KR102588397B1 - Fluorescent probe for detecting β-galactosidase and medical uses thereof - Google Patents

Abstract

The present invention relates to a fluorescent probe for detecting β-galactosidase and medical uses using the same. The fluorescent probe is a cyclic fluorescent dye that emits near-infrared light, and contains β-galactosidase in biological samples using a fluorescence microscope and fluid analyzer. It relates to a method for detecting lactosidase and cancer diagnosis technology using the same.
In particular, the fluorescent probe increases the F _red /F _yellow (F ₆₆₂ /F ₆₂₀ ) ratio by the hydrolytic enzyme reaction of β-galactosidase in the sample, resulting in stable red emission ratio measurement (ratiometric) at 620 to 662 nm. Since it is possible to diagnose cancer disease and confirm the progress stage of cancer disease by displaying fluorescence, the fluorescent probe of the present invention can provide accurate and effective cancer disease diagnosis information in cancer disease treatment.

Description

Fluorescent probe for detecting β-galactosidase and medical uses thereof}

The present invention relates to a fluorescent probe for detecting β-galactosidase and medical uses using the same. The fluorescent probe is a cyclic fluorescent dye that emits near-infrared light, and contains β-galactosidase in biological samples using a fluorescence microscope and fluid analyzer. It relates to a method for detecting lactosidase and cancer diagnosis technology using the same.

β-Galactosidase (β-gal), a useful reporter enzyme, is known to be widely used in gene expression, enzyme-linked immunosorbent assay (ELISA), transcription, transfection, and hybridization studies.

β-Galactosidase can be a powerful biomarker for various diseases, including colorectal cancer and ovarian cancer, so monitoring the distribution and activity of β-galactosidase in living samples can improve diagnosis, prognosis, and treatment. there is.

Over the past decades, various bioimaging techniques have been widely used to monitor intracellular β-galactosidase activity, but most of them have limitations due to the efficacy of the enzymatic reaction and limited spatiotemporal resolution.

Therefore, the methods for measuring the activity of β-galactosidase developed to date cannot specifically detect and image the enzyme activity in living cells, and there is no development of a fluorescent marker for detection of the enzyme, so human samples There is a need for the development of a fluorescent probe capable of ratiometrically quantifying β-galactosidase.

Republic of Korea Patent Publication No. 10-2016-0033868 (2016.03.29)

The purpose of the present invention is to develop a fluorescent probe capable of ratiometrically quantifying β-galactosidase in human samples and to provide medical uses using the same.

The present invention provides a compound represented by the following Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof:

[Formula 1]

[Formula 2]

Additionally, the present invention provides a fluorescent probe for detecting β-galactosidase containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Formula 1]

Additionally, the present invention provides a composition and kit for diagnosing cancer diseases including the fluorescent probe.

Additionally, the present invention provides a method of providing information for diagnosing cancer diseases using the fluorescent probe.

Additionally, the present invention provides a method for quantitative imaging of β-galactosidase activity using the fluorescent probe.

The fluorescent probe according to the present invention increases the F _red / F _yellow (F ₆₆₂ / F ₆₂₀ ) ratio by the hydrolytic enzyme reaction of β-galactosidase in the sample, measuring stable red emission at 620 to 662 nm (ratiometric). ) Since it is possible to diagnose cancer disease and confirm the progress stage of cancer disease by displaying fluorescence, the fluorescent probe of the present invention can provide accurate and effective cancer disease diagnosis information in cancer disease treatment.

, (7)

OtBu, (8)

