REAL-TIME FLUORESCENCE IMAGING SENSOR FOR MEASURING GLUTATHIONE IN ENDOPLASMIC RETICULUM AND METHOD USING SAME

Abstract

The present invention relates to a real-time fluorescence imaging sensor for measuring glutathione in the endoplasmic reticulum (ER) and a method for producing same. More specifically, the present invention relates to a new compound for measuring glutathione in the endoplasmic reticulum (ER), a method for producing the new compound, a real-time imaging sensor for measuring glutathione in the endoplasmic reticulum (ER) comprising the new compound, a method for producing same, and a method for measuring glutathione in the endoplasmic reticulum (ER) by using the imaging sensor.

Claims

1-29. (canceled)

30. A compound represented by the following Formula I or a pharmaceutically acceptable salt thereof: ##STR00020## wherein R.sub.1 is a 3- to 7-membered heterocycloalkyl ring containing at least one N atom, and wherein the compound represented by Formula I is represented by any one of the following Formulas IV to VII: ##STR00021##

31. The compound or pharmaceutically acceptable salt thereof according to claim 30, wherein the compound represented by Formula I exhibits a maximum emission wavelength at 550 to 680 nm in a free state, and exhibits a maximum emission wavelength at 430 to 550 nm in a thiol-bound state.

32. The compound or pharmaceutically acceptable salt thereof according to claim 31, wherein the fluorescence intensity increases or decreases at an emission wavelength ranging from 430 nm to 680 nm.

33. A composition for measurement of antioxidant activity in living cells, the composition containing, as an active ingredient, the compound or pharmaceutically acceptable salt thereof according to claim 30.

34. The composition according to claim 33, wherein, as the level of thiols in the measurement of the level of thiols increases, the fluorescence intensity at 550 to 680 nm decreases and the fluorescence intensity at 430 to 550 nm increases.

35. The composition according to claim 33, wherein the measurement of the level of thiols is performed by obtaining a ratio of the fluorescence intensity at 430 to 550 nm to the fluorescence intensity at 550 to 680 nm.

36. The composition according to claim 33, wherein the measurement of the level of thiols is quantitative or qualitative detection of the thiols in the endoplasmic reticulum (ER).

37. The composition according to claim 33, wherein the measurement of the level of thiols is real-time quantitative measurement.

38. The composition according to claim 33, wherein the measurement of the level of thiols indicates the oxidative stress or degree of oxidation of the cells.

39. The composition according to claim 33, wherein the measurement of the level of thiols indicates the degree of senescence of the cells.

40. The composition according to claim 33, wherein the thiols are glutathione (GSH), homocysteine (Hcy), cysteine (Cys), or thiols in cysteine residues of proteins.

41. A method for screening a thiol enhancer or inhibitor in living cells, the method comprising: (a) adding the composition of claim 33 and a candidate substance simultaneously or sequentially in any order to living cells; (b) obtaining a ratio of the fluorescence intensity of the living cells at 430 to 550 nm to the fluorescence intensity of the living cells at 550 to 680 nm and comparing the obtained ratio with standard data; (c) determining that the candidate substance is a thiol enhancer or inhibitor; and (d) determining that when the ratio of the fluorescence intensity at 550 to 680 nm to the fluorescence intensity at 430 to 550 nm decreases, the candidate substance is the thiol enhancer, and determining that when the ratio of the fluorescence intensity at 550 to 680 nm to the fluorescence intensity at 430 to 550 nm increases, the candidate substance is the thiol inhibitor.

42. A kit for diagnosing oxidative stress-induced disease comprising the compound or salt thereof according to claim 30.

43. A method for measuring antioxidant activity in living cells, the method comprising: (a) measuring in real time the ratio of the fluorescence intensity of the living cells at 430 to 550 nm to the fluorescence intensity of the living cells at 550 to 680 nm; (b) adding the composition of claim 4 to the living cells; (c) adding an oxidizing agent to the living cells of step (b); and (d) observing a change in the ratio of the fluorescence intensities.

44. The method according to claim 43, wherein the measuring is performed for endoplasmic reticulum (ER) which is an organelle in the living cells.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0067] FIG. 1 shows the structure of ER-FT represented by Formula IV.

[0068] FIG. 2 shows the structure of ER-FT represented by Formula V.

[0069] FIG. 3 shows the structure of ER-FT represented by Formula VI.

[0070] FIG. 4 shows the structure of ER-FT represented by Formula VII.

