WO2020036329A1 - Fluorescent probe for detecting nad(p)h in mitochondria, and detection method using same - Google Patents

Abstract

The present invention relates to a fluorescent probe for detecting NAD(P)H in mitochondria, and a detection method using same. A probe of the present invention exhibits high selectivity and sensitivity to NAD(P)H and emits strong red fluorescence in the presence of NAD(P)H. Therefore, the probe exhibits predominant accumulation in the mitochondria of living cells, is usable in the detection and quantification of NAD(P)H in the mitochondria, and can provide a selective image of the mitochondria in real time, thereby being effectively usable for biological pathway studies and the identification of pathological diagnoses and the like. In addition, the present invention can be used to differentiate cancer cells from normal cells on the basis of mapping of the intensity of NAD(P)H-dependent fluorescence in mitochondria.

Description

Fluorescent probe for detecting NAD (P) H in mitochondria and detection method using 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 phosphate esters thereof (NADPH) are essential biomolecules found in all living cells. NAD (P) H plays an important role as a coenzyme of many biological processes, including energy metabolism, immune function, biosynthesis, cell death and aging. Abnormal levels of NAD (P) H are associated with neoplasia, cancer, ischemia, diabetes and the like. In particular, NAD (P) H located in the mitochondria is essential for the maintenance of mitochondrial antioxidant defenses and the regulation of mitochondrial membrane potential, as well as ATP production by mitochondrial respiratory chain. Abnormal levels of NAD (P) H in mitochondria cause mitochondrial dysfunction, production of reactive oxygen species (ROS), DNA damage, etc., 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, the detection of NAD (P) H in vitro involves several methods, such as high-performance liquid chromatography (HPLC), enzymatic cycling assays, and capillary electrophoresis. This was used. NAD (P) H has also been used for quantification and imaging of human derived biospecimens. NAD (P) H is known to absorb light of about 340 nm and display a fluorescent signal at about 450 nm. However, the quantum yield is relatively low (Ф _F = 0.019), and short excitation and emission wavelengths are easily interfered by the 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 nonspecific manner and are not available for mitochondrial specific analysis. Accordingly, there is a need for the development of new fluorescent probes capable of detecting real-time NAD (P) H with long excitation and emission wavelengths and high quantum efficiency.

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 mitochondria, a fluorescent chemical sensor for detecting NAD (P) H, or a kit for detecting NAD (P) H.

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

Still 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 the following formula (1) or a pharmaceutically acceptable salt thereof.

The present invention also provides a composition for detecting NAD (P) H in mitochondria, a fluorescence chemical sensor for detecting NAD (P) H, or NAD comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient: Provided are (P) H detection kits.

In another aspect, the present invention provides a composition for detecting cancer cells or a kit for detecting cancer cells comprising a compound represented by the following 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 to the cell by reacting; 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. The method provides a method for detecting NAD (P) H in mitochondria.

[Formula 1]

Wherein X is a mitochondrial target moiety.

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

1 illustrates 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) upon addition of NADH (2 mM). Fluorescence spectra of probe 1 (10 μM) were confirmed with increasing time-dependent fluorescence and (d) concentration of NADH (0-3 mM).

4 shows 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).

5 shows the cytotoxicity of probe 1 in MDA-MB-231 cells.

Figure 6 shows (a) simultaneous staining of probe 1 and organelle tracker in live MDA-MB-231 cells, and (b) the degree of coexistence between probe 1 and organelle tracker.

7 is a confocal microscopy image of glucose or FCCP treated MDA-MB-231 cells, (a) probe 1 (5 μM) alone, (b) glucose (20 mM), followed by probe 1 (5) μM) addition, (c) after adding FCCP (5 μM), adding probe 1 (5 μM), and (d) fluorescence intensity of probe 1 in the cells.

Figure 8 relates to confocal microscopy images of co-culture of NIH-3T3 and MDA-MB-231 cells, (a) after addition of Calcein AM (2 μM) to NIH-3T3 cells, MDA-MB-231 cells After incubation, probe 1 (5 μM) was added, (b, c) fluorescence intensity of probe 1 in live MDA-MB-231 and NIH-3T3 cells.

9 shows the H ₂ S sensing mechanism of Compound 1 based on fluorescence (AIE) induced by aggregation.

10 is a diagram showing (a) ¹ H NMR spectrum, (b) ¹³ C NMR spectrum, (c) HR-ESI-MS spectrum of Compound 1. FIG.