OH; 1mM 아미노산, (9) Lys, (10) Arg, (11) His, (12) Asp, (13) Glu, (14) GSH; 5 U/mL 효소, (15) ALP, (16) 아미다아제, (17) CE1, (18) CE2, (19) NQO1, (20) GGT, (21) β-gal], (B) 37℃m 2시간 동안 범용 완충액 (0.1M 시트르산, 0.1M KH₂PO₄, 0.1M Na₂B₄O₇, 0.1M 트리스, 0.1M KCl에서 (빨간색) CCGal1 및 (검정색) CC1의 형광 비율(F_red/F_yellow)에 대한 pH 효과를 나타낸 것이다.
도 5는 MTT 분석을 사용하여 측정한 (A) CCGal1 및 (B) CC1의 존재 하에 SKOV-3 세포의 생존율을 나타낸 것이고, (C) CCGal1 염색 SKOV-3 세포의 형광 이미지, (D) 이미지 C의 영역 a-c에서 형광 강도의 변화를 기록한 것이다.
도 6은 CCGal1 염색 세포의 FACS 분석 결과를 나타낸 것이다[(β-gal 저발현; CCD-18Co, Huh-7, HeLa, β-gal 과발현; HT-29, SKOV-3, OVCAR-3, y축은 적색 형광 강도(Ch2, 660±15nm)를 나타내고 x축은 황색 형광 강도(Ch1, 620±15nm)를 나타냄).
도 7은 CCGal1 염색 세포주의 F_red/F_yellow 비율측정 이미지를 나타낸 것이다[(A) CCD-18Co, (B) HT-29, (C) SKOV-3 및 (D) OVCAR-3 세포의 비율 측정 이미지(F_red/F_yellow), (E) 이미지 A-D의 평균 F_red/F_yellow 비율].
도 8은 CCGal1로 염색된 인간 결장 (A) 정상 조직 및 (B) 암 조직의 비율 측정 이미지(F_red/F_yellow), (C) 평균 F_red/F_yellow 비율을 나타낸 것이다.Figure 1 shows (A) normalized absorption and emission spectra of (blue and red) CCGal1 and (black and magenta) CC1, (BD) enzymatic reaction of CCGal1 with β-gal, and (B) 2.5 U/mL in PBS. The time course fluorescence spectrum of CCGal1 (2 μM) after addition of mL β-gal, (C) average time course of F662/F620 ratio, and (D) enzyme kinetics analysis results of CCGal1 are shown.
Figure 2 shows the results of HPLC analysis to verify the formation of CC1 from CCGal1 by β-gal.
Figure 3 shows ratiometric images (F _red / F _yellow ) of SKOV-3 cells stained with CCGal1 [(a) untreated (control) and (b) treated with 100 mM D-galactose (β-gal inhibitor). Pretreatment. (c) Average F _red /F _yellow ratio of the corresponding image. Scale bar = 20 μm]
Figure 4 shows (A) the average fluorescence ratio (F _red / F _yellow ) of CCGal1 using various substances in PBS buffer (10mM, pH 7.4, 37°C) [(1) control; 200 μM ROS/RNS, (2) H ₂ O ₂ , (3) TBHP, (4) OCl ^- , (5) O ^{2 -} , (6) NO

, (7)

OtBu, (8)

OH; 1mM amino acids, (9) Lys, (10) Arg, (11) His, (12) Asp, (13) Glu, (14) GSH; 5 U/mL enzyme, (15) ALP, (16) amidase, (17) CE1, (18) CE2, (19) NQO1, (20) GGT, (21) β-gal], (B) 37 _{Fluorescence ratio ( F _} This shows the pH effect on _red /F _yellow ).
Figure 5 shows the survival rate of SKOV-3 cells in the presence of (A) CCGal1 and (B) CC1 measured using MTT assay, (C) fluorescence image of CCGal1 stained SKOV-3 cells, (D) Image C The change in fluorescence intensity in the area ac was recorded.
Figure 6 shows the results of FACS analysis of CCGal1 stained cells [(β-gal low expression; CCD-18Co, Huh-7, HeLa, β-gal overexpression; HT-29, SKOV-3, OVCAR-3, y axis is represents red fluorescence intensity (Ch2, 660 ± 15 nm) and x-axis represents yellow fluorescence intensity (Ch1, 620 ± 15 nm).
Figure 7 shows F _red / F _yellow ratio measurement images of CCGal1 stained cell lines [ratio measurement of (A) CCD-18Co, (B) HT-29, (C) SKOV-3, and (D) OVCAR-3 cells. Image (F _red /F _yellow ), (E) average F _red /F _yellow ratio of image AD].
Figure 8 shows ratiometric images (F _red /F _yellow ) and (C) average F _red /F _yellow ratio of human colon (A) normal tissue and (B) cancer tissue stained with CCGal1.

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

As a result of our diligent efforts to develop a new compound capable of ratiometrically quantitatively analyzing β-galactosidase in in vivo samples, the present inventors have developed a red-emitting fluorophore with a cyclic cyanine and a carbamate bridge capable of self-sacrificing cleavage. Discovery of a novel compound (CCGal1) consisting of a β-galactopyranosyl unit as a linked enzymatic recognition moiety, and the discovery of β-galactopyranosyl in in vivo samples through confocal fluorescence microscopy ratiometric image analysis coupled with fluorescence-activated cell sorting (FACS) flow cytometry. -The present invention was completed by discovering that galactosidase can be quantitatively analyzed ratiometrically.