[0071] FIG. 5 depicts confocal microscopic images showing the results of observing of ER-FTs represented by Formulas IV to VII in the endoplasmic reticulum of UC-MSCs.

[0072] FIG. 6 shows toxicity test results indicating the IC.sub.50 values of ER-FTs represented by Formulas IV to VII.

[0073] FIG. 7 shows the experimental results of measuring the intensity at a 510 nm wavelength, the intensity at a 580 nm wavelength, and the ratio of the intensities at the two wavelengths as a function of the retention time of ER-FTs represented by Formulas IV to VI in UC-MSCs.

[0074] FIG. 8 shows the experimental results of measuring the intensity at a 510 nm wavelength, the intensity at a 580 nm wavelength, and the ratio of the intensities at the two wavelengths as a function of the retention time of ER-FTs represented by Formulas IV, V and VI in UC-MSCs.

[0075] FIG. 9 shows confocal microscope images of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula IV.

[0076] FIG. 10 shows confocal microscope images of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula IV.

[0077] FIG. 11 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula V.

[0078] FIG. 12 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula V.

[0079] FIG. 13 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula VI.

[0080] FIG. 14 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula VI.

[0081] FIG. 15 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula VII.

[0082] FIG. 16 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula VII.

BEST MODE

[0083] An object of the present invention is to provide endoplasmic reticulum fluorescent real-time SH group-tracers (ER-FTs) represented by Formulas IV to VII.

MODE FOR INVENTION

[0084] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

[Preparation Example] Synthesis of Compounds for Measuring Antioxidant Activity of Endoplasmic Reticulum (ER)

[0085] Methods for preparing compounds (ER-FTs) used to measure the antioxidant activity of endoplasmic reticulum (ER) are as follows.

[0086] 1. Method for Preparing ER-FresH A (Formula IV)

##STR00004##

[0087] Compound 1

##STR00005##

[0088] 1-(carbobenzyloxy)-4-piperidinecarboxylic acid (0.30 g, 1.1 mmol), N-(tert-butoxycarbonyl)-ethylenediamine (0.19 mL, 1.0 equivalent), 1-hydroxybenzotriazol (HOBt; 0.23 g, 1.5 equivalents), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI; 0.29 g, 1.5 equivalents) were dissolved in 5 mL of N,N-dimethylformamide (DMF), and the solution was stirred at room temperature for 15 hours. The resulting mixture was diluted with EtOAc and then washed with an aqueous solution of NaHCO.sub.3 and a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered. The filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 1 (0.45 g, 98%).

[0089] .sup.1H NMR (500 MHz, CDCl.sub.3). δ (ppm)=7.31-7.37 (m, 5H), 6.63 (br, 1H), 5.12-5.14 (m, 3H), 4.18-4.21 (m, 2H), 3.32-3.35 (m, 2H), 3.25-3.28 (m, 2H), 2.80 to 2.85 (m, 2H), 2.22-2.27 (m, 1H), 1.80 to 1.84 (m, 2H), 1.60 to 1.67 (m, 2H), 1.42 (s, 9H).

[0090] Compound 2

##STR00006##

[0091] Palladium on activated carbon (Pd—C; 10 wt %, 45 mg) was added to a solution of compound 1 (0.45 g, 1.1 mmol) in 10 mL of MeOH, followed by stirring under H.sub.2 gas (1 atm) for 15 hours. The mixture was filtered through a celite layer, and the filtrate was distilled under reduced pressure to remove the solvent. A portion (0.15 g, 0.55 mmol) of the solid obtained by vacuum drying, cyanoacetic acid (48 mg, 1.0 equivalent), HOBt (0.11 g, 1.3 equivalents), EDCI (0.14 g, 1.3 equivalents), and N,N-diisopropylethylamine (DIEA; 0.15 mL, 1.5 equivalents) were dissolved in 5 mL of DMF, and the solution was stirred at room temperature for 14 hours. The solvent was removed by distillation under reduced pressure, and the residue was purified by SiO.sub.2 column chromatography to obtain compound 2 (0.17 g, 90%).

[0092] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=6.62 (br, 1H), 4.96 (br, 1H), 4.46-4.49 (m, 1H), 3.74-3.77 (m, 1H), 3.51 (s, 2H), 3.34-3.37 (m, 2H), 3.28-3.31 (m, 2H), 3.18-3.24 (m, 1H), 2.80-2.85 (m, 1H), 2.34-2.40 (m, 1H), 1.90-1.98 (m, 2H), 1.63-1.80 (m, 2H), 1.44 (s, 9H).