FIG. 11 is a diagram showing (a) ¹ H NMR spectrum, (b) ¹³ C NMR spectrum, and (C) HR-ESI-MS spectrum of Compound 2. FIG.

12 is a diagram showing the reaction mechanism of Compound 1 in the presence of H ₂ S.

FIG. 13 is a plot of fluorescence intensity (FI) plots at (a) absorbance, (b) fluorescent spectra, and (c) 471 nm of Compound 1 according to f _w . FIG.

14 shows (a) absorbance, (b) fluorescence spectra, and (c) fluorescence intensity (FI) plots at 500 nm according to f _w .

FIG. 15 shows (a) absorbance, (b) fluorescent spectra and (c) fluorescence intensity (FI) plots at 480 nm of Compound 2 according to f _w . FIG.

FIG. 16 shows (a) UV / Vis absorbance, (b) fluorescence spectra of compounds 1, 2 and 3 with and without 20 equivalents of NaHS (equiv.), And c) FE-SEM image, (d) a diagram showing the DLS size distribution.

FIG. 17 shows FE-SEM images and DLS size distributions of (a, b) Compound 1, (c, d) Compound 2, and (e, f) Compound 3, respectively.

FIG. 18 shows (a) fluorescence reaction of Compound 1 with various acidic reducing species (20 equivalents), (b) fluorescence changes at different concentrations of NaHS, (c) fluorescence intensity at 480 nm according to NaHS concentration, (d) Fluorescence change with time of Compound 1 with and without 20 equivalents (equiv.) of NaHS (black line).

FIG. 19 shows (a) fluorescence spectra of Compound 1 for compounds with various metal ions, anions, redox species and thiol groups, and (b) fluorescence intensity (FI) at 480 nm of Compound 1 in the presence of various metal ions and anions. The figure which shows the change.

FIG. 20 is a plot of UV / Vis absorbance of Compound 1 at different concentrations of NaHS (0-34 equivalent). FIG.

FIG. 21 shows fluorescence changes of (a) Compound 1 and (b) Compound 2 with respect to excess amount of various thiol groups.

FIG. 22 is a diagram showing a TLC analysis of Compound 1 for (a) 0.8 equivalent of NaHS and (b) 50 equivalent of NaHS.

FIG. 23 is a plot of the fluorescence intensity (FI) plot of Compound 1 against NaHS concentration at 480 nm.

FIG. 24 shows the fluorescence spectra of Compound 1 over time in the presence of a NaHS 20 equivalent. FIG.

FIG. 25 shows fluorescence changes of Compound 1 relative to NaHS 20 equivalents at pH 1-11.

FIG. 26 shows (a) a TLC plate immersed in a DCM solution of Compound 1, (b) a TLC plate coated with Compound 1 exposed to vapor comprising a volatile solution, and (c) after exposure to various vapors. Fluorescence of the coated TLC plate, (d) is a diagram showing the fluorescence change of the TLC plate coated with Compound 1 when exposed to increasing concentration of NaHS.

FIG. 27 is a fluorescence of TLC plates coated with Compounds 1, 2, and 3. FIG.

28 is a result of confirming the standard absorption (Abs.) And fluorescence (Em.) Spectrum of each compound in water containing 10% DMS0, Figure 28 (a) is Compound 1, 28 (b) is Compound 2, Figure 28 (c) is a compound 3 and Figure 28 (d) is a result of compound 4, each compound is 5 μM concentration and the inserted picture is a result of checking the visual and fluorescence color.

29 is a result of confirming the fluorescence change of Compound 1 (5 μM) in the EtOH solution containing different proportions of water, Figure 29 (a) is 0-50%, Figure 29b is 50-90% results, insertion Drawing shows fluorescence intensity (FI) vs. It is a graph showing [Water] (%). 29C is a result of confirming the degree of visualization of Compound 1 fluorescence change with increasing water, and all spectra were obtained using 350 nm excitation.

Figure 30 (a) is the result of confirming the change in the fluorescence intensity ratio of the compound 1 according to the various moisture ratio (0-90%) in the ACN, MeOH and EtOH solution, Figure 30 (b) shows the fluorescence intensity ratio in the ACN solution [ Water] (%) is a result of confirming the linear correlation, Figure 30 (c) and Figure 30 (d) is a result of confirming the linear correlation between the fluorescence intensity ratio in the MeOH solution and [Water] (%) 30 (e) and 30 (f) confirm the linear correlation between the fluorescence intensity ratio in the EtOH solution and [Water] (%), and all data were obtained at 350 nm excited state.