Accordingly, the present invention provides a compound represented by the following Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof:

[Formula 1]

[Formula 2]

Additionally, the present invention provides a fluorescent probe for detecting β-galactosidase containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Formula 1]

As shown in Scheme 1, the compound or a pharmaceutically acceptable salt thereof may react with β-galactosidase of cells, organelles, or biological tissues to exhibit fluorescence, and in particular, β-galactosidase of cells, organelles, or biological tissues. It can react with galactosidase to exhibit stable red emission ratiometric fluorescence at 620 to 662 nm.

[Scheme 1]

Additionally, the present invention provides a composition for diagnosing cancer diseases including the fluorescent probe.

Additionally, the present invention provides a kit for diagnosing cancer diseases including the fluorescent probe.

The composition for diagnosing cancer disease further includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers include, for example, excipients and diluents, and are well known in the art. The choice of carrier may be determined depending on the specific dosage form to be prepared and the specific administration method of the composition. Therefore, the composition for diagnosing cancer diseases of the present invention may exist as a preparation in a wide variety of forms. Additionally, the composition for diagnosing cancer diseases of the present invention can be administered by an appropriate administration method depending on its purpose or type of active ingredient.

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

A preferred method of administration of the composition for diagnosing cancer diseases of the present invention is parenteral, for example, bolus injection, intravenous injection, intra-arterial injection, or, if the lungs are to be contrasted, spray, for example, aerosol spray, orally or rectally. Administration can also be used, but known contrast agent administration methods are also possible. Parenteral administration preparations must be sterile, free from physiologically unacceptable substances and paramagnetic, superparamagnetic, ferromagnetic, or quasi-ferromagnetic contaminants, and contain preservatives, antibacterial agents, buffers and antioxidants commonly used in parenteral solutions, It may contain excipients and may further contain any other additives that do not interfere with molecular imaging.

In addition, the present invention includes the steps of treating a sample isolated from a human with the fluorescent probe; Irradiating excitation light to the sample treated with the fluorescent probe; and confirming the level of increase in the rate of color change occurring from the fluorescent probe in the sample irradiated with the excitation light.

The excitation light may have a wavelength ranging from 620 to 662 nm.

The color change rate may be increased by the F _red /F _yellow (F ₆₆₂ /F ₆₂₀ ) ratio due to the hydrolytic enzyme reaction of β-galactosidase in the sample.

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

The step of confirming the level of increase in the color change rate can be observed through fluorescence activated cell sorting (FACS) flow cytometry and fluorescence microscopy images.

Additionally, the present invention includes the steps of injecting the fluorescent probe into a sample isolated from a human; The fluorescent probe injected into the sample in the above step reacts with β-galactosidase in the sample to emit fluorescence; and observing the fluorescence through fluorescence activated cell sorting (FACS) flow cytometry and fluorescence microscopy images.

The fluorescent probe can react with β-galactosidase in the sample to display stable red emission ratiometric fluorescence at 620 to 662 nm, and F by the hydrolytic enzyme reaction of β-galactosidase in the sample. _{The red} /F _yellow (F ₆₆₂ /F ₆₂₀ ) ratio may increase.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Synthesis example> Synthesis of CCGal1 and CC1

Compound D was prepared using a previously known method [H. W. Lee, C. H. Heo, D. Sen, H.-O. Byun, I. H. Kwak, G. Yoon and H. M, Kim, Anal. Chem., 86, 10001], and CCGal1 and CC1 were synthesized according to Scheme 2 as follows.

[Scheme 2]

1. Synthesis of Compound A

tert -Butyl(5-oxo-5,6,7,8-tetrahydronapralen-2-yl)carbamate (2.0 g, 7.65 mmol) and potassium tert -butoxide (3.86g, 34.44 mmol) were dissolved in benzene (25 mL) at 0°C for 2 hours, then ethyl formate (1.55 mL, 19.13 mmol) was added dropwise. After addition, stirring was continued at 0°C for 4 hours. After completion of reaction, satd. NH ₄ Cl was added and extracted with DCM (3X). The organic layer thus obtained was washed with brine, dried over Na ₂ SO ₄ , and evaporated to dryness. The desired material was obtained in 82% yield through column chromatography using 25% ethyl acetate in hexane.

