KR102089393B1 - Fluorescent probe for detecting NAD(P)H in mitochondria, and method for detecting using the same - Google Patents

Abstract

The present invention relates to a fluorescent probe for detecting NAD(P)H in mitochondria and a detecting method using the same. The probe of the present invention shows high selectivity and sensitivity to NAD(P)H, and emits strong red fluorescence in the presence of the NAD(P)H. Thus, the probe shows a predominant accumulation in the mitochondria of living cells, can be used to detect and quantify the NAD(P)H in the mitochondria, can provide a selective image of the mitochondria in real time, and can be useful for revealing biological pathway research and pathological diagnosis. In addition, it can also be used to differentiate cancer cells from normal cells based on the mapping of NAD(P)H dependent fluorescence intensity in the mitochondria.

Description

Fluorescent probe for detecting NAD (P) H in mitochondria, and method for detecting using the same}

The present invention relates to a fluorescent probe for detecting NAD (P) H in mitochondria and a detection method using the same.

Reduced nicotinamide adenine dinucleotide (NADH) and its phosphate ester (NADPH) are essential biomolecules found in all living cells. NAD (P) H plays an important role as a coenzyme of several biological processes, including energy metabolism, immune function, biosynthesis, cell death and aging. Abnormal levels of NAD (P) H are associated with neoplasia, cancer, ischemia and diabetes. In particular, NAD (P) H located in the mitochondria is essential for ATP production by the mitochondrial respiratory chain, as well as maintenance of mitochondrial antioxidant defense and regulation of mitochondrial membrane potential. Abnormal levels of NAD (P) H in mitochondria cause mitochondrial dysfunction, generation of free radicals (ROS), DNA damage, and are involved in stroke, Alzheimer's disease and Parkinson's disease, and recently NAD (P) H in mitochondria Has been reported to be overexpressed in various cancers.

To date, to detect NAD (P) H in vitro, several methods such as high-performance liquid chromatography (HPLC), enzymatic cycling assay, and capillary electrophoresis Was used. In addition, NAD (P) H has been used for quantification and imaging of human-derived biospecimens. It is known that NAD (P) H absorbs light of about 340 nm and exhibits a fluorescence signal at about 450 nm. However, the quantum yield is relatively low (Ф _F = 0.019), and the short excitation and emission wavelengths are easily interfered by autofluorescence of other biomolecules. Recently, some fluorescent off-on probes have been developed for the selective detection of NAD (P) H in human cells. However, these probes operate in a non-specific manner and cannot be used for mitochondrial specific analysis. Therefore, there is a need to develop a new fluorescent probe capable of detecting real-time NAD (P) H with long excitation and emission wavelengths and high quantum efficiency.

Republic of Korea Patent Publication No. 10-2017-005658 (2017.01.16 published)

It is an object of the present invention to provide novel compounds or pharmaceutically acceptable salts thereof.

Another object of the present invention is to provide a composition for detecting NAD (P) H in a mitochondria, a fluorescent chemical sensor for detecting NAD (P) H, or a kit for detecting NAD (P) H.

Another object of the present invention is to provide a composition for detecting cancer cells or a kit for detecting cancer cells.

Another object of the present invention is to provide a method for detecting NAD (P) H in mitochondria.

In order to achieve the above object, the present invention provides a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof.

In addition, the present invention is a composition for detecting NAD (P) H in a mitochondrial, a fluorescence chemical sensor for detecting NAD (P) H, or NAD comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient A kit for detecting (P) H is provided.

In addition, the present invention provides a composition for detecting cancer cells or a kit for detecting cancer cells comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention comprises the steps of (a) treating the compound of formula 1 according to claim 1 or a pharmaceutically acceptable salt by treating the cells; And (b) measuring any one or more indicators selected from the group consisting of the fluorescence wavelength, fluorescence intensity, and optical change of the reactants reacted to provide a method for detecting NAD (P) H in mitochondria.

