KR102273736B1 - Dual recognition two-photon fluorescent probe and uses thereof - Google Patents

Abstract

The present invention relates to a tumor-specific dual recognition two-photon fluorescent probe and a cancer disease diagnosis method using the same, and more particularly, to a two-photon fluorescent probe in which D-glucosamine and β-D-galactopyranoside are combined as a double recognition site. It can be selectively absorbed into cancer cells through glucose receptors overexpressed in cancer cells, and exhibits fluorescence and color change through abnormally upregulated β-galactosidase (β-gal) hydrolase activity in cancer cells, The dual recognition two-photon fluorescent probe of the present invention provides accurate and effective cancer disease diagnosis information in cancer disease treatment as it is possible to diagnose cancer and confirm the stage of cancer disease through the color change ratio and color intensity of the two-photon fluorescent probe. can provide

Description

Dual recognition two-photon fluorescent probe and uses thereof

The present invention relates to a tumor-specific dual recognition two-photon fluorescent probe and a cancer disease diagnosis method using the same.

Colon cancer is pathologically adenocarcinoma, and is largely divided into colon cancer and rectal cancer by site. The frequency of occurrence by region is the lower colon, that is, the rectum is the highest at about 50%, and the incidence and mortality rates of colorectal cancer are increasing remarkably in Korea as well due to changes in dietary habits in recent years.

Although the cause of colorectal cancer is still unclear, genetic factors, dietary habits related to intake of high-fat and low-fiber foods, and inflammatory bowel disease are being considered. Colorectal cancer can occur in any age group, but the incidence increases with age, and it occurs frequently in those in their 50s and 60s.

The treatment of colorectal cancer is based on surgical resection, and chemotherapy and radiation therapy are combined. Diagnosis of colorectal cancer is performed by rectal digital examination, fecal occult blood test, and colonography in patients with colon-related symptoms, and if necessary, biopsy through colonoscopy is performed. Currently, the earliest detection method available is a test for fecal blood or an endoscopic procedure.

However, typically significant tumor size must be present until fecal blood is detected. The sensitivity of the fecal test based on occult blood is 26%, meaning that 74% of patients with malignant lesions remain undetected. Visualization of precancerous and cancerous lesions is the best approach for early detection, but colonoscopy has problems that can cause considerable cost as well as pain and risk to the patient. In addition, conventional methods have a problem in that diagnosis accuracy is low, and early diagnosis of patients before the onset of colorectal cancer is impossible, and inconvenient to subjects.

Cancer cells usually have a higher rate of glucose uptake due to upregulation of glucose transporters (GLUTs), resulting in a higher rate of glycolysis and more energy production. Positron emission tomography (PET) using a radiation tracer such as ^{2-deoxy-2-[ 18} F]fluoroglucose ( ¹⁸ F-FDG) is being performed to monitor glucose uptake by these cancer cells. There are spatial and temporal constraints in terms of resolution. As an alternative to solve this problem, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D- as a fluorescent tracer for single cells and real-time observation Since the discovery of glucose (2-NBDG), various small molecules have been developed to confirm glucose uptake.

Most of these probes contain a D-glucosamine unit, a D-glucose analog, to detect cancer cells by increasing fluorescence intensity through fast GLUT-specific uptake. However, their application was limited due to inaccuracies caused by experimental artifacts such as passive diffusion in normal cells and changes in laser power and probe concentration.

Republic of Korea Patent Publication No. 10-2017-0052399 (published on May 12, 2017)

An object of the present invention is to provide a two-photon fluorescent probe that selectively doubles recognition of cancer cells using a glucose receptor overexpressed in cancer cells and an intracellular β-galactosidase reaction, and a cancer disease diagnosis method using the same.

The present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, X is O, NH, S or Se,

R is selected from the group consisting of Formula 2, Formula 3, Formula 4, and Formula 5.

[Formula 2]

[Formula 3]

In Formula 3, Y is O or NH.

[Formula 4]

[Formula 5]

In Formulas 4 and 5, Y is O or NH, and n is an integer of 1 to 50.

The present invention contains a compound represented by Formula 1, wherein the compound comprises a glucose receptor (Glucose transporters; GLUT) specific recognition site, a two-photon fluorescent probe, and a β-galactosidase reaction site. A dual recognition two-photon fluorescent probe to provide.

The present invention comprises the steps of treating a sample isolated from a human with the dual recognition two-photon fluorescent probe; irradiating excitation light to the sample treated with the double recognition two-photon fluorescent probe; There is provided a method for providing information for diagnosing cancer diseases, comprising the step of confirming an increase in the intensity of color and the rate of color change generated from the fluorescent probe in the sample irradiated with the excitation light.

The present invention provides a photodynamic therapy for preventing or treating cancer diseases, comprising the dual recognition two-photon fluorescent probe and a photosensitizer as active ingredients.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer diseases containing the dual recognition two-photon fluorescent probe and an anticancer agent as active ingredients.

According to the present invention, a two-photon fluorescent probe in which D-glucosamine and β-D-galactopyranoside are combined as a double recognition site for a cancer cell target can be selectively absorbed into cancer cells through a glucose receptor overexpressed in cancer cells, It exhibits fluorescence and color change through abnormally up-regulated β-galactosidase (β-gal) hydrolase activity in cancer cells, and diagnosis of cancer and disease through the color change rate and color intensity of the two-photon fluorescent probe. As it is possible to confirm the stage of cancer disease, the dual recognition two-photon fluorescent probe of the present invention can provide accurate and effective cancer disease diagnosis information in cancer disease treatment.

