WO2017010657A1 - Advanced probe for sensing tyrosine kinase and use thereof - Google Patents

Abstract

The present invention relates to a fluorescent probe composition for sensing tyrosine kinase comprising a compound of formula 5 or analogues thereof, and a use thereof and, more specifically, to a fluorescent probe having greatly improved characteristics through a structural change of conventional compound 1 (KP1), to a cell and tissue imaging method using the same, and to a cancer diagnosing and imaging method which targets living animals. The present invention can be effectively utilized for selective imaging of cancer tissues due to advanced sensing characteristics which exhibit improved sensitivity and selectivity.

Description

Advanced detection system for detecting tyrosine kinase and its use

The present invention relates to a fluorescent probe composition for detecting tyrosine kinase, comprising the compound of formula (5) or analogs thereof, and use thereof.

In order to detect a series of intermolecular interactions such as enzyme-substrate and antigen-antibody interactions in the living body and electrical signal transmission of the nervous system, a material for efficient molecular probes and bioimaging is required. In particular, a fluorescent probe having a specific substrate to be analyzed and optionally a fluorescence change has advantages in high sensitivity, accuracy and fast signal provision.

Recently, active researches on molecular probes for detecting biomarkers of specific diseases have been conducted. Among them, the development of probes for detecting the activity of kinases, proteins directly related to various diseases, is a very important research task. Kinases are the most important phosphatase that regulates the signaling system in vivo, and are responsible for delivering gamma (γ) -phosphate groups of adenosine triphosphate (ATP) to specific substrate proteins.

Since overexpression or mutation of phosphatase is known to cause various diseases such as cancer, diabetes and brain diseases, the development of molecular probes that can effectively detect the activity of protein phosphatase has led to the development of various diseases including cancer. It can be used for diagnosis. In addition, with respect to drug development, such molecular probes can be used for drug search with a mechanism of action that inhibits the activity of phosphatase. In the case of cancer, high activity and production of specific kinases are shown in comparison with general cells and tissues, and thus, the development of molecular probes that selectively bind to specific kinases expressed in cancer can be utilized for selective fluorescence imaging and cancer diagnosis of cancer.

In terms of the photophysical properties of organic fluorescent materials required for in vivo material analysis and bioimaging, and the biotissue permeability of photostability and excitation light sources, the two-photon absorbing fluorescent material is a one-photon absorbing fluorescent material. It is advantageous compared to. Two-photon absorption is a phenomenon in which electrons in a molecule are transferred to an excited state by simultaneously absorbing two photons through a virtual transition state (within about 10 ^-16 seconds).

Accordingly, the present inventors have developed a fluorescent kinase probe (KP1) compound that interacts with tyrosine kinase in fluorescence in vivo (patent registration No. 10-1524915). However, since this KP1 compound is somewhat less sensitive to phosphatase, it is also important to create molecular probes capable of detecting PDGFRa (platelet-derived growth factor receptor) phosphatase with more effective and higher sensitivity. Was recognized as.

Meanwhile, cyclic arginine-glycine-aspartate (cRGD) peptides, folic acid, or biotin, which are known as cancer cell hitters, have recently been introduced into the detection system to detect more cancer cells. Researches that target the system have been actively conducted. In the present invention, the tyrosine kinase is more efficiently produced by introducing such a cancer cell target into the KP1 compound to prepare an efficient "cancer cell targeting" fluorescent kinase probe. We wanted to develop an advanced sensor that detects with

In addition, sensing platforms based on nanoparticles or solid-supported materials have great advantages in terms of high sensitivity, stability and surface modification. Nanoparticle-based fluorescent probes, which feature fluorescent molecular probes on the surface of nanoparticles, have a more aggregated form than single-molecule probes, providing better fluorescence signals, higher stability, and increased in vivo. It is also important to study cancer cell selectivity due to enhanced permeability and retention effect (EPR effect). The development of a detection system incorporating a fluorescent molecular probe on the surface of a solid support such as a microarray chip is also an area that requires continuous research due to the advantage that it can provide improved fluorescence signal and convenience of detection.

Thus, the development of advanced sensor systems is an important research task that must be performed by providing improved sensitivity, selectivity, accuracy and convenience in cancer diagnosis and imaging implementation.

In order to solve the above problems, the present inventors provide a fluorescent probe with improved characteristics through structural changes of the conventionally developed KP1 compound, and at the same time, to provide a fluorescent probe capable of efficiently targeting cancer cells by introducing a cancer cell target. To that end, in addition to providing a sensing platform based on nanoparticles and solid supports.

Accordingly, the present invention is to devise compounds having improved sensitivity, selectivity, accuracy, and convenience, and to provide a method for imaging cells and tissues using them and a method for diagnosing cancer in living animals.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a fluorescent probe composition for detecting tyrosine kinase, comprising a compound of Formula 5 or analogues thereof.

[Formula 5]

(In the formula 5, R ₁ and R ₂ each independently represent hydrogen, C _{1- 10} alkyl, aryl, acetate, or profile chewy; R ₃ represents an acetal or a formyl group; R ₄ is hydrogen, acetyl , Methyl or methoxymethyl group, except when R ₁ and R ₂ are methyl, R ₃ is formyl and R ₄ is hydrogen)

In one embodiment of the present invention, the analogue is characterized in that the cellular target group such as cyclic arginine-glycine-aspartate peptide, folic acid or biotin is linked to the compound of formula (5).

In another embodiment of the present invention, the analogue is linked to a solid support such as silica nanoparticles, paramagnetic nanoparticles, or ferromagnetic nanoparticles in the compound of Formula 5. It features.

In another embodiment of the present invention, the analog is characterized in that the microarray chip (microarray chip) is further connected to the compound of Formula 5 as a solid support.

In another embodiment of the present invention, the compound is characterized in that the compound of formula (2).

[Formula 2]

In another embodiment of the invention, the analog is characterized in that the compound of formula (3).

[Formula 3]

In another embodiment of the invention, the analog is characterized in that the compound of formula (4).

[Formula 4]

In another embodiment of the invention, the compound or analog is characterized in that one- or two-photon absorbing phosphor.

In another embodiment of the present invention, the tyrosine kinase is characterized in that it is selected from the group consisting of PDGFRa, Src, ABL1 (T315I), BRAF, RSK2 and TYK2.

The present invention also provides a method of imaging a cell or tissue using the fluorescent probe composition.

In one embodiment of the invention, the method is characterized in that it comprises the step of measuring the fluorescence of the tyrosine kinase overexpressed in the cell or tissue in combination with the compound.

In another embodiment of the present invention, the fluorescence is characterized by measuring by a one-photon fluorescence microscope or a two-photon fluorescence microscope.