[0093] Compound 3

##STR00007##

[0094] 10-oxo-2,3,5,6-tetrahydro-1H,4H,10H-11-oxa-3a-azabenzo[de]anthracene-9-carbaldehyde (0.12 g, 0.45 mmol), compound 2 (0.17 g, 1.1 equivalents), and piperidine (44 μL, 1.0 equivalent) were dissolved in 3 mL of 2-propanol, and the solution was heated at 60° C. for 16 hours and then cooled to room temperature. The solvent was removed by distillation under reduced pressure, and the residue was purified by SiO.sub.2 column chromatography to obtain compound 3 (0.25 g, 96%).

[0095] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=((E)-conformer) 8.63 (s, 1H), 7.94 (s, 1H), 6.99 (s, 1H), 6.55 (br, 1H), 4.97 (br, 1H), 4.29 (br, 2H), 3.28-3.38 (m, 8H), 3.06 (br, 2H), 2.86 (t, J=6.3 Hz, 2H), 2.76 (t, J=6.2 Hz, 2H), 2.36-2.42 (m, 1H), 1.94-2.00 (m, 6H), 1.71-1.80 (m, 2H), 1.44 (s, 9H).

[0096] ER-FresH A

[0097] Compound 3 (0.25 g, 0.42 mmol) was dissolved in a mixed solution of trifluoroacetic acid (TFA; 2 mL)/CH.sub.2Cl.sub.2 (2 mL), followed by stirring at room temperature for 2 hours. After the solvent was removed by distillation under reduced pressure, the remaining compound, p-toluenesulfonyl chloride (TsCl; 32 mg, 1.0 equivalent), and DIEA (58 μL, 2.0 equivalents) were dissolved in 2 mL of DMF, and the solution was stirred at room temperature for 4 hours. The solvent was removed by distillation under reduced pressure, and the residue was purified by SiO.sub.2 column chromatography to obtain ER-FReSH A (23 mg, 22%).

[0098] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=((E)-conformer) 8.63 (s, 1H), 7.92 (s, 1H), 7.72 (d, J=8.0 Hz, 2H), 7.31 (d, J=8.0 Hz, 2H), 7.00 (s, 1H), 6.52 (t, J=5.9 Hz, 1H), 5.66 (t, J=6.1 Hz, 1H), 4.26 (br, 2H), 3.32-3.38 (m, 6H), 3.02-3.08 (m, 4H), 2.85 (t, J=6.4 Hz, 2H), 2.76 (t, J=6.3 Hz, 2H), 2.41 (s, 3H), 2.36-2.42 (m, 1H), 1.86-1.99 (m, 6H), 1.68-1.77 (m, 2H).

##STR00008##

[0099] 2. Method for Preparing ER-FresH B (Formula V)

[0100] Compound 4

##STR00009##

[0101] 1-(carbobenzyloxy)-4-piperidinecarboxylic acid (0.24 g, 0.87 mmol), N-(tert-butoxycarbonyl)-4,7,10 to trioxa-1,13-tridecanediamine (0.28 g, 1.0 equivalent), HOBt (0.18 g, 1.3 equivalents), and EDCI (0.22 g, 1.3 equivalents) were dissolved in 10 mL of DMF, and the solution was stirred at room temperature for 18 hours. The resulting mixture was diluted with EtOAc and then washed with an aqueous solution of NaHCO.sub.3 and a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4 and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 4 (0.49 g, 99%).

[0102] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=7.31-7.36 (m, 5H), 6.37 (br, 1H), 5.12 (s, 2H), 4.96 (br, 1H), 4.17-4.23 (m, 2H), 3.57-3.64 (m, 10H), 3.52 (t, J=5.9 Hz, 2H), 3.35-3.39 (m, 2H), 3.19-3.23 (m, 2H), 2.81-2.86 (m, 2H), 2.20-2.26 (m, 1H), 1.73-1.83 (m, 6H), 1.61-1.68 (m, 2H), 1.43 (s, 9H).

[0103] Compound 5

##STR00010##

[0104] Pd—C (10 wt %, 49 mg) was added to a solution of compound 4 (0.49 g, 0.87 mmol) in 5 mL of MeOH, followed by stirring under H.sub.2 gas (1 atm) for 14 hours. The mixture was filtered through a celite layer, and the filtrate was distilled under reduced pressure to remove the solvent. The solid obtained by vacuum drying, cyanoacetic acid (74 mg, 1.0 equivalent), HOBt (0.17 g, 1.3 equivalents), EDCI (0.22 g, 1.3 equivalents), and DIEA (0.23 mL, 1.5 equivalents) were dissolved in 5 mL of DMF, and the solution was stirred at room temperature for 12 hours. The solvent was removed by distillation under reduced pressure, and the residue was purified by SiO.sub.2 column chromatography to obtain compound 5 (0.34 g, 79%).