FIG. 31 shows partial 1H NMR spectra of Compound 1 in CDCl ₃ containing DMSO-d ₆ at different ratios, FIG. 31 (a) 0%, FIG. 31 (b) 50%, and FIG. 31 (c) 100. % (v / v), asterisk (*) indicates residual CDCl ₃ solvent.

FIG. 32 (a) shows the fluorescence color change of the paper strip immersed in Compound 1 in EtOH, MeOH and ACN solutions containing different percentages of water, and FIG. 32 (b) shows the paper strip by simple drying. As a result of confirming the reversibility of the fluorescence change of, the fluorescence change was confirmed using a portable UV lamp providing 365 nm excitation.

33 is a schematic diagram showing a method of detecting water using a paper strip impregnated with compound 1. FIG.

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

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

[Formula 1]

Wherein 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 the following formula (2).

[Formula 2]

The present invention also provides a composition for detecting NAD (P) H in mitochondria comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Wherein 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.

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

[Formula 1]

Wherein 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.

The present invention also provides a kit for detecting NAD (P) H in mitochondria comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Wherein 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 another aspect, the present invention provides a composition for detecting cancer cells comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Wherein 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 another aspect, the present invention provides a kit for detecting cancer cells comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Wherein 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 to the cell by reacting; 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. The method provides a method for detecting NAD (P) H in mitochondria.

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

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, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with 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 Institute of Basic Science (Seoul). NMR spectral data were obtained using a Bruker (500 MHz) instrument.

Example 2: UV / Vis Absorption and Fluorescence Spectroscopy Analysis

The solvent used in the spectroscopy experiment was HPLC grade free of fluorescent impurities, and deionized water was used for water. PIPES buffer (pH7.4) contains 25 mM piperazine-N, N'-bis (2-ethanesulfonic acid) (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. It was.

Example 3: Cell Culture and Cytotoxicity Assay

For cell culture, DMEM medium, phosphated buffered saline (PBS), fetal bovine serum (FBS), try's balanced salt solution (HBSS) 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 micro plate reader (Molecular devices, San Jose, Calif.). Fluorescence images were obtained using Zeiss LSM-700. Red (probe 1) and green (tracker) 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

Breast adenocarcinoma cell line (MDA-MB-231) was cultured in a 37 ° C., 5% CO ₂ incubator using high glucose DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, and fresh medium every 2 days. Replaced with. When the cells grew to 80-90%, the medium was removed, washed with PBS, PBS was removed, 1.5 mL of trypsin 0.25% -EDTA was added and then incubated for 3 minutes in a 37 ℃, 5% CO ₂ incubator. After separating the cells from the flask, 1 mL of medium was added to the flask and mixed. The cell suspension was then 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 incubated for 24 hours (37 ° C., 5% CO ₂ ) in coverslips of 35 mm confocal dishes at a concentration of 10 ⁵ cells / mL, then the cells were washed with PBS and 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 confocal microscopy.

Example 5: Cytotoxicity Assay

MDA-MB-231 cells were dispensed in 96 well plates at 1 × 10 ⁴ cells / well concentration and incubated for 24 hours to allow cells to adhere to the plate bottom. 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 6 hours of incubation, probe 1 was removed and washed with PBS. MTT reagent (0.5 mg / mL) was then 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 and the plate was shaken gently to dissolve formazan crystals for 30 minutes. Absorbance was read at 550 nm using a Spectra Max i3x micro plate reader.

Example 6: Confocal Microscopy Image Analysis

MDA-MB-231 cells were dispensed onto glass coverslips of confocal dishes without surface treatment and attached for 24 hours. Thereafter, the medium was removed, washed with PBS, and then stained with probe 1, before 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. The cells were stained with one of the organelle trackers, and then sequentially treated with probe 1 for 90 minutes. Fluorescence images were acquired every 10 minutes using confocal microscopy.