In another embodiment of the present invention, the cell or tissue is characterized in that the cancer cells or cancer tissue.

The present invention also provides an information providing method for diagnosing cancer using the fluorescent probe composition.

In one embodiment of the present invention, after treating the fluorescent probe composition to the sample, if fluorescence is observed, characterized in that it comprises a step of determining cancer.

Since the fluorescent probe of the present invention has advanced detection characteristics indicating improved sensitivity and selectivity in detecting phosphorylase compared to conventional KP1 compounds, it can be effectively used for selective imaging of cancer tissue.

In addition, it is possible to apply to the chip-based assay (chip-based assay) by immobilized on the nanoparticles and integrated, thereby providing a material required for application as a sensing device (device).

In addition, because of the two-photon excitation characteristics, not only are they significantly affected by deep cell permeability, low cell disruption, and in vivo autofluorescent materials, but also excite only the focal region, thereby achieving very high resolution.

1 shows a transmission electron microscopy image of compound 4 (KP1 ^NP or nano-KP1).

2 shows the zetapotential measurements of compounds 4b, 4c, 4d, 4 and 4a.

FIG. 3 shows absorption in PBS buffer solution (10 mM, pH 7.4, with 1% DMSO) for compounds 1, 1a, 2, 2a, 3, 3a and 1 mg / mL at concentrations of compounds 4, 4a Spectrum (FIG. 3A) and fluorescence emission spectrum (FIG. 3B) are shown.

Figure 4 shows the fluorescence emission spectrum according to the ratio of the organic solvent of compound 4a at a concentration of 1 mg / mL.

FIG. 5 shows fluorescence emission spectra in PBS buffer solution (10 mM, pH 7.4, with 20% MeCN) for 10 μM Compound 1, 1a, 2, 2a and Compound 4, 4a at 1 mg / mL concentration will be.

FIG. 6 shows fluorescence intensities measured before and after 1 hour of reaction with phosphatase of Compound 1 (KP1) and Compound 2 (KP1 ^Pr ) at 100 μM.

FIG. 7 shows photons measured after treatment of Compound 1, 2, 3 and 1 mg / mL concentrations of Compound 4 at 10 μM concentrations in normal cells (HEK-293A) and cancer cells (MCF-7, PC3, A549). An optical microscope image is shown.

8 is a photon measured after treatment of 10 μM concentrations of Compound 1 (KP1) and Compound 2 (KP1 ^Pr ) in normal cells (PNT-2, HEK-293A) and cancer cells (MCF-7, PC3, A549). An optical microscope image and a quantitative graph of fluorescence intensity are shown.

9 is a two-photon optical microscope image and fluorescence intensity measured after treatment of 10 μM compound 3 (KP1 ^Bi ) to normal cells (PNT-2, HEK-293A) and cancer cells (MCF-7, PC3, A549) It shows a quantitative graph of.

10 is a photon measured after treatment of normal cells (PNT-2, HEK-293A) and cancer cells (MCF-7, PC3, A549) with Compound 4 (KP1 ^NP or nano-KP1) at a concentration of 1 mg / mL. An optical microscope image and a quantitative graph of fluorescence intensity are shown.

11 shows normal cells (PNT-2, HEK-293A) and cancer cells (MCF-7, PC3, A549) treated with Compound 4 at 10 μM concentrations of Compound 1, 2, 3 and 1 mg / mL. The measured two-photon optical microscope image and the quantitative graph of the fluorescence intensity are shown.

12 shows two-photon optical microscopy images observed after treatment with Compound 3 (KP1 ^Bi ) at a concentration of 10 μM for normal tissues (control) and colon cancer tissues, respectively.

FIG. 13 shows two-photon optics observed after treatment with 100 μM of Compound 1 (KP1) and 1 mg / mouse of Compound 4 (KP1 ^NP or nano-KP1) for normal and control tissues of living mice. Microscopic image (FIG. 13A) and quantitative graph of fluorescence intensity (FIG. 13B) are shown.

FIG. 14 shows optical coherence tomography (OCT) images and angiography images of normal tissues (controls) and cancerous tissues of living mice.

FIG. 15 shows brain, kidney, lung, spleen, liver and cancer of rats injected with 100 μM Compound 1 (KP1) and 5 mg / mouse Compound 4 (KP1 ^NP or nano-KP1) via tail vein injection. A two photon optical microscope image of the tissue is shown.

The present invention provides a fluorescent probe composition for detecting tyrosine kinase, comprising a compound of Formula 5 or analogues thereof.

[Formula 5]

(In the formula 5, R ₁ and R ₂ each independently represent hydrogen, C _{1- 10} alkyl, aryl, acetate, or profile chewy; R ₃ represents an acetal or a formyl group; R ₄ is hydrogen, acetyl , Methyl or methoxymethyl group, with the exception that R ₁ and R ₂ are methyl groups, R ₃ is formyl group and R ₄ is hydrogen).

The compound or analog of the present invention may include an ortho-hydroxy-benzaldehyde structure, and binds to a tyrosine kinase to form a hydrogen bond in the molecule of the ortho-hydroxy-benzaldehyde. Fluorescence can occur when intra-molecular hydrogen-bonding is broken.

In addition, the analog may be linked to a cancer cell target (eg, a cyclic arginine-glycine-aspartate peptide, folic acid, or biotin) through an amide bond or click chemistry to R ₁ or R ₂ of the compound, as shown in Formula 6 below. Can be.

[Formula 6]

This can achieve cancer cell specific targeting. In this case, the hither can be connected without limitation through a universal and suitable linker (except as specifically illustrated in the present patent).

In addition, the analog is a solid support such as silica nanoparticles, paramagnetic nanoparticles, nanoparticles such as ferromagnetic nanoparticles, microarray chips through an amide bond or click chemistry to R ₁ or R ₂ of the compound, as shown in the following formula (7) May be further connected.

[Formula 7]

This can provide higher sensitivity, stability and surface modification. That is, these nanoparticles or solid support-based fluorescent probes may exhibit improved fluorescence signal and high stability in vivo, and cancer cell selectivity due to increased permeation and residual effects (EPR effect) compared to monomolecular probes. In this case, the nanoparticles or solid support may be connected without limitation through a suitable linker (linker).

The compound of Formula 5 of the present invention is not particularly limited as a two-photon absorbing phosphor, but is preferably a compound of Formula 2 below.

[Formula 2]

In addition, analogues of the compound of formula 5 of the present invention (analogues) as a one-photon or two-photon absorbing phosphor, including all derivatives that can be synthesized by universal knowledge from the compound of formula 5 or a protected form thereof It is preferable that it is a compound of following General formula (3) or (4).

[Formula 3]

[Formula 4]

In this case, the compound of Formula 3 is a structure in which the biotin is connected to the terminal of the compound of Formula 5 via a linker derived from tetraethylene glycol.

