acid-chitosan composite nanoparticles manufacturing method comprising the following steps:
(A) mixing the bile acid and methanol and preparing a bile acid solution by adding NHS,
(B) preparing a chitosan solution by adding EDC to an aqueous solution containing glycol chitosan and methanol,
(C) adding the chitosan solution to the bile acid solution, followed by dialysis, dispersion and lyophilization. Fluorescent compound-bile acid-chitosan nanoparticles manufacturing method comprising the following steps:
(A) dissolving bile acid-chitosan composite nanoparticles in a carbonate buffer solution to prepare a bile acid-chitosan composite nanoparticle solution,
(B) dissolving the fluorescent compound according to any one of claims 1 to 4 in DMSO to prepare a fluorescent compound solution,
(C) adding the fluorescent compound solution to the bile acid-chitosan complex nanoparticle solution and dialysis lyophilization. A method for labeling a fluorescent compound comprising the step of binding the fluorescent compound according to any one of claims 1 to 4 with a labeling substance,
The label material is at least one selected from fibers, biomolecules, nanoparticles and organic compounds,
The material to be labeled includes at least one functional group selected from an amine group, a hydroxyl group and a thiol group,
The fluorescent compound labeling method characterized in that the functional compound is bonded to the functional group. The method of claim 12, wherein the biomolecule is selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins, and siRNAs. A fluorescent compound labeling method comprising the step of binding the fluorescent compound-bile acid-chitosan nanoparticles according to claim 7 with a labeling substance,
The label material is at least one selected from fibers, biomolecules, nanoparticles and organic compounds,
The material to be labeled includes at least one functional group selected from an amine group, a hydroxyl group and a thiol group,
The fluorescent compound-bile acid-chitosan nanoparticles labeling method characterized in that the fluorescent compound is bonded to the functional group. 15. The label of claim 14, wherein the biomolecule is selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins, and siRNAs. Way.

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