1 is a result of confirming the enzymatic reaction of a double recognition two-photon fluorescent probe (SGG) and β-galactosidase (β-gal), FIG. 1 (a) is SGG according to the addition of β-gal (1 unit/mL). 1(b) is the result of confirming the fluorescence response of SGG in a buffer containing various biological species when irradiated with 363 nm excitation light, and the bar indicates that each biological species was treated for 1 to 6 hours. The F ₅₂₅ /F ₄₅₃ fluorescence ratio after treatment is shown (n = 10).
2 shows colorectal cancer cells (HT-29 and HCT 116) and normal cells (CCD-18Co) treated with or without L-glucose (L-Glc) or D-glucose (D-Glc) at a concentration of 55 mM. As a result, FIG. 2(a) is a two-photon microscope (TPM) image, and FIG. 2(b) is a result of confirming the intensity of two-photon-excited fluorescence (TPEF).
3(a) and 3(b) are the results of confirming the TPM fluorescence ratio images of SGG-labeled lacZ-negative HCT 116 and lacZ-positive HCT 116, and FIG. 3(c) is SGG-labeled lacZ-negative HCT 116. , Results showing _{the F green} / F _blue ratio of lacZ-positive HCT 116 and GS-labeled HCT 116.
Figure 4 is a result of confirming the level of SGG uptake in the cell, Figure 4 (a) and Figure 4 (b) are real-time SGG (5 μM) in colonic normal cells (CCD-18Co) and colorectal cancer cells (HT-29) It is a TPM image confirming absorption, and FIG. 4(c) is a result of confirming the relative TPEF intensity of the intracellular probe at an interval of 2 seconds (n = 10).
Figure 5 is the result of confirming the TPM image of SGG in colorectal cancer cells (HT-29, RKO and HCT 116) and normal cells (CCD-18Co), Figure 5 (a) is obtained from the central channel (400-600 nm) TPM image results, Figure 5 (b) is a result of confirming the TPM fluorescence ratio image, Figure 5 (c) is a result showing a box plot of the standardized TPEF intensity, Figure 5 (d) is the standardized F _green /F _{It is a} result showing the blue ratio, and FIG. 5(e) is a result showing a plot of intensity versus ratio.
6(a) to 6(c) are TPM images confirmed in the entire channel (400-600 nm), and FIGS. 6(d) to 6(f) are TPM fluorescence ratio images (F _green /F _blue ) As a result of confirming, a to d are the results of confirming human colon normal tissue (a and d), adenoma (b and e), and carcinoma (c and f) tissue after incubation with SGG (10 μM) for 40 minutes, FIG. 6 (g) shows the standardized TPEF intensity, FIG. 6(h) is _{the result of confirming the standardized F green} /F _blue ratio, and FIG. 6(i) is the result showing the intensity versus ratio plot of each tissue.

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

The present invention may provide a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, X is O, NH, S or Se,

R is selected from the group consisting of Formula 2, Formula 3, Formula 4, and Formula 5.

[Formula 2]

[Formula 3]

In Formula 3, Y is O or NH.

[Formula 4]

[Formula 5]

In Formulas 4 and 5, Y is O or NH, and n is an integer of 1 to 50.

In more detail, the compound represented by Formula 1 may be a compound represented by Formula 6, Formula 7, Formula 8, and Formula 9 below.

[Formula 6]

In Formula 1, X is O, NH, S or Se.

[Formula 7]

In Formula 7, X is O, NH, S or Se, and Y is O or NH.

[Formula 8]

[Formula 9]

In Formulas 8 and 9, X is O, NH, S or Se, Y is O or NH, and n is an integer of 1 to 50.

The present invention contains a compound represented by Formula 1, wherein the compound comprises a glucose receptor (Glucose transporters; GLUTs) specific recognition site, a two-photon fluorescent probe, and a β-galactosidase reaction site. A dual recognition two-photon fluorescent probe comprising a can provide

More preferably, it may be a dual recognition two-photon fluorescent probe represented by the following Chemical Formula 10, but is not limited thereto.

[Formula 10]

The glucose receptor (GLUTs) specific recognition site may bind to a carboxyl group of a two-photon fluorescent probe, and may be selected from the group consisting of D-glucosamine and D-glucopyranoside.

The β-galactosidase reaction site may bind to the amine group of the two-photon fluorescent probe, more preferably β-D-galactopyranoside, but is not limited thereto.

The probe may be selectively absorbed into tumor cells through glucose transporters (GLUTs).

The probe may exhibit a color change by a hydrolase reaction of β-galactosidase (β-gal) in tumor cells.

The present invention comprises the steps of treating a sample isolated from a human with the dual recognition two-photon fluorescent probe; irradiating excitation light to the sample treated with the double recognition two-photon fluorescent probe; There is provided a method for providing information for diagnosing cancer diseases, comprising the step of confirming an increase in the intensity of color and the rate of color change generated from the fluorescent probe in the sample irradiated with the excitation light.

The excitation light may have a wavelength in the range of 400 to 600 nm.

The color change rate may be to determine the rate at which the emission color of the two-photon fluorescent probe changes from blue to green by the hydrolase reaction of β-galactosidase in the sample.

The cancer disease may be selected from the group consisting of colon cancer, breast cancer, liver cancer, lung cancer, uterine cancer, pancreatic cancer, kidney cancer, stomach cancer and brain cancer, but is not limited thereto.

The present invention provides a photodynamic therapeutic agent for preventing or treating cancer diseases, comprising the dual recognition two-photon fluorescent probe and a photosensitizer as active ingredients.

The photodynamic therapeutic agent may be in the form of a photosensitizer coupled to a dual recognition two-photon fluorescent probe composed of the compound represented by Formula 1 above.

The photosensitizer may be selected from the group consisting of porphyrins and chlorines, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer diseases containing the dual recognition two-photon fluorescent probe and an anticancer agent as active ingredients.