In addition, the compound of Formula 4 is a structure in which the silica nanoparticles are connected to the terminal of the compound of Formula 5 via a linker derived from tetraethylene glycol.

In the present invention, the tyrosine kinase is not particularly limited, and may be selected from the group consisting of PDGFRa, Src, ABL1 (T315I), BRAF, RSK2 and TYK2, but it is preferable that it is PDGFRa, Src or ABL1 (T315I). , PDGFRa is most preferred.

The present invention also provides a method for imaging cells and tissues using the fluorescent probe composition. That is, the binding of tyrosine kinase overexpressed in cells or tissues with the compound of the present invention increases fluorescence, and thus the presence and activity of kinase can be confirmed by measuring the degree of fluorescence through a one- or two-photon optical microscope. .

According to the compound developed in the present invention, it does not fluoresce itself, but when combined with a specific tyrosine kinase, the hydrogen bond in the molecule is broken, and the specific luminescent property of emitting light by hydrogen bonding between functional groups of nearby enzymes. Will be displayed. This property could be realized by introducing intramolecular hydrogen bonds into the properties of the fluorophore of the electron donor-electron acceptor structure.

Accordingly, the present invention provides a method for diagnosing cancer using the fluorescent probe composition. That is, since cancer cells or cancer tissues are overexpressed tyrosine kinase, it can be usefully used for the diagnosis of cancer through fluorescence imaging. In particular, the present invention has a feature that can diagnose cancer by imaging cells and tissues using a fluorescent probe composition in a living animal.

In the present invention, the content of the fluorescent probe as an active ingredient in the fluorescent probe composition may be appropriately adjusted according to the use form, purpose, patient condition, and the like, and is 1 to 20 mg / kg, preferably 5 to 10 mg / kg, most preferably. Preferably 10 mg / kg, but is not limited thereto.

In addition, the composition may be administered to mammals including humans, mice, rats, pigs, rabbits, guinea pigs, hamsters, dogs, cats, cows or goats by various routes. The mode of administration can be any of the routinely used forms and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. The compositions of the present invention can be used in the form of oral dosage forms, such as powders, tablets, capsules, suspensions, emulsions, and parenteral formulations in the form of transdermal, suppository, and sterile injectable solutions, respectively, according to conventional methods.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1:

7- ( Benzyl (methyl) amino Naphthalene-2- All- (7-benzyl (methyl) amino) naphthalen -2-ol); Synthesis of Compound 2c

The synthetic route of compound 2c is shown in Scheme 1 below.

Scheme 1

Specifically, a sealed container containing the starting material 2b compound (3.0 g, 18.7 mmol, Sigma-aldrich, D116408) and sodium metabisulfite (Na ₂ S ₂ O ₅ , 7.11 g, 37.4 mmol) ) Was added water (H ₂ O, 15.0 mL) and N-methylbenzylamine (N-methylbenzylamine, 4.82 mL, 37.5 mmol) and the vessel was closed. The mixture was stirred at 140 ° C. for 6 hours using a silicone oil container. After lowering the temperature of the reactant to room temperature (25 ℃), open the container, add dichloromethane (100 mL), water (100 mL), saturated brine (30 mL) and extract the organic layer using a separatory funnel. The organic layer was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 5 g). After removing the solvent under reduced pressure of 40 mbar, purified by column chromatography (silica gel, Merck-silicagel 60, 230-400 mesh) (column chromatograph, diameter 6 cm, height 15 cm) (development solution 20 % EtOAc / Hexane) White solid compound 2c (2.61 g, 53%) was obtained.

Example 2:

N- benzyl -7- ( Methoxy methoxy ) -N-methylnaphthalene-2- Amine (N-benzyl-7- (methoxymethoxy) -N-methylnaphthalen-2-amine); Synthesis of Compound 2d

The synthetic route of compound 2d is shown in Scheme 2 below.

Scheme 2

Specifically, sodium hydride (NaH, sodium hydride, 261 mg, 10.90 mmol) is added to a solvent of N, N-dimethylformamide (16.5 mL), and an argon balloon is placed in a saturated brine. The temperature was lowered to -15 ℃ using ice and. Compound 2c (2.61 g, 9.39 mmol) obtained in Example 1 was dissolved in DMF (16.5 mL) in the mixed solution, and the mixture was slowly added for about 5 minutes while maintaining the temperature. Hydrogen gas generated in this process was discharged to the outdoors after going through a silicon oil trap. After stirring for 1 hour at the same temperature, it was confirmed that no more hydrogen gas was generated in the trap. Then chloromethyl methyl ether (0.75 mL, 9.91 mmol) was added slowly for about 5 minutes. When the injection was completed, the temperature was changed to room temperature (25 ° C.), and the mixture was stirred for 6 hours. After 6 hours, ethyl acetate (EtOAc, 200 mL), water (100 mL) and saturated brine (30 mL) were added and the organic layer was extracted using a separatory funnel. The organic layer was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 5 g). Dried. After removing the solvent under reduced pressure at 40 mbar, purified by column chromatography using silica gel (silica gel, Merck-silicagel 60, 230-400 mesh) (column 6 cm, 15 cm in height) (developing solution 5 % EtOAc / Hexane) White solid compound 2d (2.43 g, 80%).

Example 3:

6- ( Benzyl (methyl) amino ) -3- ( Methoxy methoxy )-2- Naphthalaldehyde (6- (benzyl (methyl) amino) -3- (methoxymethoxy) -2-naphthaldehyde); Synthesis of Compound 2e

The synthetic route of compound 2e is shown in Scheme 3 below.

Scheme 3

Specifically, the compound 2d (1.49 g, 4.85 mmol) obtained in Example 2 was put into a 100 mL round-bottom flask, and then an argon balloon was inserted. Subsequently, diethyl ether (Et ₂ O, 24 mL) was added thereto, and the temperature was lowered to −20 ° C. using saturated brine and ice. Tertiary butyllithium (tert-BuLi, 4.28 mL, 7.28 mmol) was slowly added over about 30 minutes. At this time, the color gradually changed to dark brown, and when the injection was completed, the mixture was stirred at the same temperature for 2 hours. After stirring for 2 hours, N, N-dimethylformamide (N, N-dimethylformamide, DMF, 0.64 mL, 8.25 mmol) was slowly added dropwise over about 5 minutes. When the injection was complete, the mixture was stirred for 1 hour at the same temperature, and the color gradually changed to light yellow. After stirring for 1 hour, cold primary distilled water (10 mL) was added thereto and stirred for 10 minutes. After 10 minutes, ethyl acetate (EtOAc, 200 mL), water (100 mL) and saturated brine (30 mL) were added and the organic layer was extracted using a separatory funnel. The organic layer was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 5 g). Dried. After removing the solvent under reduced pressure at 40 mbar, purified by column chromatography using silica gel (silica gel, Merck-silicagel 60, 230-400 mesh) (column 6 cm, 15 cm in height) (developing solution 5 % EtOAc / Hexane) Yellow solid compound 2e (1.22 g, 75%) was obtained.