[Formula 1]

In the above formula, X is a mitochondrial target moiety.

The probe of the present invention shows high selectivity and sensitivity to NAD (P) H and emits strong red fluorescence in the presence of NAD (P) H. Thus, the probe shows a predominant accumulation in the mitochondria of living cells, and can be used to detect and quantify NAD (P) H in the mitochondria, and can provide a selective image of the mitochondria in real time, biological pathway research and pathology It can be useful for revealing diagnosis, etc. It can also be used to differentiate cancer cells from normal cells based on the mapping of NAD (P) H dependent fluorescence intensity in mitochondria.

1 shows a NAD (P) H detection mechanism using probe 1 of the present invention.
Figure 2 shows the synthesis of probes 1 and 2 in a scheme.
FIG. 3 shows (a) absorption of probe 1 (10 μM) in the absence and presence of NADH (2 mM) and (b) fluorescence spectrum, (c) for probe 1 (10 μM) when NADH (2 mM) is added. The fluorescence spectrum of probe 1 (10 μM) was confirmed according to the increase in time-dependent fluorescence and (d) NADH (0-3 mM) concentration.
Figure 4 confirms the relative fluorescence intensity for probe 1 (10 μM) in the presence of various biologically relevant metals, anions, reactive oxygen species, thiols, NADPH, NADH and other biomolecules (1: probe 1, 2: K ⁺ , 3: Na ⁺ , 4: Cu ⁺ , 5: Cu ²⁺ , 6: Ca ²⁺ , 7: Co ²⁺ , 8: Mg ²⁺ , 9: Zn ²⁺ , 10: Fe ²⁺ , 11: Fe ^{3 +, 12: Cl -, 13} : CN -, 14: H 2 PO 4 -, 15: OAc -, 16: OH -, 17: ClO -, 18: H 2 O 2, 19: HOO t Bu, 20: ^{_{· O 2 -, 21: ·}} OH -, 22: · O t Bu, 23: Cys, 24: GSH, 25: Hcy, 26: H 2 S, 27: FADH 2, 28: ATP, 29: ADP, 30 : Glucose, 31: NAD ⁺ , 32: NADPH, 33: NADH).
Figure 5 confirms whether the cytotoxicity of probe 1 in MDA-MB-231 cells.
FIG. 6 shows the (a) simultaneous staining of probe 1 and organelle tracker in living MDA-MB-231 cells, and (b) confirmation of the degree of coexistence between probe 1 and organelle tracker.
Figure 7 relates to confocal microscopy images of glucose or FCCP-treated MDA-MB-231 cells, (a) probe 1 (5 μM) alone, (b) glucose (20 mM), and then probe 1 (5 μM) addition, (c) FCCP (5 μM) addition, probe 1 (5 μM) addition, and (d) fluorescence intensity of probe 1 in cells was confirmed.
Figure 8 relates to confocal microscopy images in the co-culture of NIH-3T3 and MDA-MB-231 cells, (a) after adding Calcein AM (2 μM) to NIH-3T3 cells, with MDA-MB-231 cells After incubation, probe 1 (5 μM) was added, and (b, c) shows the fluorescence intensity of probe 1 in living MDA-MB-231 and NIH-3T3 cells.

The inventors of the present invention can be utilized to quantify NAD (P) H in the mitochondria of living cells as the probe developed in the present invention is reduced in the presence of non-fluorescence or NAD (P) H to show strong red fluorescence. The present invention was completed while confirming that real-time quantitative analysis is possible.

Accordingly, the present invention provides a compound represented by Formula 1 below, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In the above formula, X is a mitochondrial target moiety.

The mitochondrial target moiety may be selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine, but is not limited thereto.

Preferably, the compound or a pharmaceutically acceptable salt thereof may be represented by Formula 2 below.