[0105] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=6.53 (br, 1H), 4.95 (br, 1H), 4.43-4.47 (m, 1H), 3.75-3.79 (m, 1H), 3.57-3.66 (m, 10H), 3.53 (t, J=6.0 Hz, 2H), 3.52 (s, 2H), 3.36-3.39 (m, 2H), 3.18-3.23 (m, 3H), 2.81-2.87 (m, 1H), 2.33-2.39 (m, 1H), 1.87-1.94 (m, 2H), 1.73-1.82 (m, 5H), 1.64-1.72 (m, 1H), 1.44 (s, 9H).

[0106] Compound 6

##STR00011##

[0107] Compound 5 (0.13 g, 0.26 mmol) was dissolved in a mixed solution of TFA (2 mL)/CH.sub.2Cl.sub.2 (1 mL), followed by stirring at room temperature for 3 hours. After the solvent was removed by distillation under reduced pressure, the remaining compound and DIEA (0.12 mL, 2.6 equivalents) were dissolved in 3 mL of CH.sub.2Cl.sub.2, and the solution was cooled to 0° C. p-TsCl (63 mg, 1.3 equivalents) was added thereto, followed by stirring for 4 hours while warming to room temperature. The mixture was diluted with CH.sub.2Cl.sub.2 and then washed with a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 6 (68 mg, 47%).

[0108] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=7.77 (d, J=8.1 Hz, 2H), 7.32 (d, J=8.1 Hz, 2H), 6.62 (t, J=5.2 Hz, 1H), 5.85 (t, J=5.6 Hz, 1H), 4.42-4.46 (m, 1H), 3.67-3.73 (m, 5H), 3.62-3.64 (m, 2H), 3.58 (t, J=5.8 Hz, 2H), 3.50-3.55 (m, 6H), 3.33-3.37 (m, 2H), 3.14-3.20 (m, 1H), 3.04-3.08 (m, 2H), 2.74-2.80 (m, 1H), 2.43 (s, 3H), 2.35-2.41 (m, 1H), 1.82-1.91 (m, 2H), 1.58-1.80 (m, 6H).

[0109] ER-FresH B

[0110] 10-oxo-2,3,5,6-tetrahydro-1H,4H,10H-11-oxa-3a-azabenzo[de]anthracene-9-carbaldehyde (30 mg, 0.11 mmol), compound 6 (68 mg, 1.1 equivalents), and piperidine (11 μL, 1.0 equivalent) were dissolved in 1 mL of 2-propanol, and the solution was stirred at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the residue was purified by SiO.sub.2 column chromatography to obtain ER-FT B (77 mg, 86%).

[0111] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=((E)-conformer) 8.63 (s, 1H), 7.92 (s, 1H), 7.77 (d, J=8.2 Hz, 2H), 7.30 (d, J=8.2 Hz, 2H), 6.99 (s, 1H), 6.53 (t, J=5.3 Hz, 1H), 5.74 (t, J=5.7 Hz, 1H), 4.25 (br, 2H), 3.67-3.69 (m, 4H), 3.62-3.64 (m, 2H), 3.53-3.60 (m, 4H), 3.50 3.52 (m, 2H), 3.32-3.39 (m, 6H), 3.04-3.08 (m, 2H), 3.02 (br, 2H), 2.87 (t, J=6.5 Hz, 2H), 2.76 (t, J=6.0 Hz, 2H), 2.42 (s, 3H), 2.34-2.41 (m, 1H), 1.95-2.00 (m, 4H), 1.89-1.92 (m, 2H), 1.66-1.81 (m, 6H).

[0112] 3. Synthesis of ER-FresH C (Formula VI)

##STR00012##

[0113] Compound 7

##STR00013##

[0114] 1-(tert-butoxycarbonyl)-4-piperidinecarboxylic acid (0.30 g, 1.3 mmol), N-carbobenzyloxy-ethylenediamine hydrochloride (0.31 g, 1.0 equivalent), HOBt (0.27 g, 1.3 equivalents), EDCI (0.33 g, 1.3 equivalents), and DIEA (0.34 mL, 1.5 equivalents) were dissolved in 10 mL of DMF, and the solution was stirred at room temperature for 18 hours. The resulting mixture was diluted with EtOAc and then washed with a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 7 (0.53 g, 100%).