Example 4: Synthesis of Compound 2g

The synthetic route of compound 2g is shown in Scheme 4 below.

Scheme 4

<4-1> N-benzyl-6- (1,3-dioxan-2-yl) -7- (methoxymethoxy) -N-methylnaphthalen-2-amine (N-benzyl-6- (1 , 3-dioxan-2-yl) -7- (methoxymethoxy) -N-methylnaphthmido-2-amine; Synthesis of Compound 2f

Compound 2e obtained in Example 3 (100 mg, 0.30 mmol), triethyl orthoformate (55 μL, 0.33 mmol) and 1,3-propane diol (1,3-propane diol, 86 μL, 1.2 mmol) was dissolved in dichloromethane (1.5 mL) at 0 ° C., and then tetrabutylammonium tribromide (7.23 mg, 0.015 mmol) was added thereto. The mixture was stirred at room temperature for 1.5 hours and then extracted with ethyl acetate (2 × 10 mL). The organic layer was washed with water (10 mL), saturated brine (10 mL) and dried over anhydrous sodium sulfate. After removing the solvent under reduced pressure of 40 mbar, the next step was carried out without further purification.

<4-2> 6- (1,3-dioxan-2-yl) -7- ( Methoxy methoxy ) -N-methylnaphthalene-2- Amine (6- (1,3-dioxan-2-yl) -7- (mehtoxymethoxy) -N-methylnaphthalen-2-amine); Synthesis of Compound 2g

In a 50 mL round bottom flask, compound 2f (95 mg, 0.24 mmol) and palladium hydroxide (palladium hydroxide on carbon, 20 wt%, Degussa type, 9.5 mg) obtained through Example 4-1 were added with tetrahydrofuran ( tetrahydrofuran, THF, 6 mL), and then inserted a hydrogen balloon and stirred at room temperature for 3 hours. After the hydrogen was removed, the mixture was filtered through celite. The filtrate was removed under reduced pressure of 40 mbar, and then purified by column chromatography using silica gel (silica gel, Merck-silicagel 60, 230-400 mesh) (column chromatograph, diameter 1 cm, height 12 cm) ( The development yielded 2 g (58.5 mg, 80%) of 5% EtOAc / Hexane) clear liquid compound.

Example 5: Synthesis of Compound 2a

The synthetic route of compound 2a is shown in Scheme 5 below.

Scheme 5

<5-1> 6- (1,3-dioxan-2-yl) -7- (methoxymethoxy) -N-methyl-N- (prop-2-yn-1-yl) naphthalene-2 -Amine (6- (1,3-dioxan-2-yl) -7- (methoxymethoxy) -N-methyl-N- (prop-2-yn-1-yl) naphthalen-2-amine); Synthesis of Compound 2h

Compound 2g (836 mg, 2.75 mmol) and anhydrous potassium carbonate (potassium carbonate, K ₂ CO ₃ ) (1.9 g, 13.78 mmol) obtained in Example 4 were added to acetonitrile (acetonitrile, MeCN, 30 mL) at room temperature. After dissolving, 80% propargyl bromide (1.0 mL, 8.27 mmol) was added thereto. The mixture was stirred at reflux at 40 ° C., cooled to room temperature and extracted with ethyl acetate (2 × 50 mL). The organic layer was washed with water (20 mL), saturated brine (20 mL) and dried over anhydrous sodium sulfate. After removing the solvent under reduced pressure of 40 mbar, the next step was carried out without further purification.

<5-2> 3- ( Methoxy methoxy ) -6- ( Methyl (prop-2-yn-1-yl) amino )-2- Naphthalaldehyde (3- (methoxymethoxy) -6- (methyl (prop-2-yn-1-yl) amino) -2-naphthaldehyde); Synthesis of Compound 2a

Compound 2h (12.4 mg, 0.036 mmol) and 5M hydrochloric acid (HCl, 0.24 mL) obtained in Example 5-1 were each obtained with 2.5 isopropyl alcohol (IPA, 0.5 mL) and THF (0.2 mL). It was dissolved in the mixed solvent mixed with 1, and stirred at room temperature for 10 minutes. The mixture was neutralized with saturated sodium bicarbonate (NaHCO ₃ ), and then the solvent was removed under reduced pressure of 40 mbar. The mixture was extracted again with ethyl acetate (2 × 10 mL) and the organic layer was washed with water (10 mL), saturated brine (10 mL) and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure of 40 mbar, and 10 mg of a yellow solid compound was obtained without further purification.

¹ H NMR (CDCl ₃ , 300 MHz, 293K, δ): 10.49 (s, 1H), 8.25 (s, 1H), 7.77 (d, 1H), 7.27 (s, 1H), 7.11 (dd, 1H), 6.91 (d, 1H), 5.38 (s, 2H), 4.18 (d, 2H), 3.56 (d, 3H), 3.15 (d, 3H), 2.23 (t, 1H).

Example  6: compound 2 ( KP1 ^Pr ) Synthesis

The synthetic route of Compound 2 (KP1 ^Pr ) is shown in Scheme 6 below.

Scheme 6

<6-1> 6- (1,3-dioxan-2-yl) -7- (methoxymethoxy) -N-methyl-N- (prop-2-yn-1-yl) naphthalene-2 -Amine (6- (1,3-dioxan-2-yl) -7- (methoxymethoxy) -N-methyl-N- (prop-2-yn-1-yl) naphthalen-2-amine); Synthesis of Compound 2h

First, compound 2h was obtained in the same manner as in Example 5-1.

<6-2> 3- (hydroxy) -6- ( Methyl (prop-2-yn-1-yl) amino )-2- Naphthalaldehyde (3- (hydroxy) -6- (methyl (prop-2-yn-1-yl) amino) -2-naphthaldehyde), Synthesis of Compound 2

Compound 2h (52.4 mg, .0.018 mmol) and 5M hydrochloric acid (HCl, 1.20 mL) obtained in Example 6-1 were added with isopropyl alcohol (isopropyl alcohol, IPA, 2.5 mL) and THF (1.0 mL) at room temperature. It was dissolved in a mixed solvent mixed with 2.5: 1, and stirred at 40 ° C for 3 hours. The mixture was neutralized with saturated sodium bicarbonate (NaHCO ₃₎ and then the solvent was removed under reduced pressure of 40 mbar The mixture was extracted again with ethyl acetate (2 × 10 mL) and the organic layer was water (10 mL). Washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure of 40 mbar, followed by column chromatography using silica gel (silica gel, Merck-silicagel 60, 230-400 mesh). , 1 cm in diameter, 12 cm in height) (developing solution 5% EtOAc / Hexane) to give a yellow solid compound 2 (40.0 mg, 90%).