¹ H NMR (chloroform- d , 600 MHz): δ (ppm) 8.00 (d, J = 6.2 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.46 (s, 1H), 7.13 (d) , J = 9.0 Hz, 1H), 6.63 (s, 1H), 2.85 (t, J = 6.9 Hz, 2H), 2.54 (t, J = 6.9 Hz, 2H), 1.52 (s, 9H).

2. Compound B synthesis

Compound A (1.5 g, 5.18 mmol) dissolved in THF (25 mL) and DDQ (1.8 g, 5.18 mmol) dissolved in THF (5.0 mL) were slowly added at 0°C and reacted at room temperature for 3 hours. After completion of the reaction, the solvent was evaporated to dryness under reduced pressure, and the desired material was obtained in 61% yield through column chromatography using 25% ethyl acetate in hexane.

¹ H NMR (chloroform- d , 600 MHz): δ (ppm) 12.67 (s, 1H), 9.88 (s, 1H), 8.32 (d, J = 9.2 Hz, 1H), 8.02 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.30 (dd, J = 9.2, 2.3 Hz, 1H), 7.25 (d, J = 9.2 Hz, 1H), 6.81 (s, 1H), 1.54 (s, 9H) .

3. Compound C synthesis

Compound B (1.0 g, 3.48 mmol) and 1-(2-(1,3-dioxolan-2-yl)ethyl)-3,3-dimethyl-2-methyleneindoline [1-(2-(1,3- dioxolan-2-yl)ethyl)-3,3-dimethyl-2-methyleneindoline; (0.99 g, 3.83 mmol)] was added to EtOH (20 mL) and heated at 72°C for 48 hours. The solvent was evaporated to dryness under reduced pressure, and the desired material was obtained in 68% yield through column chromatography using 25% to 50% ethyl acetate in hexane.

¹ H NMR (chloroform- d , 600 MHz): δ (ppm) 7.96 (s, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.16-7.21 (m, 2H), 7.11 (d, J = 8.3 Hz, 1H), 7.07 (d, J = 8.3 Hz, 2H), 6.98 (dd, J = 9.1, 1.9 Hz, 1H), 6.89 (d, J = 10.3 Hz, 1H), 6.83 (t, J = 7.2 Hz, 1H), 6.63 (d, J = 7.6 Hz, 2H), 5.62 (d, J = 10.3 Hz, 1H), 4.83 (t, J = 4.8 Hz, 1H), 3.82-4.04 (m, 7H) , 3.44-3.49 (m, 1H), 3.26-3.31 (m, 1H), 1.97-2.01 (m, 1H), 1.87-1.93 (m, 1H), 1.51 (s, 9H), 1.28 (s, 3H) , 1.17 (s, 3H).

4. CC1 synthesis

Compound C (0.2 g, 0.38 mmol) was added to DCM (30 mL) and cooled to -78°C, 1.0M BBr ₃ solution dissolved in DCM was added dropwise over a period of 15 min and stirring continued for 4 h at -78°C. did. Slowly, the temperature was increased to room temperature and stirring was continued overnight. Excess BBr ₃ from the dark orange reaction mass was quenched slowly at 0° C. using aqueous NH ₄ Cl and extracted with DCM (3X) to obtain a dark blue reaction mixture. The organic layer was washed with brine, dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography using 3% to 20% methanol in CHCl ₃ to obtain the desired CC1 as a dark blue solid in 15% yield.

^1H NMR (methanol- d _4,600MHz ): δ (ppm) 8.27 (s, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.67 (d, J = 6.9 Hz, 1H), 7.62 (d) , J = 7.6 Hz, 1H), 7.57 (td, J = 7.6, 1.4 Hz, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.31 (d, J = 9.0 Hz, 1H), 7.13 (d , J = 9.0 Hz, 1H), 6.95 (dd, J = 9.0, 2.1 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H), 5.54-5.57 (m, 1H), 4.68-4.71 (m, 1H), 4.27 (td, J = 13.9, 4.4 Hz, 1H), 3.00-3.04 (m, 1H), 2.76-2.63 (1H), 1.83 (s, 3H), 1.77 (s, 3H). MS (Q-TOF): m/z calcd. for [C ₂₅ H ₂₃ N ₂ O] ⁺ :367.1805,found:367.1804.

5. CCGal1 synthesis

1) Step 1

Six drops of pyridine were added to CC1 (20 mg, 54 μmol) dissolved in DCM (2.0 mL). p-nitrophenyl chloroformate (44 mg, 0.2 mmol) dissolved in THF (0.5 mL) at 0°C was added, and stirring was continued at the same temperature, followed by stirring at room temperature for 3 hours. Next, Compound D (0.44 g, 0.98 mmol) dissolved in THF (2.0 mL) was slowly added at room temperature and stirring was continued for 8 hours. The reaction mixture was evaporated to dryness and CHCl ₃ Purified by column chromatography using 3% to 15% methanol, the desired intermediate was obtained as a dark red solid in 45% yield.