The pharmaceutical composition may be in a form in which a photosensitizer is bound to a dual recognition two-photon fluorescent probe composed of a compound represented by Formula 1.

The anticancer agent may be used without limitation as long as it is used for conventional anticancer treatment, and the connection between the anticancer agent and the probe may be performed through methods known in the art, for example, covalent bonding, treatment, and the like.

The composition for diagnosing or treating cancer diseases of the present invention further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes, for example, an excipient, a diluent, and the like, and is well known in the art. The selection of the carrier may be determined according to the specific dosage form to be prepared and the specific administration method of the composition. Accordingly, the composition for diagnosing or treating cancer diseases of the present invention may exist as a formulation in a wide variety of forms. In addition, the composition for diagnosing or treating cancer diseases of the present invention may be administered by an appropriate administration method according to its use or type of active ingredient.

The dosage of the composition for diagnosing or treating cancer diseases of the present invention may vary depending on the type of disease to be diagnosed or treated, the degree of disease, the target tissue or organ to be diagnosed, and the characteristics of the diagnostic device, and the dosage may be It may increase or decrease depending on the age, sex, weight, race, etc. of the subject to be administered.

A preferred method of administering the composition for diagnosing or treating cancer diseases of the present invention is parenteral, for example, bolus injection, intravenous injection, intra-arterial injection, or a spray, for example, spraying an aerosol when the lungs are to be contrasted, Oral or rectal administration can also be used, but it is also possible with a known contrast medium administration method. Formulations for parenteral administration must be sterile and free from physiologically unacceptable substances and paramagnetic, superparamagnetic, ferromagnetic, or semiferromagnetic contaminants, including preservatives, antibacterial agents, buffers and antioxidants commonly used in parenteral solutions; It may contain excipients, and may further contain other optional additives that do not interfere with molecular imaging.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Reference example>

Reactions were performed in flame-dried round-bottom flasks and magnetic stirrers under a nitrogen atmosphere. All compounds were purchased from Sigma-Aldrich, and solvents were distilled before use. Thin layer chromatography (TLC) glass pates (TLC Silica gel 60 F254, 1.05715.0001, Merck) were used to monitor the reaction process.

The final product was purified by medium-pressure liquid chromatography (AI-580S, YAMAZEN) using a general-purpose or reversed-phase C-18 column cartridge.

An NMR spectrum was obtained using a 600 MHz NMR spectrometer (JNM-ECZ 600R, JEOL). High-resolution mass spectrometry was performed using the HR-MS system (Accela UPLC/LTQ-Orbitrap XL, Thermo Fisher Scientific) of Gyeonggi-do Business & Science Accelerator.

<Synthesis Example> Synthesis of compound SGG

Compounds 1, 2 and 4 were prepared by previously reported methods (CS Lim, et. al., Chem. Soc. 2011, 133, 11132-11135; JA Carmona, et. al., Org. Biomol. Chem. 2017, 15, 2993-3005; HW Lee, et. al., Anal. Chem. 2014, 86, 10001-10005.), and other compounds were synthesized according to the following scheme.

[Scheme 1]

1. Synthesis of compound GS

Compound 1 (14.8 mg, 0.044 mmol, 1.0 eq.) and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate [N,N, N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HBTU, 25.2 mg, 0.066 mmol, 1.5 eq.] in DMF (0.4 mL) was treated with DIPEA (15 μL, 0.09 mmol, 2 eq.), and the reaction mixture was stirred at 25° C. for 1 hour. D-(+)-glucosamine hydrochloride [D-(+)-glucosamine hydrochloride; 10.4 mg, 0.048 mmol, 1.1 eq.] and further stirred at 25° C. for 3 hours.

The reaction mixture was _{treated with saturated aqueous NaHCO 3} to terminate the reaction and diluted with EtOAc. After separating the layers, the aqueous layer _{was extracted with a premixed solution (CHCl 3} :i-PrOH = 3:1). The combined organic layer was washed with brine, dried over an excess of anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude residue was purified by column chromatography (10 to 20% MeOH in DCM) to obtain GS compound (25.3 mg, 71%) in the form of a yellow semi-solid.

¹ H NMR (DMSO-d ₆ , 600 MHz): δ (ppm) 8.68 (s, 1H), 8.44 (s, 1H), 8.20 (d, J = 6.9 Hz, 1H), 8.05-8.01 (m, 3H) ), 7.83 (d, J = 9.6 Hz, 1H), 7.73 (d, J = 9.6 Hz, 1H), 7.04 (d, J = 6.9 Hz, 1H), 6.73 (s, 1H), 6.48 (d, J) = 4.1 Hz, 1H), 6.43 (t, J = 4.8 Hz, 1H), 5.12 (d, J = 4.1 Hz, 1H), 4.97 (d, J = 3.4 Hz, 1H), 4.75 (d, J = 5.5) Hz, 1H), 4.45 (d, J = 5.5 Hz, 1H), 3.86-3.76 (m, 2H), 3.66 (m, 2H), 3.55-3.42 (m, 1H), 3.23-3.20 (m, 1H) , 2.82 (d, J = 4.1 Hz, 3H); HRMS (ESI ⁺ ): m/z calculated for [C ₂₅ H ₂₆ O ₆ N ₃ S] ⁺ : 496.1537, found: 496.1545.

2. Synthesis of compound 3

Compound 1 (432 mg, 1.29 mmol, 1.0 eq.), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride [N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; EDCI, 297 mg, 1.55 mmol, 1.2 eq.] and 1-hydroxybenzotriazole hydrate [1-hydroxybenzotriazole hydrate; HOBt, 209 mg, 1.55 mmol, 1.2 eq.] in DMF (6.4 mL) was treated with DIPEA (1.12 mL, 6.47 mmol, 5.0 eq.) and stirred at 25° C. for 30 minutes.