¹ H NMR (CDCl ₃ , 300 MHz, 293K, δ): 10.51 (s, 1H), 9.95 (s, 1H), 7.96 (s, 1H), 7.76 (d, 1H), 7.09 (dd, 1H), 7.06 (s, 1H), 6.84 (d, 1H), 4.22 (d, 2H), 3.17 (s, 3H), 2.27 (t, 1H). ¹³ C NMR (CDCl ₃ , 300 MHz, 293K, δ): 195.57, 156.83, 150.08, 140.36, 137.62, 130.89, 121.42, 119.67, 114.91, 109.44, 105.46, 78.68, 72.47, 42.09, 38.50. HRMS: m / z calcd for C ₁₅ H ₁₃ NO ₂ , 239.27; found, 239.0945.

Example 7: Synthesis of Compound 3a

The synthetic route of compound 3a is shown in Scheme 7 below.

Scheme 7

Specifically, Example 3 and the compound 3c (21.3 mg, 0.0480 mmol) in which a linker and a biotin each comprising an amine-azide end derived from tetraethylene glycol are connected via an amide bond. Compound 2a (13.6 mg, 0.0480 mmol) was dissolved in a mixed solvent of distilled water (1.5 mL) and THF (1.5 mL) in a 1: 1 ratio at room temperature, and then copper acetate monohydrate (1.9 mg, 9.6). μmol) and sodium ascorbate (3.8 mg, 19.2 μmol) were added thereto, followed by stirring for 3 hours while blocking light.

After stirring for 3 hours, ethyl acetate (EtOAc, 10 mL), water (10 mL) and saturated brine (10 mL) were added, and the organic layer was extracted using a separatory funnel. The organic layer was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 500 mg). )). After removing the solvent under reduced pressure of 40 mbar, purified by column chromatography (silica gel, Merck-silicagel 60, 230-400 mesh) by column chromatography (column chromatograph, diameter 1 cm, height 8 cm) (developing solution 5 % MeOH / CH ₂ Cl ₂ ) to give a yellow solid compound 3a (5 mg).

¹ H NMR (CDCl ₃ , 300 MHz, 293K, δ): 10.46 (s, 1H), 8.22 (s, 1H), 7.73 (dd, 1H), 7.48 (s, 1H), 7.21 (s, 1H), 7.12 (dd, 1H) 7.09 (d, 1H), 6.82 (m, 1H), 5.94 (s, 1H), 5.37 (s, 2H), 5.03 (s, 1H), 4.78 (s, 2H), 4.49 ( m, 3H), 3.83 (m, 2H), 3.55 (s, 3H), 3.50 (m, 12H), 3.16 (s, 3H), 3.12 (m, 1H), 2.90 (m, 1H), 2.86 (d , 1H), 2.20 (m, 2H), 1.72 (m, 6H).

Example  8: biotin based compound 3 ( KP1 ^Bi ) Synthesis

The synthetic route of compound 3 (KP1 ^Bi ) is shown in Scheme 8 below.

Scheme 8

Specifically, Example 3 and the compound 3c (21.3 mg, 0.0480 mmol) in which a linker and a biotin each comprising an amine-azide end derived from tetraethylene glycol are connected through an amide bond. Compound 2 (19.4 mg, 0.0810 mmol) was dissolved in a mixed solvent of distilled water (1.5 mL) and THF (1.5 mL) in a 1: 1 mixture at room temperature, and then copper acetate monohydrate (3.2 mg, 16.2). μmol) and sodium ascorbate (6.4 mg, 32.4 μmol) were added thereto, followed by stirring for 3 hours while blocking light.

After stirring for 3 hours, ethyl acetate (EtOAc, 10 mL), water (10 mL) and saturated brine (10 mL) were added, and the organic layer was extracted using a separatory funnel. The organic layer was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 500 mg). )). After removing the solvent under reduced pressure of 40 mbar, purified by column chromatography (silica gel, Merck-silicagel 60, 230-400 mesh) by column chromatography (column chromatograph, diameter 1 cm, height 8 cm) (developing solution 5 % MeOH / CH ₂ Cl ₂ ) to give a yellow solid compound 3 (5 mg).

¹ H NMR (CDCl ₃ , 300 MHz, 293K, δ): 10.59 (s, 1H), 9.89 (s, 1H), 7.91 (s, 1H), 7.69 (d, 1H), 7.52 (s, 1H), 7.09 (dd, 1H), 6.95 (s, 1H), 6.73 (d, 1H), 6.72 (m, 1H), 6.18 (s, 1H), 5.23 (s, 1H), 4.78 (s, 2H), 4.49 (m, 2H), 4,29 (m, 1H), 3.83 (m, 2H), 3.80 (m, 12H), 3.38 (s, 3H), 3.18 (m, 1H), 2.96 (m, 1H), 2.85 (m, 1 H), 2.20 (m, 2 H), 2.15 (m, 6H). ¹³ C NMR (CDCl ₃ , 300 MHz, 293K, δ): 195.75, 173.50, 163.96, 157.18, 150.55, 144.75, 140.86, 137.98, 131.43, 123.08, 121.27, 119.61, 114.81, 109.27, 104.29, 70.84, 70.61, 70.61, 70.61 , 70.23, 69.77, 62.06, 60.45, 55.77, 50.63, 48.49, 40.85, 39.44, 39.11, 36.19, 30.04, 28.45, 28.39, 25.85. HRMS: m / z calcd for C ₃₃ H ₄₅ N ₇ O ₇ S, 683.82; found, 684.3179.

Example 9 Synthesis of Silica Nanoparticle-Based Compound 4a

The synthetic route of compound 4a is shown in Scheme 9 below.

Scheme 9

Specifically, a silica containing an isocyanate end derived from a linker and tetraethyl orthosilicate (TEOS) each containing an amine-azide end derived from tetraethylene glycol. Compound 4d (27 mg) in which the nanoparticles were connected through an amide bond and Compound 2a (102 mg, 0.360 mmol) obtained through Example 5 were diluted with distilled water (1.5 mL), THF (1.5 mL), and tert-butanol ( tert-butanol (1.5 mL) is dissolved in a mixed solvent of 1: 1: 1, and then copper sulfate (19.0 mg, 0.120 mmol) and sodium ascorbate (95.0 mg, 0.480 mmol) are added. After stirring for 24 hours while blocking the light. After stirring for 24 hours, the mixture was separated using a centrifugal separator (15000 rpm, 20 min), washed with distilled water and ethanol to obtain a yellow solid compound 4a.