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 in glass coverslips of confocal dishes. Mixed cells were stained with probe 1 for 2 hours and visualized with confocal fluorescence microscopy (Zeiss LSM-700, Axio Observer), and fluorescence images were obtained using 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

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 precursor 3 (1.00 g, 3.63 mmol). Added. The resulting reaction mixture was stirred overnight and refluxed. After cooling to rt, the solvent was evaporated and CH ₂ Cl ₂ layer was added by adding CH ₂ Cl ₂ (3 × 100 mL) and brine (100 mL). 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 rt for 3 h. The yellow precipitate was then filtered off, washed with CH ₂ Cl ₂ and diethyl ether and 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  1: in the mitochondria NAD (P) H  Fluorescence for detection Of probe  effect

Synthetic 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.). The 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 PIPES buffer (pH 7.4, 25 mM) with 2% (v / v) DMSO. Probe 1 did not exhibit unique 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 showed a visual color change from yellow to purple, confirming that visual inspection of NADH was possible (FIG. 3A). Probe 1 at 570 nm showed a new emission band at 615 nm and showed a visual color change with bright red fluorescence (FIG. 3B). In addition, fluorescence quantum yield was measured using crystal violet perchlorate (Ф _F = 0.22) as a standard, and probe 1 responded to the cell NAD (P) H, causing fluorescence off-on in the far infrared emission region. It was found to show a change, which means that it can be easily detected without the interference of its own fluorescence, which does not depend on the analyte.

The time dependent fluorescence change of probe 1 was observed in the presence of NADH. Addition of NADH (2 mM) to the Probe 1 (10 μM) solution confirmed that the 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. 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). Plots of NADH concentration versus fluorescence intensity at 615 nm showed good linearity for NADH concentrations between 0 and 800 μM with a detection limit of NADH of 1.5 μM (insertion of FIG. 3D).

Analysis of 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 revealed that probe 1 was evident at 615 nm in the presence of NADH and NADPH. An increase in fluorescence was shown (FIG. 4).

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

Probe 1 showed excellent selectivity with respect to NADH, it was intended to demonstrate the cell activity of probe 1 in the detection of NADH in mitochondria of live breast cancer cells (MDA-MB-231). Initially, cells were treated with various concentrations of probe 1 and incubated for 6 hours and 24 hours to assess cytotoxicity.

As a result, when treated for 6 hours at a high concentration of 30 μM, no cytotoxicity was observed, it was confirmed that the cytotoxicity slightly increased with 80% viability when treated for 24 hours (Fig. 5).

To determine whether probe 1 selectively targets mitochondria in living cells, probe 1 and organelle markers were co-stained respectively. As a result, the merged image of probe 1 (red) and the mitochondrial marker (green) shows that the two stains coexist in most cells, while the merged image of probe 1 and lysosomal marker, or probe 1 and vesicle marker, No coexistence of stains was observed.

Analysis of Pearson's correlation coefficient (PCC) values showed statistically significant correlation (r = 0.98) between probe 1 and mitochondrial markers, while probe 1 and lysosomal markers, or probe 1 and endoplasmic reticulum markers, were statistically significant. There was no significant correlation (0.63, 0.81) (FIG. 6).

These results demonstrate that probe 1 correlates strongly with commercially available mitochondrial marker dyes 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 under 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 preincubated with glucose in the NAD (P) H boosting group.

On the other hand, carbonyl cyano 4-trifluoromethoxyphenylhydrazone [carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone; FCCP] interferes with ATP synthesis by delivering protons through the mitochondrial lining, pre-culturing MDA-MB-231 cells with FCCP in a group depleted of NAD (P) H to induce oxidation of mitochondrial NADH through NADH dehydrogenase It was.

As a result, cells pretreated with FCCP showed lower fluorescence intensity than control cells, whereas cells pretreated with glucose showed strong fluorescence signal (FIG. 7).

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

Because cancer cells grow faster and differentiate than normal cells, the fluorescent signals of probe 1 were compared in mitochondria of living cancer cells (MDA-MB-231) and fibroblasts (NIH-3T3).

As a result, the signal of the fluorescence image showed a clear difference between the MDA-MB-231 cells and the NIH-3T3 cells (FIG. 8). Compared with MDA-MB-231 cells, NIH-3T3 cells stained with Calcein AM (green) showed weak red fluorescence signal of probe 1.

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

In addition, the present inventors have synthesized a novel compound for aggregation-induced emmission luminogen (AIEgen) induced by aggregation reaction capable of detecting hydrogen sulfide (H ₂ S), which is a thiol such as GSH, Cys and Hcy. It can selectively detect H ₂ S even if it reacts with interferences such as compounds having a) group, redox species (ROS), metal ions and anions, and can also be used as a sensor that can detect H ₂ S gas. It was found that the present invention was completed.

The present invention provides a compound represented by the following formula (3) or a pharmaceutically acceptable salt thereof.