^1H NMR (methanol -d _4,600MHz ): δ (ppm) 8.35 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.05 (s, 1H), 7.72 (t, J = 7.2 Hz) , 2H), 7.56-7.62 (m, 3H), 7.50 (d, J = 8.3 Hz, 1H), 7.39 (q, J = 4.4 Hz, 3H), 7.02 (d, J = 9.0 Hz, 2H), 5.62 (q, J = 5.5 Hz, 1H), 5.43 (d, J = 3.4 Hz, 1H), 5.33 (dd, J = 10.3, 8.3 Hz, 1H), 5.29 (d, J = 8.3 Hz, 1H), 5.23 (dd, J = 10.0, 3.8 Hz, 1H), 5.16 (s, 2H), 4.78 (dd, J = 15.1, 4.8 Hz, 1H), 4.33-4.38 (m, 1H), 4.27 (t, J = 6.5 Hz, 1H), 4.15 (d, J = 6.2 Hz, 2H), 3.05-3.08 (m, 1H), 2.76 (qd, J = 12.4, 5.5 Hz, 1H), 2.15 (s, 4H), 2.03 (s) , 3H), 1.99 (s, 3H), 1.95 (s, 3H), 1.86 (s, 3H), 1.80 (s, 3H).

2) Step 2

The previously obtained intermediate (10 mg, 11 μmol) was dissolved in methanol (1.0 mL) at 0°C, a 1.0M NaOMe solution in methanol (0.11 mL, 0.1 mmol) was added dropwise, and stirring was continued at the same temperature for 1 hour. The crude mixture was evaporated under reduced pressure and purified by preparative-HPLC using 30%-70% acetonitrile in water to give CCGal1 as a dark red solid in 38% yield.

^1H NMR (methanol -d _4,600MHz ): δ (ppm) 8.36 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.05 (s, 1H), 7.73 (t, J = 7.2 Hz) , 2H), 7.61 (q, J = 6.4 Hz, 2H), 7.57 (d, J = 9.6 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H) ), 7.37 (d, J = 9.0 Hz, 2H), 7.10 (d, J = 8.3 Hz, 2H), 5.63 (q, J = 5.5 Hz, 1H), 5.15 (s, 2H), 4.78 (dd, J = 15.1, 4.8 Hz, 1H), 4.36 (t, J = 14.1 Hz, 1H), 3.88 (d, J = 2.4 Hz, 1H), 3.71-3.79 (m, 4H), 3.67 (t, J = 5.9 Hz) , 1H), 3.55 (dd, J = 9.6, 3.4 Hz, 1H), 3.08 (d, J = 6.2 Hz, 1H), 2.76 (qd, J = 12.3, 5.2 Hz, 1H), 1.86 (s, 3H) , 1.80 (s, 3H). HRMS (ESI ⁺ ): m/z calcd. for [C ₃₉ H ₃₉ N ₂ O ₉ ] ⁺ : 679.2650, found: 679.2642.

<Experimental example>

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

1. Materials used and general analysis methods

All reactions of the present invention were performed under a nitrogen atmosphere in a flame-dried round bottom flask. All chemicals were purchased from Sigma-Aldrich and solvents were appropriately distilled. The reaction process was monitored using a thin layer chromatography (TLC) plate (TLC Silica gel 60 F254, 1.05715.0001, Merck). The product was purified using medium pressure liquid chromatography (AI-580S, YAMAZEN) with a general purpose or C18 column cartridge. NMR spectra were obtained using a 600 MHz NMR spectrometer (JNM-ECZ 600R, JEOL). High-performance liquid chromatography (HPLC) was performed using a Waters 2695 module (Waters, Milford, MA, USA) using a Waters X-Bridge column (C18, 5 μm, 4.6x250 mm). Preparative HPLC was performed on a Waters 2525 Binary Gradient module using a Waters Mass data were obtained using UPLC/TripleTOF 5600+, AB Sciex).

2. Spectroscopic analysis

Absorption spectra were acquired on a S-3100 UV-Vis spectrophotometer and emission spectra were recorded on a FluoroMate FS-2 fluorometer using a 1.0 cm optical path Hellma quartz cell with a Teflon stopper. The relative fluorescence quantum yield was set using cresyl violet (Φ = 0.54 in methanol) as the reference compound.