Example  10: silica nanoparticle-based compound 4 ( KP1 ^NP  or nano -KP1) Synthesis

The synthetic route of compound 4 (KP1 ^NP or nano-KP1) is shown in Scheme 10 below.

Scheme 10

Specifically, a silica containing an isocyanate end derived from a linker and tetraethyl orthosilicate (TEOS) each containing an amine-azide end derived from tetraethylene glycol. Compound 4d (14.4 mg) and the compound 2 (46.1 mg, 0.192 mmol) obtained through Example 6, wherein the nanoparticles were connected through an amide bond, were distilled water (0.8 mL), THF (0.8 mL), and tert-butanol ( tert-butanol (0.8 mL) is dissolved in a mixed solvent of 1: 1: 1, and then copper sulfate (10.0 mg, 64.0 μmol) and sodium ascorbate (51.0 mg, 256 μmol) are added. After stirring for 24 hours while blocking the light. After stirring for 24 hours, the mixture was separated using a centrifugal separator (15000 rpm, 20 min x 3), washed with distilled water and ethanol to obtain a yellow solid compound 4 (KP1 ^NP or nano-KP1).

Example 11 Characterization of Silica Nanoparticle-Based Fluorescence Probes

In order to determine the properties of the silica nanoparticle compounds 4a, 4b, 4c, 4d and silica nanoparticle-based fluorescent probe compound 4, the following experiment was performed.

First, in order to confirm the size of the synthesized silica nanoparticles and silica nanoparticle-based fluorescent probe, Compound 4 (20 μL) dispersed in distilled water was treated on a carbon-coated copper grid and a transmission electron microscope (Transmission electron microscopy, TEM) measured the size of the nanoparticles using JEOL JEM-2100, the results are shown in FIG. The size of the measured nanoparticles was found to be 61.6 ± 6.2 nm, which is suitable for bioapplication.

In addition, to confirm the surface modification of the synthesized silica nanoparticles and silica nanoparticle-based fluorescent probe, Compound 4b (hydroxy-terminated silica nanoparticles), Compound 4c (isocyanate-terminated silica nanoparticles), compounds dispersed in distilled water Charge 4d (azide terminated silica nanoparticles), 4a and 4 (20 μL each) and distilled water into a Malverns folded capillary cell and charge the nanoparticle surface using a Zetasizer Nano Z instrument (Malven Instruments, Malvern, UK). The surface modification change was qualitatively confirmed by measuring, and each zetapotential (ZP) is shown in FIG. 2.

Example 12 Confirmation of Absorption and Fluorescence Properties of the Fluorescent Probe

In order to determine the fluorescence characteristics of Compound 2 (KP1 ^Pr ), Compound 3 (KP1 ^Bi ), and Compound 4 (KP1 ^NP ) of the present invention, the following experiment was performed. In this case, as a control, the following known compound 1 (KP1) was used.

[Compound 1]

Specifically, in order to confirm the absorption and fluorescence characteristics of the two-photon absorbing phosphor, PBS (phosphate buffer silane) of compounds 1, 1a, 2, 2a, 3, 3a and 1 mg / mL concentration of compounds 4, 4a at 10 μM concentration Buffer solution (containing 10 mM, pH 7.4, 1% dimethyl sulfoxide (DMSO)) in a 1 cm pass length quartz cell to measure the UV / Vis absorption spectrum and measure the wavelength (x-axis) Absorbance (y-axis) according to the results are shown in Figure 3a. In addition, the fluorescence emission spectrum (Photon Technical International Fluorescence System) was measured, and the results of fluorescence intensity (y-axis) according to the wavelength (x-axis) are shown in FIG. 3B.

In this case, compounds 1a, 2a, 3a, and 4a are comparative substances in which alkyls are introduced into -OH of compounds 1, 2, 3, and 4 to remove intramolecular hydrogen bonds. As shown in FIG. 3A, compounds 1, 2, 3 and 4 all exhibited the highest absorption values at 370-390 nm. In addition, as shown in FIG. 3B, compounds 1a, 2a, 3a, and 4a emit strong fluorescence, while compounds 1, 2, 3, and 4 show little fluorescence. In addition, in the case of Compound 2 (KP1 ^Pr ) and 3 (KP1 ^Bi ), a greater fluorescence increase was observed in comparison to Control Compound 1 (KP1), which was developed in the past, which provides improved sensitivity in cancer diagnosis and imaging. It means you can.

Also, 100% PBS buffer solution (100 mM, pH 7.4), PBS buffer solution with 1% acetonitrile (MeCN), PBS buffer solution with 10% acetonitrile, PBS with 20% acetonitrile The fluorescence spectrum of Compound 4a at a concentration of 1 mg / mL in the buffer solution was measured, and the fluorescence intensity (y-axis) according to the wavelength (x-axis) is shown in FIG. 4. As a result, the fluorescence intensity increased as the proportion of the organic solvent increased, which confirms that the fluorescence intensity of the fluorescent probe increases as the hydrophilic environment is changed from the hydrophilic environment.

In addition, the fluorescence emission spectrum of PBS buffer solution (10 mM, pH 7.4, 20% MeCN) of compound 1, 1a, 2, 2a and compound 4, 4a at concentrations of 10 μM is shown in FIG. 5. Indicated. As a result, in spite of the increase of the ratio of the organic solvent, compared to the control compound 1 (KP1) developed in the past, the greater fluorescence in the case of the present compound 2 (KP1 ^Pr ) and compound 4 (KP1 ^NP or nano-KP1) An increase could be identified, which means that the compounds of the present invention can provide improved sensitivity in cancer diagnosis and imaging implementation.

Example  13: compound 2 ( KP1 ^Pr )of PDGFRa  Phosphorylase Detection Characteristics

The binding selectivity of PDGFRa phosphatase with respect to compound 2 (KP1 ^Pr ) and control compound 1 (KP1) of the present invention was compared and confirmed by fluorescence change, and specific experimental methods are as follows.