[Formula 3]

(In Chemical Formula 3,

R ¹ is selected from the group consisting of hydroxy, amine, (C 1 -C 4) alkylamine, (C 1 -C 4) alkyl, (C 1 -C 4) alkoxy and halogen,

R ² , R ³ and R ⁴ may each be the same or different and are selected from the group consisting of hydrogen, amine, (C 1 -C 4) alkylamine, (C 1 -C 4) alkyl, (C 1 -C 4) alkoxy and halogen.)

In this case, R ¹ is selected from the group consisting of hydroxy, amine, (C1 ~ C4) alkylamine, (C1 ~ C4) alkyl, (C1 ~ C4) alkoxy and halogen, R ² , R ³ and R ⁴ is It may be hydrogen.

In addition, R ¹ is selected from the group consisting of hydroxy, amine and (C1 ~ C4) alkylamine, R ² , R ³ and R ⁴ may be hydrogen. Preferably, R ¹ is hydroxy and R ² , R ³ and R ⁴ may be hydrogen.

In addition, the present invention provides a composition for detecting hydrogen sulfide comprising the compound according to Formula 3 or a pharmaceutically acceptable salt thereof.

The present invention also provides a fluorescent probe for detecting hydrogen sulfide comprising the compound according to Formula 3 or a pharmaceutically acceptable salt thereof.

The present invention also provides a sensor kit for detecting hydrogen sulfide comprising the compound according to Formula 3 or a pharmaceutically acceptable salt thereof.

In this case, the sensor kit may detect hydrogen sulfide by forming a dimer of two compounds according to Chemical Formula 3 or a pharmaceutically acceptable salt thereof fluorescence through disulfide bonds in the presence of hydrogen sulfide, but is not limited thereto.

In addition, the hydrogen sulfide may be hydrogen sulfide present in an aqueous solution or gas phase, but is not limited thereto.

In addition, the present invention comprises the steps of reacting a sample containing hydrogen sulfide, and the two compounds according to the formula (3) or a pharmaceutically acceptable salt thereof by contacting; And it provides a hydrogen sulfide detection method comprising the step of measuring one or more indicators selected from the group consisting of the fluorescence signal, absorbance and optical change of the reacted reactant.

In this case, the fluorescent signal provides a hydrogen sulfide detection method characterized in that the presence of hydrogen sulfide, two compounds according to the formula (3) or a pharmaceutically acceptable salt thereof is generated by the dimer form obtained through disulfide bonds, the hydrogen sulfide May be, but is not limited to, hydrogen sulfide present in an aqueous solution or gas phase.

AIEgen novel compounds (compound 1) comprising disulfide bonds according to the invention can form a special AIEgen dimerization reaction via a thiol-disulfide exchange between molecules for the selective detection of H ₂ S in aqueous and gaseous phases. . As shown in FIG. 9, Compound 1 was composed of disulfide bonds as sites of the dimerization reaction for tetraphenylethene (TPE) and H ₂ S as AIEgen. Initially, compound 1 showed weak fluorescence. However, when H ₂ S is present, Compound 1 shows a strong blue fluorescence showing Compound 2, a TPE dimer, due to the increased Aggregation-induced emmission effect. Thereafter, when H ₂ S was overreacted, compound 2 was modified to non-fluorescent TPE-NH ₂ .

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 the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art to which the present invention pertains. Will be self-evident.