[Formula 2]

In addition, the present invention provides a composition for detecting NAD (P) H in mitochondria comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the above formula, X is a mitochondrial target moiety.

The mitochondrial target moiety may be selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine, but is not limited thereto.

In addition, the present invention provides a fluorescence chemical sensor for detecting NAD (P) H in mitochondria comprising the compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the above formula, X is a mitochondrial target moiety.

The mitochondrial target moiety may be selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine, but is not limited thereto.

In addition, the present invention provides a kit for detecting NAD (P) H in mitochondria comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the above formula, X is a mitochondrial target moiety.

The mitochondrial target moiety may be selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine, but is not limited thereto.

In addition, the present invention provides a composition for detecting cancer cells comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the above formula, X is a mitochondrial target moiety.

The mitochondrial target moiety may be selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine, but is not limited thereto.

In addition, the present invention provides a cancer cell detection kit comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the above formula, X is a mitochondrial target moiety.

The mitochondrial target moiety may be selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine, but is not limited thereto.

In addition, the present invention comprises the steps of (a) treating the compound of formula 1 according to claim 1 or a pharmaceutically acceptable salt by treating the cells; And (b) measuring any one or more indicators selected from the group consisting of the fluorescence wavelength, fluorescence intensity, and optical change of the reactants reacted to provide a method for detecting NAD (P) H in mitochondria.

The indicator may be measured by using any one or more methods selected from the group consisting of UV-Vis (Ultraviolet-visible) spectrophotometer, fluorescence photometer, electrospray ionization mass spectrometry, and confocal microscopy, but is not limited thereto. State.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Synthetic materials and methods

All reagents were purchased from Alfa (Alfa, Heysham, LA3 2XY, United Kingdom), Aldrich (Aldrich, St. Louis, MO, USA) and TCI (TCI, Tokyo, Japan) and used without further purification. All solvents were used for analytical or HPLC. HR-ESI-MS data were obtained using a liquid chromatography mass spectrometer (LC / MS) from the Korea Basic Science Institute (Seoul). NMR spectrum data was obtained using a Bruker (500 MHz) instrument.

Example 2: UV / Vis absorption and fluorescence spectroscopy analysis

As the solvent used in the spectroscopy experiment, HPLC grade without fluorescent impurities was used, and deionized water was used for water. The PIPES buffer (pH7.4) deionized 25 mM piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES) and 100 mM NaCl. The stock solution of the synthetic probe was prepared using dimethyl sulfoxide (DMSO) All spectra of the probe were PIPES buffer (25 mM, pH) with 2% (v / v) DMSO added 7.4) Absorption and fluorescence spectroscopy data were obtained using a spectrophotometer (UV-2600, Shimadzu Corporation, Kyoto, Japan), measured with an excitation wavelength of 570 nm and an excitation / emission slit width of 5 nm. Did.

Example 3: Cell culture and cytotoxicity analysis

For cell culture, DMEM medium, phosphate buffered saline (PBS), fetal bovine serum (FBS), HBSS (Hank's balanced salt solution) added with trypsin 0.25% -EDTA and penicillin / streptomycin Was used. MitoTracker Green FM (Mitochondria-Tracker) and ER-Tracker Green dye (endoplasmic reticulum (ER) -Tracker) and LysoTracker Green DND-26 (Lysosome-Tracker) were purchased from Invitrogen (Carlsbad, CA). MTT was purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. UV-Vis absorbance was performed using a Spectra Max i3x microplate reader (Molecular devices, San Jose, CA). Fluorescence images were obtained using a Zeiss LSM-700. Red (probe 1) and green (tracer) fluorescence were obtained using excitation at 488 nm and 555 nm and band-path emission filters at 300-578 nm and 568-800 nm, respectively.