The solvent used in the experiment was a buffer solution (10 mM HEPES buffer, pH 7.4), and the kinase was used as a product of Carna Biosciences, and each kinase was a solvent provided by the manufacturer (50 mM Tris-HCl, 150). mM NaCl, 0.1% CHAPS, 1 mM DTT, 10% Glycerol, pH 7.5) and stored at -80 ° C. The ATP and MgCl ₂ used were made from Sigma. The plate used for fluorescence observation was a 96-well fluorescence assay plate (SPL Life Science Co., Ltd.), and the observation equipment was a VCITOR 3 multilabel counter (Perkin Elmer-Wellesley Co., Ltd.). A 355 nm filter was used as the excitation wavelength of the observation equipment, and a 535 nm filter was used as the emission wavelength. The incubation was used NB-205Q model (N-BIOTEK Co., Ltd.), and proceeded with shaking at 170 rpm for 1 hour at 37 ℃. Compound 1 (KP1) and Compound 2 (KP1 ^Pr ) were used in 10 mM dissolved in DMSO solution and controlled to have the same amount of DMSO (less than 1%) under the solvent conditions used. The Mg ^{2 +} and ATP used was 100 μM each condition. Each experimental value was based on the average of five experiments conducted through the same experimental preparation process, and the amount of phosphatase (PDGFRa) used was 0.2 μg / mL.

As a result of measuring the fluorescence intensity before and after 1 hour of reaction with the phosphatase of Compound 1 and Compound 2 at 100 μM concentration, as shown in FIG. Since the fluorescence intensity represented by Compound 1 (KP1) itself before the reaction is greater than that represented by Compound 2 (KP1 ^Pr ) itself, consequently, when Compound 2 (KP1 ^Pr ) is applied to bioimaging, it is clearer than Compound 1 (KP1). You will be able to get the video.

Example 14: Photon and Two-Photon Cell Imaging by Treatment of Compounds 2, 3, and 4

For Compounds 2, 3, 4 and Control Compound 1 of the present invention, various normal cells (PNT-2, HEK-293A) and cancer cells (MCF-7, PC3, A549) were treated with a one-photon or two-photon optical microscope. The fluorescence on phenomenon was confirmed, and the specific experimental method is as follows.

First, the photon microscopy fluorescence image consists of an upright confocal microscope (TCS SP5 II MP with SMD, Leica Microsystems Ltd.) and a 40x objective (HCX PL APO 40x / 1.10 W CORRCS, 506341, Leica, Germany). It was taken at 405 nm photon excitation wavelength. In this case, the horizontal and vertical length of the image is 123 μm or 369 μm, and the scale bar is 25 μm or 75 μm.

The two-photon optical microscope consists of an upright microscope (BX51, Olypmpus) and a 20x objective (XLUMPLEN, NA 1.0, Olympus). ) Was used, and a laser power of 6 mW and a two-photon excitation wavelength of 780 nm were observed. In this case, the horizontal and vertical lengths of the image are 150 μm, respectively, and the scale bar is 50 μm.

PNT-2 (human prostate epithelial cell), HEK-293A (human kidney cell), two kinds of normal cells, MCF-7 (human breast cancer cell), PC3 (human prostate cancer) Cells), A549 (human lung cancer cells) was used. Each cell was incubated at 37 ° C. in Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute medium (RPMI). The cultured cells were incubated at 37 ° C. for 24 hours to produce a cell number of about 1 × 10 ⁵ again in a 12 mm petri dish. Thereafter, 10 μM of Compounds 1, 2, 3 and 1 mg / mL of Compound 4 were treated, followed by an additional 1 hour of incubation, and then the culture medium was removed from the cells and 4% formaldehyde solution was added to the cells. Fixed on.

As a result, as shown in Figs. 7, 8 and 11, not only increased fluorescence selectively appeared in the cancer cells treated with Compound 2 (KP1 ^Pr ), but also Compound 2 (KP1 ^Pr ) than Compound 1 (KP1) in cancer cells. It was confirmed that the fluorescence intensity of) increased more significantly. It can be seen that the compound 2 (KP1 ^Pr ) of the present invention binds to phosphatase present in cancer cells better and exhibits strong fluorescence, thereby confirming excellent sensitivity of cancer diagnosis and imaging.

In addition, as shown in Figures 7, 9 and 11, not only did fluorescence increase selectively for cancer cells treated with Compound 3 (KP1 ^Bi ), but also Compound 3 (KP1 ^Bi ) than Compound 1 (KP1) in cancer cells. It was confirmed that the fluorescence intensity of increased significantly. It can be seen that the compound 3 (KP1 ^Bi ) of the present invention binds to phosphatase present in cancer cells better and exhibits strong fluorescence, thereby confirming excellent sensitivity of cancer diagnosis and imaging.

In addition, as shown in FIGS. 7, 10 and 11, not only did the fluorescence increase selectively in cancer cells treated with Compound 4 (KP1 ^NP or nano-KP1), but also Compound 4 in the cancer cells than Compound 1 (KP1). It was confirmed that the fluorescence intensity of increased significantly. It can be seen that Compound 4 of the present invention binds to phosphatase present in cancer cells better and exhibits strong fluorescence, thereby confirming excellent sensitivity of cancer diagnosis and imaging.

Example  15: compound 3 ( KP1 ^Bi Two-photon Optical Microscopy Imaging of Treated Mouse Tissues

For the compound 3 (KP1 ^Bi ) of the present invention, the following experiment was performed for two-photon optical microscopy imaging of the tissue of the animal model.

First, rats were reared for about 8 weeks by treatment with azoxymethane (AOM) and dextran sodium sulfate (DSS) to express colorectal cancer. The colon tissue formed was extracted. Then spread well using surgical scissors to reveal the inner lumen of colon tissue and buffer PBS with 100 μM Compound 3 (KP1 ^Bi ) (100 mM, pH 7.4, 1% DMSO) The solution was soaked for about 30 minutes to allow it to be absorbed and soaked into the tissue. Then, washed three times with PBS solution to remove the compound 3 (KP1 ^Bi ) remaining on the surface. The prepared colonic tissue was spread well so that the inner surface of the colonic tissue was visible on the slide glass, and then covered with a coverslip having a thickness of 170 μm and subjected to two-photon excitation laser-based microscopy. As a control, a normal colon colon tissue was also prepared and photographed. At this time, the two-photon optical microscope image was observed with a laser output of 6 mW and a two-photon excitation wavelength of 780 nm, the horizontal and vertical length of the image is 300 μm, respectively, the scale bar bar means a length of 100 μm.

As a result, as shown in Figure 12, bright fluorescent images from cancer tissues were observed as compared to a normal tissue treated with Compound 3 (KP1 ^Bi), thus compound 3 (KP1 ^Bi) is with optionally kinases in the cancer tissue The binding was found to be strongly observed fluorescence.

Example  16: compound 1 (KP1) and compound 4 (KP1 ^NP  Or nano-KP1)  Two-photon Optical Microscope Imaging Of Managed Living Mouse Tissues

For Compound 4 (KP1 ^NP or nano-KP1) and Compound 1 (KP1) of the present invention, the following experiments were performed for two-photon optical microscopy imaging of tissues of living animal models.