[0115] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=7.31-7.38 (m, 5H), 6.24 (br, 1H), 5.22 (br, 1H), 5.10 (s, 2H), 4.08-4.14 (m, 2H), 3.33-3.39 (m, 4H), 2.68-2.73 (m, 2H), 2.14-2.20 (m, 1H), 1.73-1.76 (m, 2H), 1.52-1.60 (m, 2H), 1.46 (s, 9H).

[0116] Compound 8

##STR00014##

[0117] Pd—C (10 wt %, 53 mg) was added to a solution of compound 7 (0.53 g, 1.3 mmol) in 10 mL of MeOH, followed by stirring under H.sub.2 gas (1 atm) for 2 hours. The mixture was filtered through a celite layer, and the filtrate was distilled under reduced pressure to remove the solvent. A portion (0.10 g, 0.37 mmol) of the solid obtained by vacuum drying and DIEA (0.13 mL, 2.0 equivalents) were dissolved in 3 mL of CH.sub.2Cl.sub.2, and pentafluorobenzoyl chloride (52 μL, 1.0 equivalent) was added to the solution, followed by stirring for 2 hours. The mixture was diluted with CH.sub.2Cl.sub.2 and then washed with an aqueous solution of NaHCO.sub.3 and a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 8 (0.12 g, 73%).

[0118] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=7.33 (br, 1H), 6.28 (t, J=5.3 Hz, 1H), 4.10 to 4.15 (m, 2H), 3.58-3.61 (m, 2H), 3.49-3.53 (m, 2H), 2.70 to 2.75 (m, 2H), 2.25 (tt, J=11.6 Hz, J=3.7 Hz, 1H), 1.77-1.80 (m, 2H), 1.52-1.61 (m, 2H), 1.45 (s, 9H). .sup.19F NMR (470 MHz, CDCl.sub.3): δ (ppm)=−140.8 (2F), −150.6 (1F), −159.9 (2F).

[0119] Compound 9

##STR00015##

[0120] Compound 8 (0.12 g, 0.27 mmol) was dissolved in 2 mL of HCl solution (4 M, dioxane), followed by stirring at room temperature for 2 hours. After the solvent was removed by distillation under reduced pressure, the remaining compound, cyanoacetic acid (24 mg, 1.0 equivalent), HOBt (56 mg, 1.3 equivalents), EDCI (70 mg, 1.3 equivalents), and DIEA (73 μL, 1.5 equivalents) were dissolved in 2 mL of DMF, and the solution was stirred at room temperature for 17 hours. The mixture was diluted with CH.sub.2Cl.sub.2 and then washed with a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 9 (44 mg, 37%).

[0121] .sup.1H NMR (500 MHz, DMSO-d6): δ (ppm)=8.94 (t, J=5.7 Hz, 1H), 7.91 (t, J=5.6 Hz, 1H), 4.24-4.28 (m, 1H), 3.97-4.09 (m, 2H), 3.63-3.67 (m, 1H), 3.30-3.34 (m, 2H), 3.18-3.21 (m, 2H), 3.00-3.05 (m, 1H), 2.64-2.69 (m, 1H), 2.31-2.38 (m, 1H), 1.68-1.72 (m, 2H), 1.51-1.59 (m, 1H), 1.34-1.43 (m, 1H). .sup.19F NMR (470 MHz, DMSO-d6): δ (ppm)=−141.9 (2F), −153.0 (1F), −161.4 (2F).

[0122] ER-FresH C

[0123] Compound 9 (44 mg, 0.10 mmol) and 10-oxo-2,3,5,6-tetrahydro-1H,4H,10H-11-oxa-3a-azabenzo[de]-anthracene-9-carbaldehyde (30 mg, 1.1 equivalents) were dissolved in 1 mL of DMF, and chlorotrimethylsilane (TMSCl; 39 μL, 3.0 equivalents) was added to the solution, followed by stirring at 130° C. for 5 hours. The mixture was cooled to room temperature, diluted with CH.sub.2Cl.sub.2, and then washed with a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain ER-FT C (33 mg, 48%).