Example  9: Materials and Equipment

All fluorescence and UV / Vis absorbance data were obtained using RF-6000 (Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) and UV-2600 (Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) spectrophotometers, respectively. ¹ H and ¹³ C NMR spectra were obtained using a Brucker 500 MHz spectrometer on CDCl ₃ (Cambridge Isotope Laboratories, Cambridge, Mass.). HR-ESI-MS data were obtained using a liquid chromatography mass spectrometer (LC / MS) from the Korea Basic Science Institute (Seoul). Field emission scanning electron microscopy (FE-SEM) images were obtained using a JEOL JSM-7600F instrument at an acceleration voltage of 10 kV. Samples of SEM images were loaded with a few drops of agglomerated solution onto the bonded carbon tape and lyophilized for 3 days using an Operon lyophilizer. Then, it was prepared by Pt coating for 40 seconds with 108 auto (Cressington Sputter Coater). Particle size and distribution were measured using a particle size analyzer (Microtrac, UAP-150) dynamic light scattering (DLS). Silica gel 60 (Merck, 0.063-0.2 mm) was used for column chromatography. Analytical thin layer chromatography was performed using Merck60 F ₂₅₄ silica gel (precoated sheet, 0.25 mm thick). All reactions were carried out under a nitrogen atmosphere. Piperazine, acetonitrile, chloride Na ⁺ , K ⁺ , Ca ^{2 +} , Mg ^{2 +} , Ba ^{2 +} , Cd ^{2 +} , Co ^{2 +} , Pb ^{2 +} , Hg ²⁺ , Ni ^{2 +,} Cu ^{2 +,} Zn ^{2 +,} Fe ^{2 +,} Fe ^{3 +} ions with a metal, tetrabutyl ammonium as (tetrabutylammonium; TBA) salt of _{^{H 2 PO 4 -, HSO 4}} -, ClO 4 -, AcO -, CN ^{-, OH -, F -,} Cl -, I - anions, such as ion, cysteine (cysteine; Cys), homocysteine (homocysteine; Hcy), glutathione (glutathione; GSH), sodium hydro sulfide, (sodium for H ₂ S generation All reagents, including compounds with thiol groups such as hydrosulfide (NaHS) and other chemical and buffer solutions for synthesis, were purchased from Aldrich, Alfa and TCI. All solvents used analytical reagents. DMSO for spectrophotometric assays is an HPLC reagent free of fluorescent impurities, and water is used as deionized water.

Example  10: UV / Vis Absorbance  And fluorescence spectroscopy methods

Stock solutions of synthetic compounds were prepared in DMSO. The anion of TBA salt and the metal ion stock solution of chloride were prepared in DMSO and water, respectively. H ₂ O ₂ , tert-butylhydroperoxide ( tert- butylhydroperoxide; HOO ^t Bu) and sodium hyperchloride (NaOCl) were used in 35%, 70% and 11-14% aqueous solutions, respectively. Hydroxy radicals (OH) and tert- butoxy radicals ( ^t BuO˙) reacted with 10 mM H ₂ O ₂ and HOO ^t Bu in 10 mM Fe (ClO ₄ ) ₂ , respectively. Was caused by. Stock solutions of compounds with thiol groups were prepared in 20 mM pH 7.4 HEPES buffer solution. Samples for absorbance and emission measurements were measured in quartz cuvettes (4 mL volume). Excitation and emission Slit widths 5 and 10 nm with excitation were measured at 320 nm.

Example  11: Synthesis of Compound 1

Pyridine (0.1 mL, for mixing 4-nitrophenyl chloroformate (58 mg, 0.3 mmol) in compound 3 (50 mg, 0.1 mmol) and 10 mL dichloromethane (DCM) 1.2 mmol) was added dropwise. The solution was mixed under nitrogen gas for 1 hour. Reaction completion was confirmed by TLC. 2,2'-dithiodiethanol (0.1 mL, 0.8 mmol) and diisopropylethylamine (DIPEA) in DCM / THF (v / v = 1: 1) mL, 0.1 mmol) was added to the solution. Thereafter, the mixture was mixed at room temperature overnight. After removal of the solvent, the crude product was dissolved in DCM, rinsed thoroughly in water and dried over Na ₂ SO ₄ . After evaporation of the solvent, the crude product was purified by silica gel column chromatography using ethylacetate and hexane (v / v = 1: 3) as eluent to give yellow color in 55% yield (25mg). Solid compound 1 was obtained. HR-ESI-MS m / z [M + Na] ⁺ calcd 550.14790, found 550.14917. ¹ H NMR (CDCl ₃ , 500 MHz): δ 2.90 (t, 2H, J = 7.5 Hz); 2.97 (t, 2H, J = 7.5 Hz); 3.89 (s, 2 H); 4.40 (t, 2H, J = 7.5 Hz); 6.73 (s, 1 H); 6.95-7.10 (m, 19 H) ppm. ¹³ C NMR (CDCl ₃ , 125 MHz): δ 29.7, 37.5, 41.6, 60.3, 126.4, 127.6, 127.7, 131.3, 131.4, 132.1, 135.8, 139.1, 140.2, 140.7, 143.6, 143.7, 143.7 ppm. (FIG. 10A , b, c).