Example 4: In vitro fluorescence microscopy analysis

The breast adenocarcinoma cell line (MDA-MB-231) was cultured in a 37%, 5% CO ₂ incubator using high glucose DMEM medium with 10% fetal calf serum and 1% penicillin / streptomycin added, and fresh medium every 2 days. Was replaced with. When the cells grew to 80-90%, the medium was removed, washed with PBS, PBS was removed, and 1.5 mL of trypsin 0.25% -EDTA was added, followed by incubation for 3 minutes in a 37 ° C, 5% CO ₂ incubator. After separating the cells from the flask, 1 mL of medium was added to the flask and mixed. Thereafter, the cell suspension was centrifuged at 1000 rpm for 3 minutes to remove the supernatant, and the pellet was resuspended by adding 3 mL of fresh medium to the cell pellet. MDA-MB-231 cells were cultured for 24 hours in a coverslip of a 35 mm confocal dish at a concentration of 10 ⁵ cells / mL (37 ° C., 5% CO ₂ ), and then the cells were washed with PBS and 5 μM. Probe 1 (1 mM stock solution) was added to the cells. Fluorescence images of the cells were acquired every 10 minutes for 2 hours using a confocal microscope.

Example 5: Cytotoxicity analysis

MDA-MB-231 cells were dispensed into a 96 well plate at a concentration of 1 X 10 ⁴ cells / well and incubated for 24 hours to allow the cells to adhere to the bottom of the plate. Thereafter, the medium was removed, and probe 1 was added to the cells at concentrations of 1, 3, 5, 10, 15, 20, 30 μM, and only the medium was added to the control group. After incubation for 6 hours, probe 1 was removed and washed with PBS. Then MTT reagent (0.5 mg / mL) was added to each well and further incubated for 3 hours. After 3 hours, the MTT reagent was removed and DMSO (100 μl / well) was added to each well, then the plate was gently shaken and the formazan crystals were dissolved for 30 minutes. Absorbance was read at 550 nm using a Spectra Max i3x microplate reader.

Example 6: Confocal microscopy image analysis

MDA-MB-231 cells were dispensed onto the glass cover slip of the confocal dish without surface treatment and attached for 24 hours. Then, after removing the medium and washing with PBS, before staining with probe 1, the organelle tracker MitoTracker® Green FM (Mitochondria, 100 nM), ER-Tracker ™ Green dye (ER, 100nM) and LysoTracker® Green DND- 26 (Lysosome, 100 nM) was treated and stained for 30 minutes. Cells were stained with one of the organelle trackers, and subsequently probe 1 was stained for 90 minutes. Fluorescence images were acquired every 10 minutes using a confocal microscope.

Prior to mixing with MDA-MB-231 cells, NIH-3T3 cells were stained with Calcein AM and the mixture of cells was co-cultured for 24 hours on a glass cover slip of a confocal dish. The mixed cells were stained with probe 1 for 2 hours, visualized with a confocal fluorescence microscope (Zeiss LSM-700, Axio Observer), and the fluorescence image was obtained using a Plan-Apochromat 63X / 1.40 oil immersion lens. Mito-Tracker Green FM, ER-Tracker Green dye, and Lyso-Tracker Green DND-26 were excited at 488 nm, and emission fluorescence was obtained at 300-578 nm using a band emission filter. Image analysis was performed using ZEN software and Image J software.

Example 7: Synthesis of Probe 1

Precursor 3 (1.00 g, 3.63 mmol) was added to a mixture of (3-bromopropyl) triphenylphosphonium bromide (1.68 g, 3.61 mmol) and NaI (544 mg, 3.62 mmol) dissolved in CH ₃ CN (10 mL). Was added. The resulting reaction mixture was stirred overnight and refluxed. After cooling to room temperature, the solvent was evaporated and CH ₂ Cl ₂ (3 X 100 mL) and brine (100 mL) were added to obtain a CH ₂ Cl ₂ layer. The CH ₂ Cl ₂ layer was dried over Na ₂ SO ₄ , filtered and concentrated, and silica gel chromatography using CH ₂ Cl ₂ / MeOH / ethyl acetate (v / v / v, 8: 1: 1) as eluent. Purification by chromatography gave a brown solid (1) (270 mg, 13%).