3. Determination of water solubility

A small aliquot of each compound was dissolved in DMSO to obtain a stock (10.0 mM) of compound solution. Stock solutions were diluted to 0.1-10.0 μM in a quartz cell containing 2.0 mL of PBS buffer (10 mM, pH 7.4). The concentration of DMSO in the PBS buffer was maintained below 0.1%. The plot of fluorescence intensity versus compound concentration was linear at low concentrations and showed a descending curvature at some specific concentrations. The highest concentration in the linear region was taken as the aqueous solubility, and the aqueous solubilities of CCGal1 and CC1 in PBS buffer were determined to be 2.0 and 1.0 μM, respectively.

4. Cell culture conditions

All cells were seeded in glass bottom dishes (Wuxi NEST Biotechnology) for 2 days before imaging and maintained in a CO ₂ incubator (Water Jacketed 3010, Thermo Fisher Sci-entific) at 37°C in a humidified atmosphere of 5/95 (v/v). I ordered it. After staining, the growth medium was removed and replaced with glucose-free medium. Cells were incubated with 2.0 μM CCGal1 or CC1 for an additional 3 h, washed twice with PBS (Gibco, Thermo Fisher Scientific), and then imaged. The growth medium for each cell is as follows.

CCD-18Co (human colon normal cell line, CRL-1459, American Type Culture Collection): DMEM (WelGene) supplemented with 10% FBS (WelGene, Korea), penicillin (100 units/mL), and streptomycin (100 μg/mL). , Korea).

Huh-7 (human hepatocellular carcinoma cell line, 60104, Korean Cell Line Bank): low-glucose DMEM supplemented with 10% FBS (WelGene, Korea), penicillin (100 units/mL), and streptomycin (100 μg/mL) WelGene, Korea).

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

OVCAR-3 (human ovarian adenocarcinoma cell line, 30161, Korea Cell Line Bank), SKOV-3 (human ovarian adenocarcinoma cell line, 30077, Korea Cell Line Bank), HT-29 (human colorectal adenocarcinoma cell line, HTB-38, American Type Culture Collection ): RPMI (WelGene, Korea) supplemented with 10% FBS (WelGene, Korea), penicillin (100 units/mL), and streptomycin (100 μg/mL).

5. Cell viability analysis

Cell viability was analyzed using the MTT kit (AbCareBio CL) analysis. After the cells were cultured on a 96-well plate for 24 hours, various concentrations of compounds were added. After culturing for 2 hours, the culture medium was replaced with serum-free medium containing 10% MTT and cultured for another 2 hours. The MTT-containing medium was removed, DMSO was added to dissolve the formed formazan precipitate, and the absorbance was measured at 600 nm.

6. Fluorescence microscopy analysis

Fluorescence microscopy images of CCGal1 and CC1 stained cells were analyzed with a spectroscopic confocal microscope (Leica TCS SP8 MP) with a ×40 oil objective and a numerical aperture (NA) of 1.30. Fluorescence microscopy images were obtained with a DMI6000B microscope (Leica) by exciting CCGal1 or CC1 with a 488 or 552 nm laser. Live cell imaging was performed using a live cell culture device (Chamlide IC system, Live Cell Instrument, Korea) to maintain a stable cell environment.

7. Enzyme kinetic analysis

Fluorescence-based enzyme kinetic analysis was performed using a Varioskan Flash multiple detection microplate reader (Thermo Fisher Scientific) at 552 nm excitation. CCGal1 solutions of various concentrations (0–50 μM) were prepared in PBS buffer (10 mM, pH 7.4, 37°C). β-gal was used at a final concentration of 79.4 mg/L, and fluorescence intensity was recorded at 1-minute intervals from 0 to 30 minutes at 620 nm and 662 nm. Changes in fluorescence ratio values were converted to rates and plotted by nonlinear fitting using the hyperbolic function (OriginPro 8.0, OriginLab), and the parameters of Michaelis-Menten dynamics were calculated.

8. Flow cytometry

FACS flow cytometry data were acquired on a Cytek Aurora-3 spectral flow cytometer (Cytek Biosciences, USA) equipped with three lasers operating at 405, 488, and 640 nm. Data were collected and analyzed using SpectroFlo software (Version 2.2.0, Cytek Bio-sciences, USA) at the 3D Immune System Imaging Core Facility of Ajou University.