Example  12: Synthesis of Compound 2

Compound 1 (0.8 mg, 0.2 mmol) was dissolved in 4 mL of acetonitrile (ACN), to which 6 mL of 20 mM HEPES buffer (pH 7.4) containing NaHS (7 mg, 0.1 mmol) was added. The solution was mixed for 30 minutes and the reaction was confirmed by TLC. The crude product was diluted with DCM, rinsed thoroughly in water and dried over Na ₂ SO ₄ . After evaporation of the solvent, the crude product was purified by silica gel column chromatography using ethyl acetate and hexane (v / v = 1: 3) as eluent to obtain white solid compound 2 in 30% yield (41 mg). . HR-ESI-MS m / z [M + Na] ⁺ calcd 923.29707, found 923.29579. ¹ H NMR (CDCl ₃ , 500 MHz): δ 2.95 (t, 4H, J = 7.5 Hz); 4.40 (t, 4H, J = 7.5 Hz); 6.95-7.02 (m, 15 H); 7.03-7.11 (m, 26H) ppm. ¹³ C NMR (CDCl ₃ , 125 MHz): δ 37.3, 62.6, 117.9, 126.4, 126.5, 127.6, 127.7, 131.3, 131.4, 132.1, 140.2, 140.7, 143.6, 143.7 ppm. (FIGS. 11A, b, c).

Experimental Example  2: Mechanism of reaction of compound 1 in the presence of hydrogen sulfide

As shown in FIG. 12, Compound 1, when H ₂ S is present, breaks the disulfide bond of Compound 1 to form TPE-SH, and then Compound 1 undergoes another thiol-disulfide bond exchange, thereby replacing the intramolecular cyclization reaction. To AIEgen dimerization reaction. This resulted in the formation of Compound 2, a TPE dimer with significant fluorescence. These AIEgen dimers resulted from the agglomeration of compound 1 by hydrophobic bonding of a portion of TPE in aqueous solution. In other words, in the aggregation state, the result of the dimerization reaction from thiol-disulfide exchange is more favorable than the intramolecular cyclization reaction. In addition, the dimerization reaction of AIEgen showed strong fluorescence due to the increased AIE effect. However, when H ₂ S was excessively reacted, Compound 2, which is a TPE dimer, was broken by disintegration reaction in the molecule to generate TPE-NH ₂ which showed very weak fluorescence.

Experimental Example  3: Fluorescence of Compounds 1, 2 and 3

AIE phenomena in water-DMSO mixtures were analyzed with different proportions of water (f _w ) in compounds 1, 2 and 3 (FIGS. 13-15). As f _w increased from 50% to 90%, disulfide-bonded AIEgen Compound 1 showed increased fluorescence at 471 nm. In Compound 3, which is TPE-NH ₂ , an increase in fluorescence intensity was observed at 500 nm when f _w increased from 60% to 90%. However, compound 2, a TPE dimer, stabilized when f _w > 30% and f _w reached 60%, and increased fluorescence was observed. In addition, it was observed that the increased fluorescence intensity was more than 16 times higher than that of Compounds 1 and 3. This revealed that the two TPEs of Compound 2 showed a greater AIE effect than Compound 1 and Compound 3. In addition, when H ₂ S is present, the dimerization reaction of Compound 1 (formation of Compound 2) revealed a strong AIE-based fluorescence increase.

Experimental Example  4: Characteristics of the reaction to hydrogen sulfide  analysis

Changes in absorbance and fluorescence of Compound 1 were observed with and without H ₂ S in HEPES buffer (pH 7.4) with 40% DMSO (v / v) (FIGS. 16A, B). The solution of Compound 1 showed an emission band at 314 nm and a luminescence reaction at 464 nm, but when 20 equivalents of NaHS (H ₂ S donor) were added to this solution, the absorption and emission bands showed a strong fluorescence increase. , 342 and 480 nm, respectively (Fig. 16B insertion). The spectra of these results matched more closely to the spectra of Compound 2, an independent TPE dimer, than TPE-NH ₂ Compound 3 under similar conditions (FIGS. 13-15).

In addition, the aggregation reaction of Compound 1 with and without NaHS was determined using FE-SEM and DLS (FIG. 16C, d). In the absence of NaHS, Compound 1 aggregated to give an average diameter of 1,533 nm (D _ave ) (FIG. 17A, b). However, in the presence of NaHS, the aggregated _ave decreased to 1,070 nm. In addition, D _ave of Compound 2 aggregate, which is an independent TPE dimer, exhibited 826 nm (FIG. 17C, d). In contrast, TPE-NH ₂ Compound 3 showed a relatively large aggregate with D _ave = 1,791 nm (FIG. 17E, f). These results showed that aggregate size decreased with increasing AIEgen's AIE effect in aqueous solution. This proved that Compound 1 led to the formation of TPE dimer Compound 2, which is a high emitter in the presence of H ₂ S. Therefore, compound 1 can be used as a fluorescent probe based on AIE for detecting H ₂ S in aqueous solution.