First, about 5 × 10 ⁶ A549 (human lung cancer cells) cells obtained by the method of Example 14 were left thighed on thymus-depleted mice (5 weeks old, female, BALB / c-nude mice, Orient BIO Inc.). Cancer was expressed by subcutaneous injection into the leg, and imaging studies were performed 7 days after cancer cell injection. All experiments were conducted with respiratory anesthesia maintained, either Compound 1 (100 μM / 100 μL) or PBS buffer solution (100 mM, pH 7.4, 1) dissolved in PBS buffer solution (100 mM, pH 7.4, with 1% DMSO). Compound 4 (1 mg / 100 μL, 1 mg / mouse) dispersed in% DMSO was injected into the rat via tail vein injection. Two-photon-excited laser-based microscopy was performed 15 minutes after injection of Compound 1 or Compound 4, and tail vein injection of PBS buffer solution (100 mM, pH 7.4, 1% DMSO) was injected into cancer-expressing mice in the same manner as a control. Tissue imaging was performed. At this time, the two-photon optical microscope image was observed with a laser output of 52.5 mW and a two-photon excitation wavelength of 780 nm, the horizontal and vertical length of the image is 300 μm, respectively, the scale bar bar is 50 μm or 100 μm length.

As a result, as shown in Fig. 13, the mice injected with tail vein of PBS buffer solution as a control showed little change in fluorescence in normal tissue and cancer tissue. On the other hand, much brighter fluorescence images were observed in cancer tissues compared to the normal tissues of rats treated with Compound 1 or Compound 4, so Compound 1 or Compound 4 was selectively combined with phosphorylation enzymes in cancer tissues, resulting in strong fluorescence. It could be known. In addition, when comparing the results of Compound 1 and Compound 4 studies, Compound 4 (KP1 ^NP or nano-KP1), an advanced form of Compound 1 (KP1), was shown to have cancer cell hits due to increased permeation and residual effect (EPR effect). targeting) effect and fluorescence increase effect was higher than compound 1.

Example  17: of living rat tissue with cancer Optical coherence  Tomography and Angiography

A wavelength swept source (SSOCT-1310, AXSUN Technologies) was used as the light source for optical coherence tomography in living rat tissues.The wavelength was 500 μm × 500 μm × 225 μm with a central wavelength of 1310 nm and a wavelength of 107 nm. An image of the size was obtained. Vascular images were obtained by analyzing the previously obtained images with a complex differential variance algorithm.

As a result, as shown in Figure 14, it can be seen that the structural image and the blood vessel image of the normal tissue (control) appears in a certain manner. On the other hand, structural images of cancer tissues are irregular, and blood vessel images show that blood vessels are overexpressed around cancer tissues. For this reason, it turns out that the injected cancer cell is effectively expressed in the mouse.

Example  18 compound 1 (KP1) and compound 4 (KP1 ^NP  Or nano-KP1)  Two-photon Optical Microscope Imaging of Treated Mouse Tissues

The following experiments were performed for two-photon optical microscopy imaging of rat tissues treated with Compound 4 (KP1 ^NP or nano-KP1) of the present invention.

First, after treating Compound 4 (experimental group) and PBS buffer solution (control group) from Example 16 to obtain a two-photon optical microscope image, the rats were immediately killed by cervical dislocation, and then 4% paraformaldehyde. Tissues are fixed by perfusion from the heart with (paraformaldehyde) solution. The brain, kidney, lung, spleen, liver and cancer tissues of rats were dissected and washed three times with PBS buffer solution before tissue imaging. At this time, the two-photon optical microscope image was observed with a laser output of 35 mW and a two-photon excitation wavelength of 780 nm, the horizontal and vertical length of the image is 300 μm, respectively, the scale bar bar means a length of 50 μm.

The fluorescence intensity of the experimental group treated with Compound 4 relative to the fluorescence intensity of the control tissue treated with the PBS buffer solution is shown in FIG. 15. As a result, it was confirmed that Compound 4 was selectively distributed in the cancer tissue.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Since the fluorescent probe of the present invention has advanced detection characteristics indicating improved sensitivity and selectivity in detecting phosphatase compared to conventional KP1 compounds, it can be effectively used for selective imaging of cancer tissues and immobilized on nanoparticles. This can be applied to chip-based assays, and from this, it has the effect of providing a material necessary for application as a sensing device.

In addition, due to its two-photon excitation properties, it is not only susceptible to deep cell permeability, low cell disruption, and interference from autofluorescent materials in vivo, but also excites only the focal region, so that a very high resolution can be realized. Can be used.

Claims (15)

A fluorescent probe composition for detecting tyrosine kinase, comprising a compound of Formula 5 or analogues thereof. [Formula 5]

In Chemical Formula 5, R ₁ and R ₂ each independently represent hydrogen, C _{1- 10} alkyl, aryl, acetate, or chewy profile; R ₃ represents a formyl group or acetal group; R ₄ represents hydrogen, acetyl, methyl, or methoxymethyl group; With the exception that R ₁ and R ₂ are methyl, R ₃ is formyl, and R ₄ is hydrogen. The method of claim 1, wherein the analogue is characterized in that the cyclic arginine-glycine-aspartate peptide, folic acid or biotin is further linked to the compound of formula (5), fluorescent probe composition. The method of claim 1, wherein the analogue is characterized in that the silica nanoparticles (silica nanoparticles), paramagnetic nanoparticles (paramagnetic nanoparticles), or ferromagnetic nanoparticles (ferromagnetic nanoparticles) is further connected to the compound of Formula 5 Probe composition. The fluorescent probe composition of claim 1, wherein the analog further has a microarray chip connected to the compound of Formula 5 as a solid support. According to claim 1, wherein the compound is characterized in that the compound of Formula 2, fluorescent probe composition. [Formula 2]

According to claim 2, The analog is characterized in that the compound of formula 3, fluorescent probe composition. [Formula 3]

The fluorescent probe composition of claim 3, wherein the analog is a compound represented by the following Chemical Formula 4. [Formula 4]

The fluorescent probe composition of claim 1, wherein the compound or analog is a one or two photon absorbing phosphor. The fluorescent probe composition of claim 1, wherein the tyrosine kinase is selected from the group consisting of PDGFRa, Src, ABL1 (T315I), BRAF, RSK2, and TYK2. A method of imaging a cell or tissue using the fluorescent probe composition of claim 1. The method of claim 10, wherein the method comprises measuring fluorescence in combination with a tyrosine kinase overexpressed in the cell or tissue and the compound. The method of claim 11, wherein the fluorescence is measured by a one-photon optical microscope or a two-photon optical microscope. The method of claim 10, wherein the cells or tissues are cancer cells or cancer tissues. Method for providing information for cancer diagnosis using the fluorescent probe composition of claim 1. 15. The method of claim 14, comprising treating the fluorescent probe composition to a sample and then determining the cancer if fluorescence is observed.

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