[0124] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=((E)-conformer) 8.61 (s, 1H), 7.89 (s, 1H), 7.48 (br, 1H), 6.99 (s, 1H), 6.62 (br, 1H), 4.29 (br, 2H), 3.97-4.09 (m, 2H), 3.57-3.61 (m, 2H), 3.48-3.52 (m, 2H), 3.33-3.39 (m, 4H), 3.03 (br, 2H), 2.73-2.86 (m, 4H), 2.40-2.47 (m, 1H), 1.89-2.04 (m, 6H), 1.72-1.80 (m, 2H). .sup.19F NMR (470 MHz, CDCl.sub.3): δ (ppm)=−140.7 (2F), −150.8 (1F), −159.9 (2F).

[0125] Synthesis of ER-FresH D (Formula VII)

##STR00016##

[0126] Compound 10

##STR00017##

[0127] N-(tert-butoxycarbonyl)-4,7,10 to trioxa-1,13-tridecanediamine (0.10 g, 0.31 mmol) and DIEA (0.1 mL, 2.0 equivalents) were dissolved in 3 mL of CH.sub.2Cl.sub.2, and pentafluorobenzoyl chloride (44 μL, 1.0 equivalent) was added to the solution, followed by stirring for 2 hours. The mixture was diluted with CH.sub.2Cl.sub.2 and then washed with an aqueous solution of NaHCO.sub.3 and a saturated aqueous solution of NaCl. The organic solvent was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 10 (0.15 g, 94%).

[0128] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=7.18 (br, 1H), 4.88 (br, 1H), 3.65 (t, J=5.8 Hz, 2H), 3.58-3.61 (m, 6H), 3.46-3.52 (m, 6H), 3.17-3.21 (m, 2H), 1.88-1.92 (m, 2H), 1.70 1.75 (m, 2H), 1.41 (s, 9H). .sup.19F NMR (470 MHz, CDCl.sub.3): δ (ppm)=−140.7 (2F), −151.8 (1F), −160.6 (2F).

[0129] Compound 11

##STR00018##

[0130] Compound 10 (0.15 g, 0.29 mmol) was dissolved in 2 mL of HCl solution (4 M, dioxane), followed by stirring at room temperature for 2 hours. After the solvent was removed by distillation under reduced pressure, the remaining compound, 1-(tert-butoxycarbonyl)-4-piperidinecarboxylic acid (69 mg, 1.0 equivalent), HOBt (59 mg, 1.3 equivalents), EDCI (73 mg, 1.3 equivalents), and DIEA (76 μL, 1.5 equivalents) were dissolved in 3 mL of DMF, and the solution was stirred at room temperature for 16 hours. The resulting mixture was diluted with EtOAc and then washed with an aqueous solution of NaHCO.sub.3 and a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 11 (0.17 g, 93%).

[0131] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=7.31 (br, 1H), 6.24 (br, 1H), 4.07-4.12 (m, 2H), 3.53-3.63 (m, 12H), 3.49-3.52 (m, 2H), 3.33-3.37 (m, 2H), 2.70-2.75 (m, 2H), 2.18 (tt, J=11.6 Hz, J=3.6 Hz, 1H), 1.88-1.92 (m, 2H), 1.73-1.81 (m, 4H), 1.52-1.60 (m, 2H), 1.43 (s, 9H). .sup.19F NMR (470 MHz, CDCl.sub.3): δ (ppm)=−140.7 (2F), −151.8 (1F), −160.6 (2F).

[0132] Compound 12

##STR00019##

[0133] Compound 11 (0.17 g, 0.27 mmol) was dissolved in 2 mL of HCl solution (4 M, dioxane), followed by stirring at room temperature for 1 hour. After the solvent was removed by distillation under reduced pressure, the remaining compound, cyanoacetic acid (23 mg, 1.0 equivalent), HOBt (54 mg, 1.3 equivalents), EDCI (67 mg, 1.3 equivalents), and DIEA (70 μL, 1.5 equivalents) were dissolved in 2 mL of DMF, and the solution was stirred at room temperature for 18 hours. The resulting mixture was diluted with CH.sub.2Cl.sub.2 and then washed with an aqueous solution of NaHCO.sub.3 and a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain compound 12 (0.11 g, 69%).

[0134] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=7.16 (br, 1H), 6.39 (br, 1H), 4.41-4.46 (m, 1H), 3.73-3.77 (m, 1H), 3.53-3.63 (m, 12H), 3.50-3.52 (m, 4H), 3.33-3.37 (m, 2H), 3.17-3.23 (m, 1H), 2.81-2.86 (m, 1H), 2.35 (tt, J=10.8 Hz, J=4.0 Hz, 1H), 1.87-1.96 (m, 4H), 1.71-1.78 (m, 3H), 1.60 1.68 (m, 1H). .sup.19F NMR (470 MHz, CDCl.sub.3): δ (ppm)=−140.7 (2F), −151.6 (1F), −160.5 (2F).