Compound 2 (981 mg, 1.55 mmol, 1.2 eq.) was added to the reaction mixture, and after stirring at 25° C. for an additional 17 hours, the reaction mixture was quenched with _{saturated aqueous NaHCO 3 , diluted with EtOAc and} extracted. The combined organic layer was washed with brine, dried over an excess of anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

The residue was purified by column chromatography (2% MeOH in DCM) to obtain compound 3 (948 mg, 80%) in the form of a green semi-solid.

¹ H NMR (CDCl ₃ , 600 MHz): δ (ppm) 8.29 (s, 1H), 8.14-8.12 (m, 3H), 8.06-8.05 (m, 2H), 7.97-7.92 (m, 5H), 7.81 (d, J = 8.2 Hz, 1H), 7.67-7.63 (m, 2H), 7.59-7.53 (m, 3H), 7.50-7.40 (m, 7H), 7.36-7.30 (m, 4H), 6.88 (d , J = 8.2 Hz, 1H), 6.78-6.75 (m, 2H), 6.29 (d, J = 8.2 Hz, 1H), 5.96-5.87 (m, 2H), 5.13 (d, J = 9.6 Hz, 1H) , 4.67 (dd, J = 12.3, 2.7 Hz, 1H), 4.54 (q, J = 5.5 Hz, 1H), 4.38 (t, J = 4.8 Hz, 1H), 2.96 (s, 3H) ; HRMS (ESI ⁺ ): m/z calculated for [C ₅₃ H ₄₂ O ₁₀ N ₃ S] ⁺ : 912.2585, found: 912.2562.

3. Synthesis of compound SGG

Step i: DCM (4.1 mL) was dissolved and a cooled solution of compound 3 (740 mg, 0.811 mmol, 1.0 eq.) was treated with pyridine (0.2 mL, 2.44 mmol, 3.0 eq.) and stirred at the same temperature for 30 minutes. . Chloroformate 4 (637 mg, 1.23 mmol, 1.5 eq.) dissolved in DCM (25 mL) was added to the mixed solution and stirred at 0° C. for 1 hour.

Then, the reaction mixture was quenched with saturated aqueous NaHCO _{3 , diluted with DCM and extracted.}

The combined organic layers were washed with brine, dried over an excess of anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography (2 to 3% MeOH in DCM) to obtain a yellow semi-solid intermediate (719 mg, 64%).

Step ii: The intermediate (121 mg, 0.094 mmol, 1.0 eq. _{) was dissolved in MeOH:H 2} O (5:1), TEA was added and stirred at 100° C. for 8.5 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure.

The residue was purified by a prepared C18 column (2 × 25 cm, 25 % distilled water/0.1% TFA in MeOH) to obtain SGG TFA salt (40.1 mg) as a yellow solid.

A solution in which the SGG TFA salt (40.1 mg) was dissolved in MeOH (30 mL) was stirred, and a base resin (Amberlite® IRA-67 free base, 200 mg, 500 wt% of SGG TFA salt) was added, and the resulting mixture was 25 The mixture was stirred at ℃ for 30 minutes.

The mixture was filtered through a pad of Celite, filtered, and concentrated in vacuo to obtain the compound SGG (23.7 mg, 34%) in a semi-solid state. ¹ H NMR (DMSO-d ₆ , 600 MHz): δ (ppm) 8.82 (s, 1H), 8.73 (s, 1H), 8.24 (d, J = 8.3 Hz, 1H), 8.17-8.14 (m, 3H) ), 8.11 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.94 (s, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.31 (d, J) = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 5.13 (m, 2H), 5.10 (s, 2H), 4.99 (d, J = 7.6 Hz, 1H), 4.82 (m, 3H) ), 4.63 (d, J = 5.2 Hz, 1H), 4.48 (d, J = 4.8 Hz, 1H), 3.69 (m, 2H), 3.57-3.47 (m, 10H), 3.38 (s, 3H), 3.08 (m, 2H); HRMS (ESI ⁺ ): m/z calculated for [C ₃₉ H ₄₂ O ₁₄ N ₃ S] ⁺ : 808.2382, found: 808.2408.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Spectroscopy Analysis

One-photon (OP) fluorescence emission experiment was performed using a FluoroMate FS-2 (SCINCO, Korea) spectrometer and a temperature controller (-10 ~ 80℃), and the one-photon absorption experiment was performed with S-3100 (SCINCO, Korea) was performed using a UV-Vis spectrometer. The irradiation experiment using a spectrometer was performed by preparing a 1.0 cm quartz cuvette (HE.111.650QG, Hellma Analytics).

A multi-detection microplate reader (Varioskan Flash, Thermo Fisher Scientific) and 96-well microplate for fluorescence (164588, Thermo Fisher Scientific) were used for selectivity experiments, enzyme kinetics analysis and pH dependence experiments.

The relative quantum yield (Φ) was previously reported using 9,10-diphenylanthracene (9,10-diphenylanthracene; Φ = 0.93 in cyclohexane) (AM Brouwer, Pure Appl. Chem. 2011, 83, 2213-2228). .) was confirmed.

2. Check solubility in water

Prepare DMSO solutions (10 mM) of SGG and GS, dilute to 0.05-10.0 µM and place in a 1.0 cm quartz cuvette (HE.111.650QG, Hellma Analytics) containing 2.0 mL of PBS buffer (10 mM, pH 7.4). It was added using a syringe.

For all samples, the final concentration of DMSO in the buffer was maintained at 0.1%.