¹ H NMR (DMSO- d ₆ , 500 MHz): δ 1.09 (s, 6H), 2.48-2.56 (m, 2H), 2.62 (s, 2H), 2.67 (s, 2H), 3.78-3.84 (m, 2H), 5.04 (t, J = 7.5 Hz, 2H) , 7.05 (s, 1H), 7.37 (d, J = 16 Hz, 1H), 7.64 (d, J = 16.5 Hz, 1H), 7.74-7.78 (m, 5H), 7.86-7.90 (m, 10H), 8.06-8.09 (m, 1H), 8.78 (d, J = 8 Hz, 1H), 9.02 (d, J = 6 Hz, 1H), 9.60 (s, 1H) ppm. ¹³ C NMR (DMSO- d ₆ , 125 MHz): δ 18.2, 18.6, 24.0, 28.0, 32.3, 38.6, 42.7, 55.6, 60.7, 79.6, 113.1, 113.9, 118.0, 118.7, 125.9, 130.0, 130.93, 134.2, 135.6, 136.8, 143.1, 144.1, 154.1, 170.4 ppm. HR-ESI-MS m / z [M] ²⁺ calc. 289.63962, obs. 289.64233.

Example 8: Synthesis of probe 2

To a solution of precursor 3 (270 mg, 0.98 mmol) dissolved in CH ₂ Cl ₂ (25 mL) was added methyl trifluoromethanesulfonate (0.55 mL, 4.89 mmol). The resulting reaction mixture was stirred at room temperature for 3 hours. Thereafter, the yellow precipitate was filtered, washed with CH ₂ Cl ₂ and diethyl ether, and then dried under vacuum to give a pale yellow solid (2) (182 mg, 63%).

¹ H NMR (DMSO- d ₆ , 500 MHz) ^: δ 1.04 (s, 6H), 2.55 (s, 2H), 2.68 (s, 2H), 4.35 (s, 3H), 6.97 (s, 1H), 7.36 (d, J = 16.5 Hz, 1H), 7.71 (d, J = 16 Hz, 1H), 8.14-8.17 (m, 1H), 8.75 (d , J = 8 Hz, 1H), 8.89 (d, J = 6 Hz, 1H), 9.29 (s, 1H) ppm. ¹³ C NMR (DMSO- d ₆ , 125 MHz): δ 27.9, 32.2, 38.4, 42.6, 48.6, 79.8, 113.1, 113.8, 119.9, 122.4, 126.0, 128.1, 129.9, 135.8, 136.4, 142.4, 144.9, 153.9, 170.3 ppm. HR-ESI-MS m / z [M] ⁺ calc. 290.16517, obs. 290.156801H NMR (DMSO- d ₆ , 500 MHz): δ 1.04 (s, 6H), 2.55 (s, 2H), 2.68 (s, 2H), 4.35 (s, 3H), 6.97 (s, 1H), 7.36 (d, J = 16.5 Hz, 1H), 7.71 (d , J = 16 Hz, 1H), 8.14-8.17 (m, 1H), 8.75 (d, J = 8 Hz, 1H), 8.89 (d, J = 6 Hz, 1H), 9.29 (s, 1H) ppm. ¹³ C NMR (DMSO- d ₆ , 125 MHz): δ 27.9, 32.2, 38.4, 42.6, 48.6, 79.8, 113.1, 113.8, 119.9, 122.4, 126.0, 128.1, 129.9, 135.8, 136.4, 142.4, 144.9, 153.9, 170.3 ppm. HR-ESI-MS m / z [M] ⁺ calc. 290.16517, obs. 290.15680.