[0135] ER-FresH D

[0136] Compound 12 (57 mg, 96 μmol) and 10-oxo-2,3,5,6-tetrahydro-1H,4H,10H-11-oxa-3a-azabenzo[de]-anthracene-9-carbaldehyde (28 mg, 1.1 equivalents) were dissolved in 1 mL of DMF, and TMSCl (16 μL, 1.3 equivalents) was added to the solution, followed by stirring at 130° C. for 14 hours. The resulting mixture was cooled to room temperature, diluted with CH.sub.2Cl.sub.2, and then washed with a saturated aqueous solution of NaCl. The organic layer was separated, dried with Na.sub.2SO.sub.4, and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The residue was purified by SiO.sub.2 column chromatography to obtain ER-FT D (31 mg, 38%).

[0137] .sup.1H NMR (500 MHz, CDCl.sub.3): δ (ppm)=((E)-conformer) 8.61 (s, 1H), 7.88 (s, 1H), 7.35 (br, 1H), 6.99 (s, 1H), 6.44 (br, 1H), 4.26 (br, 2H), 3.51-3.63 (m, 14H), 3.33-3.39 (m, 6H), 3.07 (br, 2H), 2.82-2.87 (m, 2H), 2.73-2.78 (m, 2H), 2.31-2.40 (m, 1H), 1.87-2.04 (m, 8H), 1.68-1.79 (m, 4H). .sup.19F NMR (470 MHz, CDCl.sub.3): δ (ppm)=−140.6 (2F), −151.8 (1F), −160.5 (2F).

[0138] The ER-FresH A/B/C/D synthesized in the Preparation Example are classified by R.sub.3 as shown in Table 1 below.

TABLE-US-00001 TABLE 1 R.sub.3 Compound -(CH.sub.2).sub.m-R.sub.4 Formula IV (ER-FresH A) -(CH.sub.2).sub.n-(CH.sub.2OCH.sub.2).sub.p- Formula V (ER-FresH B) (CH.sub.2).sub.q-R.sub.4 -(CH.sub.2).sub.m-R.sub.5 Formula VI (ER-FresH C) -(CH.sub.2).sub.n-(CH.sub.2OCH.sub.2).sub.p- Formula VII (ER-FresH D) (CH.sub.2).sub.q-R.sub.5

[Experimental Example] Experimental Methods

[0139] 1. In Vitro Reaction of Endoplasmic Reticulum Fluorescent Real-Time SH Group-Tracer (ER-FT) Derivative Compound (Hereinafter Referred to as any One of Formulas IV to VII) with Thiol Compound

[0140] FIG. 5 depicts confocal microscope images showing the results of observing ER-FTs represented by Formulas IV to VII in the endoplasmic reticulum of UC-MSCs.

[0141] (1) Umbilical cord mesenchymal stem cells (UC-MSCs) and HeLa cells were seeded into confocal dishes (UC-MSC: 5×10.sup.4, HeLa cells: 2×10.sup.5) and then cultured for 24 hours. (2) 2 ml of each of ER-FTs (Formulas IV to VII) was prepared for a medium for each cell type at a concentration of 10 to 20 μM. (3) Each ER-FT was added to each dish, followed by staining and then incubation in an incubator at 37° C. for 30 minutes. (4) 2 μl of 1 mM ER tracker Red was added to each dish and mixed well, followed by incubation for 30 minutes. (5) After medium suction, 2 ml of HBSS was added.

[0142] The cells were imaged with a confocal microscope, and the results are shown in FIG. 5.

[0143] 2. Cytotoxicity Test

[0144] FIG. 6 shows toxicity test results indicating the IC.sub.50 values of ER-FTs represented by Formulas IV to VII.

[0145] (1) UC-MSCs (4×10.sup.3 cells/well) were cultured in 96-well dishes for 24 hours. (2) The cells were treated with 2 ml of each of ER-FT derivative compounds (Formulas IV to VII) at concentrations of 5, 10, 20 and 40 μM and then incubated at 37° C. for 24 hours. (3) 10 μl of 10× EZ-cytox was added to each well, followed by incubation for 3 hours. (4) The absorbance at 450 nm was measured using a plate reader (reference wavelength: 600 to 650 nm).

[0146] As a result of the cytotoxicity test, as shown in FIG. 6 and Table 2 below, it was confirmed that Formula IV (ER-FreSH A) had the lowest toxicity.