<Example 1> Compound design, synthesis and properties

1. Compound design and synthesis

CCGal1 is composed of a cyclic cyanine as a red-emitting fluorophore and a β-galactopyranosyl unit as the enzymatic recognition moiety linked to a self-immolative cleavable carbamate bridge. The fluorophore (CC1) was a push-pull cyanine with a C=C conjugate bridge incorporated into the cycle to achieve photostability.

Figure 1 shows (A) normalized absorption and emission spectra of (blue and red) CCGal1 and (black and magenta) CC1, (B-D) enzymatic reaction of CCGal1 with β-gal, and (B) 2.5 U/mL in PBS. The time course fluorescence spectrum of CCGal1 (2 μM) after addition of mL β-gal, (C) average time course of F662/F620 ratio, and (D) enzyme kinetics analysis results of CCGal1 are shown.

2. Analysis of photophysical data of compounds

The water solubilities of CCGal1 and CC1 in PBS buffer (10 mM, pH 7.4) were measured by fluorescence titration and were approximately 2.0 and 1.0 μM, respectively. In PBS buffer solution, CCGal1 and CC1 have maximum absorption wavelengths at 510 nm (molar extinction coefficient, ε = 1.90 × 10 ⁴ M ^{- 1} cm ^-1 ) and 549 nm (ε = 1.76 × 10 ⁴ M ^-1 cm ^{-1 )} . λ _ex ) and the maximum emission wavelength (λ em ) at 620 nm (relative fluorescence quantum yield, Φ = 0.18) and 662 _nm (Φ = 0.10) (Figure 1A, B and Table 1).

[Table 1]

<Example 2> Enzymatic reaction of CCGal1

The enzymatic reaction product between CCGal1 and β-gal was mainly CC1, as shown by the change in fluorescence spectrum in Figure 1c, and was confirmed using high-performance liquid chromatography (HPLC) (see Figure 2).

Referring to Figure 1, when β-gal (2.5 U/mL) was added to 2 μM CCGal1 in PBS, the fluorescence spectrum showed a gradual decrease at the 620 nm peak and a simultaneous increase at 662 nm (Figure 1c), and the F662/F620 ratio This increased 3.8 times. The plot of fluorescence ratio changed rapidly over time and the enzymatic reaction was completed within 15 min (Figure 1D).

In addition, according to Figure 3, the hydrolysis of CCGal1 by β-gal was strongly inhibited by D-galactose, a known β-gal inhibitor in living cells, which means that the shift in the fluorescence ratio of CCGal1 is entirely due to β-gal activity. It was suggested that it was caused by

Enzyme kinetics calculated using ^the Michaelis-Menten equation showed that the catalytic efficiency ^constant (kcat/Km) of CCGal1 ^{was 1.21} The gal probe, fluorescein di-β-D-galactopyranoside [fluorescein di-β-D-galactopyranoside; FDG] was confirmed to be similar to the value of [FDG].

<Example 3> Evaluation of selectivity and stability of CCGal1

To assess selectivity and stability, the F ₆₆₂ /F ₆₂₀ ratio of CCGal1 was monitored in excess of reactive oxygen/nitrogen species, amino acids, GSH and other enzymes and found that the fluorescence ratio increased only in the presence of β-gal (Figure 4A). CCGal1 and CC1 were highly stable in the biocompatible pH range of 3.5–10.0 (Figure 4B). CCGal1 showed low cytotoxicity and high photostability in the MTT cell viability assay (Figures 5A and 5B) and recurrent laser irradiation (Figures 5C and 5D). The limit of detection (LOD) for CCGal1 relative to β-gal was found to be 0.24 nM after plotting the change in the F ₆₆₂ /F ₆₂₀ ratio at various concentrations of β-gal. Therefore, it was confirmed that CCGal1 can be used to quantitatively monitor β-gal activity in living cells with excellent selectivity, stability, and sensitivity.

<Example 4> Distinguishing cancer cells from normal cells using flow cytometry

To confirm the compatibility of CCGal1 using flow cytometry, various cell lines with different levels of β-gal expression were analyzed using FACS. In Figure 6, the analysis gates for Ch1 (F _yellow , bottom right) and Ch2 (F _red , top left) were assigned polygons to reflect the dominance of the corresponding channels. Therefore, the higher the Ch2 density, the higher the expression of β-gal, and the lower the Ch1 density, the lower the expression. Similar to the fluorescence ratio, FACS results showed a distinct distribution of cells with upregulated β-gal expression in Ch2 (92.61% in HT-29, 35.03% in SKOV-3, and 77.25% in OVCAR-3); Consistent with previous studies, CCD-18Co, Huh-7, and HeLa cells showed low to moderate β-gal expression (less than 1.5% in Ch2).