Experimental Example: Effect of a fluorescent probe for NAD (P) H detection in mitochondria

The synthetic reaction schemes of probes 1 and 2 are shown in FIG. 2. Probes 3, 4 and (3-bromopropyl) triphenyl-phosphonium bromide were prepared with reference to conventional methods (Sens. Actuators B Chem. 2016, 222, 48-54., Cryst. Growth Des. 2013, 13 , 1978-1987., Biomaterials 2016, 107, 33-43.). Chemical structures of probes 1 and 2 were confirmed by ¹ H, ¹³ C NMR spectroscopy and HR-ESI-MS.

Absorption and fluorescence changes of probe 1 in the presence of NADH were performed in a PIPES buffer (pH 7.4, 25 mM) with 2% (v / v) DMSO added. Probe 1 did not exhibit distinct absorption and emission bands in the visible and far infrared regions. However, in the presence of NADH, probe 1 showed a new absorption band at 570 nm, and visual color change from yellow to purple was confirmed, and it was confirmed that visual inspection of NADH was possible (FIG. 3A). Probe 1 at 570 nm excitation showed a new emission band at 615 nm, and showed a visual color change with bright red fluorescence (FIG. 3B). In addition, the fluorescence quantum yield was measured using crystal violet perchlorate (Ф _F = 0.22) as a standard, and probe 1 reacted with cellular NAD (P) H, resulting in fluorescence off-on in the far-infrared emission region. It was confirmed that it exhibits a change, which means that it can be easily detected without interference of its own fluorescence that does not depend on the analyte.

The time dependent fluorescence change of probe 1 was observed in the presence of NADH. When NADH (2 mM) was added to the probe 1 (10 μM) solution, a fluorescence signal was detected at 615 nm and gradually increased at 37 ° C. for 23 hours (FIG. 3C).

Quantitative analysis of NADH was performed by dissolving probe 1 in PIPES buffer (pH 7.4, 25 mM) with 2% (v / v) DMSO added. When the concentration of NADH was increased from 0 to 3 mM, it was confirmed that the fluorescence intensity gradually increased at 615 nm (FIG. 3D). The plot of NADH concentration versus fluorescence intensity at 615 nm showed good linearity for NADH concentrations between 0 and 800 μM, and the detection limit of NADH was 1.5 μM (insertion in FIG. 3D).

As a result of analyzing the relative fluorescence intensity for probe 1 (10 μM) in the presence of various biologically relevant metals, anions, reactive oxygen species, thiols, NADPH, NADH and other biomolecules, probe 1 was apparent at 615 nm in the presence of NADH and NADPH. Fluorescence increased (FIG. 4).

This shows that Probe 1 produces a highly selective fluorescence image for cellular NAD (P) H without interference from other biologically relevant species.

When it was confirmed that Probe 1 exhibited excellent selectivity for NADH, it was intended to prove the cell activity of Probe 1 in detecting NADH in mitochondria of living breast cancer cells (MDA-MB-231). First, cells were treated with various concentrations of Probe 1 and cultured for 6 and 24 hours to evaluate cytotoxicity.

As a result, it was confirmed that when treated at a high concentration of 30 μM for 6 hours, cytotoxicity was not observed, and when treated for 24 hours, cytotoxicity slightly increased with 80% viability (FIG. 5).

To determine whether probe 1 selectively targets mitochondria in living cells, probe 1 and organelle markers were each stained simultaneously. As a result, looking at the combined image of probe 1 (red) and mitochondrial marker (green), it was confirmed that the two stains coexist in most cells, whereas in the combined image of probe 1 and lysosomal marker, or probe 1 and vesicle marker No coexistence of staining was observed.

As a result of analyzing the Pearson correlation coefficient (PCC) value, a statistically significant correlation (r = 0.98) was found between the probe 1 and the mitochondrial marker, but the probe 1 and the lysosomal marker, or the probe 1 and the vesicle marker were statistically Non-significant correlations (0.63, 0.81) were shown (Fig. 6).