The maximum concentration in the linear region in the fluorescence intensity versus concentration plot of each compound was chosen as the solubility. Linear fitting was performed using a linear function (OriginPro 8.0, OriginLab), and the solubility of SGG and GS in the buffer was about 5.0 μM and 3.0 μM, respectively.

3. Confirmation of Two-Photon Cross Section

Two-photon (TP) absorption efficiency (δ) was confirmed according to the general method (S. K. Lee et al., Org. Lett. 2005, 7, 323-326.). SGG and GS (1.0 μM) were dissolved in buffer (10 mM PBS, pH 7.4), and two-photon excitation emission was confirmed using Rhodamine 6G with well-established TP properties as a reference compound.

The TPEF intensity of each sample and rhodamine 6G was confirmed through the sample excitation wavelength (720 to 880 nm). In addition, the two-photon absorption efficiency was determined using δ = δ _r (S _s Ф _r Ф _r C _r )/(S _r Ф _s Ф _s C _s ). Wherein r and s represent a reference and a sample, respectively, and δ _r is the TP absorption efficiency of rhodamine G6; S is a TPEF signal collected using a CCD system (Monora 320i monochromator with DV401A-BV detector, DONGWOO OPTRON, Korea); Ф denotes the fluorescence quantum yield, Ф denotes the overall fluorescence collection efficiency of the experimental system, and C denotes the concentration of each sample.

4. HPLC analysis

High-performance liquid chromatography (HPLC) was performed using an HPLC system (Alliance 2796, Waters).

5. Selectivity Experiment

The day-photon fluorescence ratio of SGG (1.0 μM) in a buffer containing various biological species was confirmed at 363 nm excited state using a multi-detection microplate reader, and the reagents used are as follows.

1) Enzyme (1 unit/mL): β-galactosidase (β-galactosidase; G5636, Sigma-Aldrich), hNQO1 (DT Diaphorase, D1315, Sigma-Aldrich), ALP (alkaline phosphatase, P4252, Sigma-Aldrich) ), NTR (nitroreductase, N9284, Sigma-Aldrich), Ami (amidase, A6691, Sigma-Aldrich), MAO-B (monoamine oxidase B, M7441, Sigma-Aldrich), hCE1 (human carboxylesterase 1, E0287, Sigma-Aldrich) ), hCE2 (human carboxylesterase 2, C4749, Sigma-Aldrich), AChE (acetylcholinesterase, C1682, Sigma-Aldrich), BChE (butyrylcholinesterase, B4186, Sigma-Aldrich) and β-glucosidase (β-glucosidase; G0395, Sigma) - Aldrich).

2) Reactive species (200 μM): H ₂ O ₂ (hydrogen peroxide), OCl ^- (hypochlorite from NaOCl), OH (hydroxyl radical from Fe ²⁺ + H ₂ O ₂ ), O ₂ ^- (superoxide) from KO ₂ ) and GSH (glutathione, G4251, Sigma-Aldrich).

3) Amino acids (1 mM): Ala (L-alanine, A7627, Sigma-Aldrich), Glu (L-glutamic acid, G1251, Sigma-Aldrich), Gly (glycine, G8898, Sigma-Aldrich), Leu (L- leucine, L8000, Sigma-Aldrich) and Asp (L-aspartic acid, A9256, Sigma-Aldrich)

4) Metal ions (1 mM): Mg ²⁺ , Na ⁺ , Ca ²⁺ and K ⁺ (from the corresponding metal chlorides).

6. Enzyme inhibition analysis and detection limit confirmation

Enzyme inhibitory activity was confirmed in the presence of D-galactose (0, 10, 50 and 100 mM) and an excess of D-glucose (100 mM). The ratio of OP fluorescence of SGG in buffer (10 mM PBS, pH 7.4, 37° C.) was confirmed under 363 nm excitation.

7. Confirmation of enzyme reaction rate and pH effect

Enzyme kinetics analysis was performed using a multi-detection microplate reader. Various concentrations of SGG solutions (0 to 40 μM) were prepared as buffers (10 mM PBS, pH 7.4, 37° C.), β-gal at a final concentration of 2.0 mg/L was used, and the fluorescence intensity was measured at 525 nm for 1 minute. Recordings were made at intervals of 0 to 60 minutes.

The parameters of the Michaelis-Menten reaction rate were obtained by converting the change in intensity into velocity and plotting it as a nonlinear joint through the hyperbolic function (OriginPro 8.0, OriginLab).

8. Cell Culture

All cells were cultured in glass-bottomed dishes (Wuxi NEST Biotechnology) 2 days prior to image acquisition, in 5/95 (v/v) CO ₂ humidified conditions and 37° C. CO ₂ incubator (Water Jacketed 3010, Thermo Fisher Scientific). kept

For labeling, the growth medium was removed and replaced with a glucose-free medium. Cells were incubated with 5 μM SGG or GS for an additional 30 min, washed twice with PBS (Gibco, Thermo Fisher Scientific) and images were obtained.

The growth medium for each cell is as follows.

HT-29 (human colorectal adenocarcinoma cell line, HTB-38, American Type Culture Collection) cells: containing 10% FBS (WelGene, Korea), penicillin (100 units/mL), and streptomycin (100 μg/mL) RPMI (WelGene, Korea) medium.

RKO (human colon carcinoma cell line, CRL-2577, American Type Culture Collection), SW-837 (human rectum adenocarcinoma cell line, CCL-235, American Type Culture Collection), and CCD-18Co (human colon normal cell line, CRL) -1459, American Type Culture Collection) cells: DMEM (WelGene, Korea) medium containing 10% FBS (WelGene, Korea), penicillin (100 units/mL), and streptomycin (100 μg/mL).