TABLE-US-00002 TABLE 2 Formula Formula Formula Formula IV V VI VII IC.sub.50 (vM) 43.4 36.39 33.44 13.28

[0147] 3. Measurement of Intracellular Retention Time of Each ER-FT Derivative Compound

[0148] FIG. 7 shows the experimental results of measuring the intensity at a 510 nm wavelength, the intensity at a 580 nm wavelength, and the ratio of the intensities at the two wavelengths as a function of the retention time of ER-FTs represented by Formulas IV to VI in UC-MSCs.

[0149] FIG. 8 shows the experimental results of measuring the intensity at a 510 nm wavelength, the intensity at a 580 nm wavelength, and the ratio of the intensities at the two wavelengths as a function of the retention time of ER-FTs represented by Formulas IV, VI and VII in UC-MSCs.

[0150] (1) UC-MSCs were seeded into 96-well plates at a density of 4,000 cells/well and then cultured 24 hours. (2) Cell media containing each of ER-FTs (Formulas IV to VI) at concentrations of 10, 15 and 20 μM were prepared. (3) After medium suction, ER-FT staining was performed, followed by incubation in an incubator at 37° C. for 1 hour. (4) After medium suction, 100 μl of HBSS was added to each well.

[0151] As a result of Operetta imaging, as shown in FIGS. 7 and 8, Formulas IV and V showed constant fluorescence intensity in UC-MSCs. The fluorescence intensities of Formulas VI and VII were high at the initial stage, but decreased gradually during the initial 20 minutes, and then were similar to that of the other ER-FT.

[0152] As shown in FIGS. 7 and 8, taking all the results together, Formulas VI and VII remained in the cells for a long time, and the time to wash was required. In addition, after they were completely washed, they showed similar fluorescence intensities.

[0153] 4. Measurement of Reactivity of Each ER-FT Derivative Compound

[0154] FIG. 9 shows confocal microscope images of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula IV.

[0155] FIG. 10 shows confocal microscope images of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula IV.

[0156] FIG. 11 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula V.

[0157] FIG. 12 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula V.

[0158] FIG. 13 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula VI.

[0159] FIG. 14 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula VI.

[0160] FIG. 15 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide was added after treatment with the ER-FT represented by Formula VII.

[0161] FIG. 16 shows confocal microscope images of the endoplasmic reticulum of UC-MSCs to which diamide and DTT were added after treatment with the ER-FT represented by Formula VII.

[0162] (1) Cells (UC-MSC: 5×10.sup.4) were seeded into confocal dishes and then cultured for 24 hours. (2) 2 ml of each cell type medium containing each of ER-FTs (Formulas IV to VI) at a concentration of 10 to 20 μM was prepared. (3) After medium suction from the dishes, ER-FT staining was performed, followed by incubation in an incubator at 37° C. for 30 minutes. (4) 2 μl of 1 mM ER tracker Red was added to each medium and mixed well, followed by incubation for 30 minutes. (5) After medium suction, 2 ml of HBSS was added. (6) After the ER targeting experiment, 10 μl of 100 mM diamide was pipetted into the cells where the laser did hit. (7) Changes in the cells were observed through confocal imaging. (8) 20 μl of 1M DTT was pipetted in the same way as above. (9) Changes in the cells were observed through confocal imaging.

[0163] As shown in FIGS. 9 to 16, the four types of ER-FTs (Formulas IV to VII) all showed a decrease in the fluorescence intensity at 510 nm and an increase in the fluorescence intensity at 580 nm when treated with diamide, and showed an increase in the fluorescence intensity at 510 nm and a decrease in the fluorescence intensity at 580 nm when treated with DTT.

[0164] Using the compound or composition according to the present invention, it is possible to measure the antioxidant activities of the endoplasmic reticulum (ER) and Golgi apparatus, which are organelles in living cell. When the compound or composition is applied to stem cells, it is possible to screen highly active stem cells based on the results of measurement of antioxidant activity in stem cells, thereby increasing the efficiency of cell therapy products.

[0165] All the references, articles, publications, patents and patent applications cited in this specification are incorporated herein in their entirety. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiments disclosed herein.

INDUSTRIAL APPLICABILITY

[0166] The present invention relates to a real-time fluorescence imaging sensor for measuring glutathione in the endoplasmic reticulum (ER) and a method using the same. More specifically, the present invention relates to a novel compound for measuring glutathione in the endoplasmic reticulum (ER) and a method of measuring glutathione in the endoplasmic reticulum (ER) using the novel compound.