These results demonstrate that probe 1 has a strong positive correlation with the commercially available mitochondrial marker dye and is located in the mitochondria of living cells.

Next, an experiment was performed to prove that the fluorescence signal obtained from probe 1 was induced by NAD (P) H of mitochondria. Two groups of cells with different conditions were prepared and transient fluorescence images were obtained. Since glucose-mediated NAD (P) H production occurs through glycolysis, pyruvic acid oxidation, and citric acid circulation, MDA-MB-231 cells were pre-incubated with glucose as an NAD (P) H boosting group.

On the other hand, carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone; FCCP] interferes with ATP synthesis by delivering protons through the mitochondrial inner membrane, thereby inducing the oxidation of mitochondrial NADH through NADH dehydrogenase by pre-culturing MDA-MB-231 cells with FCCP as a group depleted of NAD (P) H. Did.

As a result, cells pre-treated with FCCP showed lower fluorescence intensity than control cells, whereas cells pre-treated with glucose showed strong fluorescence signals (FIG. 7).

These results show that probe 1 generates a fluorescence signal dependent on mitochondrial NAD (P) H.

Since cancer cells grow and differentiate faster than normal cells, the fluorescence signal of probe 1 was compared in the mitochondria of living cancer cells (MDA-MB-231) and fibroblasts (NIH-3T3).

As a result, the signal of the fluorescence image between MDA-MB-231 cells and NIH-3T3 cells showed a clear difference (Fig. 8). When compared to MDA-MB-231 cells, NIH-3T3 cells stained with Calcein AM (green) confirmed that the red fluorescence signal of probe 1 was weak.

These results indicate that probe 1 can be used to detect cancer cells surrounded by normal cells by mapping differences in fluorescence intensity.

The specific parts of the present invention have been described in detail above, and it is clear that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (15)

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

In the above formula, X is a mitochondrial target moiety. The compound of claim 1, wherein the mitochondrial targeting moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine, or a pharmaceutically acceptable compound thereof. Possible salts. According to claim 1, wherein the compound or a pharmaceutically acceptable salt thereof is a compound or a pharmaceutically acceptable salt thereof, characterized in that represented by the following formula (2):
[Formula 2]

A composition for detecting NAD (P) H in mitochondria comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In the above formula, X is a mitochondrial target moiety. 5. The NAD (P) H in mitochondria according to claim 4, wherein the mitochondrial targeting moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine. Detection composition. Fluorescence chemical sensor for NAD (P) H detection in mitochondria comprising a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In the above formula, X is a mitochondrial target moiety. 7. The NAD (P) H in mitochondria according to claim 6, wherein the mitochondrial target moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine. Fluorescence chemical sensor for detection. Kit for detecting NAD (P) H in mitochondria comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In the above formula, X is a mitochondrial target moiety. 9. The NAD (P) H in mitochondria according to claim 8, wherein the mitochondrial targeting moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine. Detection kit. A composition for detecting cancer cells comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In the above formula, X is a mitochondrial target moiety. 11. The composition for detecting cancer cells according to claim 10, wherein the mitochondrial targeting moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine. Cancer cell detection kit comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In the above formula, X is a mitochondrial target moiety. The kit for detecting cancer cells according to claim 12, wherein the mitochondrial targeting moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine. (A) the step of reacting the compound of formula 1 according to claim 1 or a pharmaceutically acceptable salt to the cell; And
(b) measuring any one or more indicators selected from the group consisting of fluorescence wavelength, fluorescence intensity, and optical change of the reacted reactant; a method for detecting NAD (P) H in a mitochondrial comprising a. 15. The method of claim 14, wherein the indicator is characterized by measuring any one or more selected from the group consisting of UV-Vis (Ultraviolet-visible) spectrophotometer, fluorescent photometer, electrospray ionization mass spectrometry and confocal microscopy NAD (P) H detection method in mitochondria.

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