HeLa (human cervical adenocarcinoma cell line, CCL-2, American Type Culture Collection) cells: MEM (WelGene) containing 10% FBS (WelGene, Korea), penicillin (100 units/mL) and streptomycin (100 μg/mL) , Korea) badge.

9. Check Cell Viability

According to the manufacturer's instructions using the CCK-8 kit (Cell Counting kit-8, Dojindo), the effect of SGG on the viability of HT-29 and HCT 116 cells was confirmed.

10. Two-photon microscopy (TPM) analysis

TPM imaging experiments of cells and tissues labeled with excited SGG or GS were performed using an ultrafast Ti:sapphire laser (Mai Tai HP, Spectra-Physics) and a multiphoton microscope (TCS SP8 MP with DMI6000B, Leica Microsystems).

The TP excitation wavelength was 740 nm with an average power of 2478 mW, which corresponds to ^{approximately 4.10×10 8} mW cm ^{−2 in the focal plane.}

TPM fluorescence signals were obtained from whole channels (400-600 nm) and ratio channels (Ch _blue : 400-450 nm, Ch _green : 500-600 nm). Ratiometric result processing and analysis were performed using microscope automation and image analysis software (MetaMorph NX, Molecular Devices).

During the TPM experiment, a cell culture system system (Chamlide IC, Live Cell Instrument) was used to maintain suitable conditions (temperature, humidity, CO ₂ and O ₂ ) for long-term imaging and for stability of living cells.

11. Check photostability

The photostability of SGG was confirmed by monitoring the change in TPEF intensity over time of selected SGG-labeled (5 μM) HT-29 cells without bias.

Cells were incubated with 5 μM SGG for 30 min (sufficient β-gal reaction period), washed twice with PBS (Gibco, Thermo Fisher Scientific), and then images were obtained.

The average TPEF intensity was _{almost constant for F blue} (400-450 nm) and F _green (500-600 nm) for 1 hour, indicating that SGG has very high photostability.

12. Glucose Competitive Analysis

In order to determine whether D-glucose is involved in the intracellular uptake of a glucosamine-derived moiety (GS), a glucose competition experiment was performed.

To observe competitive intracellular uptake, various cancer cell lines (SW837, HCT 116, RKO, HeLa) were incubated with GS (5 μM) for 30 min in media containing 0 or 55 mM D-glucose.

The TPEF intensity of the entire channel (400-600 nm) in the intracellular region was compared.

In addition, glucose competition analysis was performed on SGG (5 μM) in colorectal cancer cells and normal cell lines by a similar procedure.

13. lacZ experiment

HCT 116 human colorectal cancer cells (CCL-247, American Type Culture Collection) were treated with MEM (WelGene, Korea) containing 10% FBS (WelGene, Korea), penicillin (100 units/mL) and streptomycin (100 μg/mL). ) in the culture medium.

Two days before imaging, cells were plated on glass bottom dishes (Wuxi NEST Biotechnology). One day before imaging, pcDNA3.1 ⁺ (lacZ-positive cells, Plasmid #29729, Addgene) and BoxB-inserted pcDNA3 (lacZ-negative cells, Plasmid #29727, Addgene) were transduced into cells one day before imaging. .

For labeling, cells and SGG (5 μM) were incubated for 30 min in glucose-free medium. Cells were washed with PBS (PBS, Gibco, Thermo Fisher Scientific) and images were obtained.

14. TPM Image Analysis

Human colon tissues were obtained from 7 different patients at Ajou University Medical Center.

Biopsy sampling was performed according to the guidelines of the Institutional Bioethics Committee (IRB) of Ajou University Hospital (AJIRB-BMR-SMP-17-372).

Experiments were performed to evaluate the progression of cancer using three different types of tissue (normal, adenoma, and carcinoma), and the disease stage was confirmed by a pathologist.

Before imaging, the tissue sections were stained with SGG (10 μM) for 40 min, and for precise analysis, 50 sections with a depth of 80-180 μm in the z-direction were analyzed, and 7,500 ratiometric TPM images were obtained for each tissue section. .

Ratiometric TPM images were obtained from F _blue (400-450 nm) and F _green (500-600 nm) at 40× magnification.

<Example 1> Fabrication of dual recognition two-photon fluorescence probe

A dual recognition two-photon (TP) fluorescent probe (SGG) was synthesized to detect cancer cells in human colorectal cancer tissues and to discriminate between various stages of colorectal cancer (normal, adenoma and tumor).

D-glucosamine and β-D-galactopyranoside were selected as dual recognition units for cancer cell targets, expecting that a dual recognition unit with a ratiometric measurement system in a small molecule would be a sensitive tool for cancer cell analysis. It has been reported that the human β-galactosidase (β-gal) is a hydrolase (EC 3.2.1.23) and is overexpressed in various cancer cells, and exhibits abnormally upregulated activity in colorectal cancer cells.

[Scheme 2]

Accordingly, as shown in Scheme 2 above, SGG is a TP fluorescent probe (1), a GLUT-specific absorption moiety, D-glucosamine unit (2), and a β-gal reaction site, a β-D-galactopyranoside unit ( 4), and the two recognition parts were expected to improve the probe's identification ability in cancer detection.

First, the cell loading capacity of SGG may be higher in cancer cells than in normal cells, due to GLUT-mediated intracellular uptake, which may facilitate the probes to differentiate between normal cells and cancer cells by increasing the fluorescence intensity. Next, further screening of cancer cells is possible by the color change and release rate due to high β-gal enzyme activity.