<Example 5> Ratio measurement image analysis of normal and cancer cells using a fluorescence microscope

To confirm the potential of CCGal1 to distinguish between normal and cancer cells using fluorescence microscopy, CCD-18Co (human colon normal) cells were used as β-gal negative cells, HT-29 (human colon cancer), SKOV-3 , OVCAR-3 (human ovarian cancer) cells were used as β-gal positive cells.

As shown in Figure 7, the F _red /F _yellow (F ₆₆₂ /F ₆₂₀ ) ratio of CCGal1 was 0.79 ± 0.06 in normal cells, 1.74 ± 0.21, and 1.75 ± 0.16 in cancer cells (HT-29, SKOV-3, OVCAR-3). , was found to be 1.79 ± 0.22. These results indicate that CCGal1 can distinguish cancer cells from normal cells with a high F _red / F _yellow ratio in ratiometric fluorescence microscopy.

<Example 6> Differentiation of normal and cancerous human colon tissues using ratiometric imaging

To further verify real-time monitoring of β-gal activity in living samples, normal and human colon cancer tissues were obtained from Ajou University Hospital with patient consent.

As shown in Figure 8, β-gal activity could be visualized in differentiated stages of colon cancer through ratiometric fluorescence imaging. Colon cancer tissue stained with CCGal1 had an F _red / F _yellow ratio more than twice that of normal tissue, and the ratio of CCGal1 was 0.80 in normal tissue and 1.72 in cancer tissue (Figure 8C). These results suggest that CCGal1 is useful for both fluorescence microscopy and flow cytometry and can accurately determine various stages of colon cancer.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A compound represented by Formula 1 or Formula 2 below, or a pharmaceutically acceptable salt thereof:
[Formula 1]

[Formula 2]
Fluorescent probe for detecting β-galactosidase containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]
The fluorescent probe according to claim 2, wherein the compound or a pharmaceutically acceptable salt thereof reacts with β-galactosidase of cells, organelles, or biological tissues to exhibit fluorescence. The method of claim 2, wherein the compound or a pharmaceutically acceptable salt thereof reacts with β-galactosidase of cells, organelles, or biological tissues to exhibit stable red emission ratiometric fluorescence at 620 to 662 nm. Characterized by a fluorescent probe. A composition for diagnosing cancer diseases comprising a fluorescent probe according to claim 2. A kit for diagnosing cancer disease comprising a fluorescent probe according to claim 2. Processing a sample isolated from a human with the fluorescent probe according to claim 2;
Irradiating excitation light to the sample treated with the fluorescent probe; and
A method of providing information for diagnosing a cancer disease, comprising the step of confirming an increase in the rate of color change occurring from a fluorescent probe in a sample irradiated with the excitation light. The method according to claim 7, wherein the excitation light has a wavelength ranging from 620 to 662 nm. The method of claim 7, wherein the color change rate is characterized in that the F _red / F _yellow (F ₆₆₂ / F ₆₂₀ ) ratio increases by the hydrolytic enzyme reaction of β-galactosidase in the sample. The method of claim 7, wherein the cancer disease is selected from the group consisting of colon cancer, ovarian cancer, breast cancer, liver cancer, lung cancer, uterine cancer, pancreatic cancer, kidney cancer, stomach cancer, and brain cancer. The method according to claim 7, wherein the step of confirming the level of increase in the color change rate is observed through fluorescence activated cell sorting (FACS) flow cytometry and fluorescence microscopy images. Injecting the fluorescent probe according to claim 2 into a sample isolated from a human;
The fluorescent probe injected into the sample in the above step reacts with β-galactosidase in the sample to emit fluorescence; and
Observing the fluorescence through fluorescence activated cell sorting (FACS) flow cytometry and fluorescence microscopy images
Quantitative imaging method of β-galactosidase activity, including. The method of claim 12, wherein the fluorescent probe reacts with β-galactosidase in the sample and exhibits stable red emission ratiometric fluorescence at 620 to 662 nm. The method of claim 13, wherein the F _red / F _yellow (F ₆₆₂ / F ₆₂₀ ) ratio of the fluorescent probe increases due to the hydrolytic enzyme reaction of β-galactosidase in the sample.