On the other hand, as shown in Scheme 2, the recognition site is located on the opposite side of the bipolar dye (1) to minimize the interaction between the units. As shown in Scheme 2a, the electron donating ability of the amino group is reduced by the carbamate linker, and thus, the donor efficiency recovered after enzymatic hydrolytic cleavage enhances the internal charge transfer, as shown by the red-shifted emission change. Thus, as shown in Scheme 2b, cancer cells can be distinguished by dual recognition probes based on emission intensity and color change.

1-1. Synthesis and characterization of SGG and glucosamine inducing moieties (GS)

Briefly, SGG was synthesized in a three-step method as shown in Scheme 2.

First, the coupling reaction between Compound 1 and O-acyl amine hydrochloride 2 was smoothly performed by EDCI treatment to obtain amide 3 (80%).

Subsequent coupling reaction of compound 3 with chloroformate 4 gave the corresponding carbamate in 64% yield, followed by successful total deacylation through methanolization to produce SGG, and reaction of SGG with enzyme and GS The product was used as a control.

The solubility of SGG and GS in water in buffer (10 mM PBS, pH 7.4) was found to be about 5 and 3 μM, respectively. SGG and GS showed maximum absorption at 332 nm (molar extinction coefficient, ε = 2.10 × 10 ⁴ M ^-1 cm ^-1 ) and 373 nm (ε = 1.87 × 10 ⁴ M ^-1 cm ^-1 ), respectively, and 453 nm (fluorescence quantum yield, Φ = 1.00) and 540 nm (Φ = 0.10) showed the maximum emission rates, respectively.

The larger stroke shift of GS (163 nm for GS and 121 nm for SGG) is due to the stronger electron-donating methylamine, which makes the charge-transfer excited state more stable. _{The TP fluorescence efficiencies (Φδ max} ) of SGG and GS in buffer were found to be about 12 and 15 GM (1 GM = 10 ⁻⁵⁰ cm ⁴ photon−1) at 730 and 740 nm, respectively, and the values were measured by two-photon microscopy (TPM). can provide vivid images.

From high-performance liquid chromatography (HPLC) and single-photon emission spectra as shown in FIG. 1A , it was confirmed that the major enzyme reaction product of β-gal and SGG was GS. The emission spectrum of SGG (1 μM) after addition of β-gal (1 unit/mL) to the buffer showed a gradual decrease in the peak intensity at 453 nm with an increase in the peak intensity at 525 nm, and the ratio of _{F 525} /F _{453 was} 45-fold increase.

Fluorescence change over time confirmed that SGG reacted with β-gal within 30 min. Enzymatic hydrolysis of SGG was dose-dependently inhibited by D-galactose, known as a β-gal inhibitor, and it was confirmed that it was specifically dependent on β-gal enzyme through an increase in the fluorescence ratio of SGG.

In addition, the F ₅₂₅ /F ₄₅₃ ratio versus enzyme concentration plot showed a linear relationship, and the detection limit of SGG for β-gal was calculated as low as 0.04 nM.

The enzyme reaction rate was expressed using the Michaelis-Menten equation, and the catalytic efficiency constant (k _cat /K _m ) was calculated. As a result, it was confirmed as ^{0.10 ± 0.03 μM -1} s ^-1, The K _m is 011.7 ± 2.7 μM.

Referring to FIG. 1b, in the presence of various biological species such as other enzymes, reactive oxygen species, amino acids and metal ions, the F ₅₂₅ /F ₄₅₃ ratio of SGG over 6 hours showed a negligible change, but β- In the presence of gal there was a 45-fold increase.

In addition, SGG and GS were not affected by pH in the biologically relevant pH range, and as a result of cell viability and photostability experiments, SGG and low toxicity and high photostability were confirmed.

From the above results, it was confirmed that SGG can visualize β-gal enzyme activity through excellent sensitivity, selectivity and stability.

<Example 2> Confirmation of the usefulness of SGG in the cellular environment

A glucose competition assay was performed to determine whether the intracellular uptake of the probe was correlated with GLUT-mediated signaling.

First, to identify GS having only D-glucosamine units, SW837, HCT 116, RKO and HeLa cancer cell lines and GS were incubated for 30 minutes in the presence or absence of D-glucose, followed by TPM imaging.

As a result, the relative intracellular uptake of GS in the presence of D-glucose was reduced by 50%, 44%, 47%, and 54% in SW837, HCT 116, RKO and HeLa cells, respectively, and similar results were obtained in SGG as shown in FIG. 2 . Confirmed. Referring to FIG. 2 , when the colon cell line was cultured with SGG in the absence of glucose, bright images were obtained from HT-29 and HCT 116 colon cancer cells compared to CCD-18Co normal cells. In addition, in the presence of D-glucose, compared with the absence of Glc (100%), the uptake of SGG was reduced by 30% in HT-29 cells and 45% in HCT 116 cells, whereas no change was observed in the presence of L-glucose. did not appear

From the above results, it was confirmed that SGG is useful for confirming GLUT-mediated intracellular glucose uptake.

In addition, in order to confirm the enzymatic activity of β-gal in SGG living cells, lacZ, a lac operon gene encoding β-gal enzyme expression, was transduced (lacZ-positive) or not transduced as shown in FIGS. 3A and 3B . Non- (lacZ-negative) HCT 116 cells were labeled with SGG (5 μM) and a ratiometric TPM imaging study was performed.

As a result, as shown in Figure 3C, the average TPM ratio of SGG in lacZ-negative HCT 116 cells [F _green /F _blue , the F _blue and F _green are two photons from Ch1 (400-450 nm) and Ch2 (500-600 nm) Integration of excitation fluorescence intensity] was 1.0, while lacZ-positive HCT 116 was significantly increased to 1.7. In addition, HCT 116 cells labeled with the reaction product showed an average ratio of 3.0.

From the above results, it was confirmed that the TPM ratio of SGG actually exhibited β-gal enzyme activity in living cells.

Meanwhile, in order to confirm whether the probe can distinguish cancer cells from normal cells, three types of colorectal cancer cell lines (HT-29, HCT 116 and RKO) and normal colorectal cells (CCD-18Co) were selected, and Fig. As shown in 4a and 4b, the intracellular uptake efficiency of SGG in CCD-18Co and HT-29 cells was monitored and compared by performing real-time TPM imaging analysis.

As a result, the plots showing the relative TPEF intensity at 2-second intervals, as shown in FIG. 4C, indicate that the intracellular uptake of SGG is faster in cancer cells than in normal cells, and through observation of the fluorescence intensity of the sample, the probe separates normal and cancer cells It has been confirmed that identifiable

Next, β-gal activity was confirmed in colorectal cancer cells.

Referring to FIGS. 5A and 5C , as confirmed from the average TPEF intensity in the entire detection channel (400-600 nm) of SGG, various cancer cells exhibited very bright signals compared to normal cells. The results were consistent with competitive and real-time cell imaging results.

_{In addition, a significant increase in the F green} /F _blue ratio was confirmed in cancer cells than in normal cells at the same time as in FIGS. 5B and 5D , and this result may be due to the increased β-gal activity.

Although SGG can distinguish colorectal cancer cells from normal cells by increasing the intensity and ratio, a combination analysis of intensity and ratio was applied, and as a result, the intensity versus ratio plot as shown in FIG. 5E shows not only cancer cells but also cancer cells in normal cells. It has been found that different types of attention can be clearly distinguished.

<Example 3> Detection of colon cancer cells in colon tissue using SGG

Biopsy samples of colonic tissues were obtained from patients who underwent surgical operations at Ajou University Hospital. According to cancer progression, colon tumor samples of three different cancer stages were used in the order of normal, adenoma (pre-tumor stage) and carcinoma (malignant tumor or cancer), and the samples were subjected to conventional hematoxylin & eosin staining. obtained from the same lesion identified. To observe the uptake of SGG, tissues were incubated with SGG (10 μM) in glucose-free medium and images were immediately acquired at 5 s intervals at 740 nm excitation.

As a result, it was confirmed that the rapid absorption rate of the probe appeared in the order of carcinoma, adenoma, and normal tissue through TPEF intensity over time, and these results were consistent with the results of cell experiments.

Meanwhile, referring to FIGS. 6A to 6C , the average TPEF intensity of carcinoma was significantly higher than that of normal and adenoma, whereas the TPEF intensity of adenoma was increased twice that of normal tissue as shown in FIG. 6G .

From the above results, it was confirmed that carcinomas in lesions can be remarkably distinguished through TPEF intensity, but it is not easy to distinguish adenomas from normal tissues.

_{Next, the F green} / F _blue ratio was analyzed in the TPM images of normal intestinal tissue, adenoma, and carcinoma as shown in FIGS. 6D to 6F . As a result, it was confirmed that the average fluorescence ratio increased as the normal tissue progressed to adenoma and carcinoma as shown in FIG. 6H, and it was possible to clearly distinguish the adenoma from the normal tissue through the ratio. Referring to FIG. 6I, the intensity vs. Through the plot of the ratio, it was confirmed that the stages of normal, adenoma, and carcinoma were clearly distinguished in each area.

From the above results, it was confirmed that SGG, a dual recognition probe, can clearly discriminate colorectal cancers of various stages in human colon tissues.

This method can overcome the limitations of histological experiments, such as inter-observer variability, a time-consuming process, and diagnostic differences according to the obtained tissue location.

In addition, compared with histological experiments, SGG can provide more accurate diagnostic information for determining colorectal cancer treatment methods such as surgery or endoscopic treatment.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]

It contains a compound represented by the following formula (1), wherein the compound comprises a glucose receptor (Glucose transporters; GLUT) specific recognition site, a two-photon fluorescent probe, and a β-galactosidase reaction site. A dual recognition two-photon fluorescent probe:
[Formula 1]

The dual recognition two-photon fluorescent probe of claim 2, wherein the glucose receptor (GLUT)-specific recognition site binds to a carboxyl group of the two-photon fluorescent probe. The dual recognition two-photon fluorescent probe according to claim 2, wherein the glucose receptor (GLUT)-specific recognition site is selected from the group consisting of D-glucosamine and D-glucopyranoside. The dual recognition two-photon fluorescent probe of claim 2, wherein the β-galactosidase reaction site binds to an amine group of the two-photon fluorescent probe. The dual recognition two-photon fluorescent probe according to claim 2, wherein the β-galactosidase reaction site is β-D-galactopyranoside. The dual recognition two-photon fluorescent probe according to claim 2, wherein the probe is selectively absorbed into tumor cells through glucose transporters (GLUTs). The dual recognition two-photon fluorescent probe according to claim 2, wherein the probe exhibits a color change by a hydrolase reaction of β-galactosidase (β-gal) in tumor cells. treating a sample isolated from a human with the dual recognition two-photon fluorescent probe of claim 2;
irradiating excitation light to the sample treated with the double recognition two-photon fluorescent probe;
The method for providing information for diagnosing colorectal cancer, comprising the step of confirming an increase in the intensity of color and the rate of color change generated from the fluorescent probe in the sample irradiated with the excitation light. The method of claim 9, wherein the excitation light has a wavelength in the range of 400 to 600 nm. The colorectal cancer diagnosis according to claim 9, wherein the color change rate is determined by determining the rate at which the emission color of the two-photon fluorescent probe changes from blue to green by the hydrolase reaction of β-galactosidase in the sample. How to provide information for delete delete delete

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