Experimental Example  5: Interference  Selective reaction analysis of compound 1 in the presence of hydrogen sulfide

Incubation of compounds with various metal ions, anions, reactive oxygen species (ROS), and thiol groups in HEPES buffer containing 40% DMSO (v / v) at 37 ° C. for 1 hour After the fluorescence reaction was investigated. As can be seen in FIG. 18A, when the 20 equivalent of NaHS (H ₂ S donor) (equiv.) Was added, Compound 1 showed a significant increase in fluorescence intensity at 480 nm. In contrast, negligible fluorescence changes were observed when other redox species, including GSH, Cys, Hcy, ClO ⁻ ,, O 2 ⁻ , ˙OH, and ^t BuOOH, were present. In ^{addition, Na +, K +, Ca} 2 +, Mg 2 +, Ba 2 +, Cd 2 +, Co 2 +, Pb 2 +, Hg 2 +, Ni 2 +, Cu 2+, Zn 2 +, Fe 2 ⁺ and Fe ^{3 +} other metal ions and H ₂ PO containing ^{_{4 -, HSO 4 -, ClO}} 4 -, AcO -, CN -, OH -, F -, Cl - as the anion containing exist ^- and I No fluorescence change was observed (FIG. 19).

As a result, Compound 1 found that H ₂ S showed a highly selective fluorescence increase response to other potential interferences.

Experimental Example  6: Characterization of reaction according to hydrogen sulfide concentration

For quantitative analysis of H ₂ S detection using Compound 1, the absorption and fluorescence changes of Compound 1 were observed at different concentrations of NaHS. As the concentration of NaHS increased, the absorption band at 341 nm red-shifted to 342 nm due to the formation of Compound 2, a TPE dimer (FIG. 20). In contrast, when the concentration of added NaHS was increased, the fluorescence intensity gradually increased (53 times) at 480 nm and became saturated at 20 equivalents (Fig. 18b, c). Gradual fluorescence increase was also seen under the small UV lamp (Figure 18C inset). However, when NaHS was exceeded (1000 equivalent), Compound 1 showed a reduced fluorescence intensity due to the weak luminescence formation of TPE-NH ₂ Compound 3 via the breakdown of disulfide bonds (FIG. 21A). Formation of compound 3 was confirmed using silica gel TLC analysis (FIG. 22). In addition, similar fluorescence decreases were observed when excess NaHS was present in compound 2, a TPE dimer, but negligible fluorescence changes were observed in compound 2 when excess compounds with other thiol groups, including GSH, Cys and Hcy, were present (FIG. 21b). As a result, it was found that Compound 1 can produce TPE-NH ₂ Compound 3 through the formation of Compound 2, which is optionally a TPE dimer, in the presence of H ₂ S. To achieve the H ₂ S-selective fluorescent probe, the concentration of fluorescence of Compound 1 was concentrated in the 0-34 equivalent (0-170 μM) of NaHS. In addition, the fluorescence intensity increased proportionally between 0 and 100 μM NaHS at 480 nm, which is a range of general physiological values of H ₂ S (FIG. 23). In addition, the detection limit (LOD, 3σ / s) of Compound 1 to NaHS was 84 nM, which allows Compound 1 to detect 0.01-3 μM of intracellular H ₂ S levels.

Experimental Example  7: Characterization of Compound 1 over Hydrogen Sulfide Over Time

Fluorescence change over time of compound 1 in reaction with H ₂ S was observed in HEPES buffer (pH 7.4, 20 mM) containing 40% DMSO (v / v) at 37 ° C. In the presence of 20 equivalents (equiv.) Of NaHS (H ₂ S donor), the fluorescence intensity increased at 480 nm and reached a steady state within 1 hour (red line in FIG. 18D, FIG. 24). In contrast, in the absence of NaHS, Compound 1 did not show fluorescence even after 2 hours (black lines in FIG. 18D). As a result, it was found that Compound 1 stabilizes in the absence of H ₂ S, but reacts very sensitively to H ₂ S to provide a fluorescent signal even under physiological conditions.

Experimental Example  8: Analysis of the Effect of pH on Hydrogen Sulfide Detection of Compound 1

When NaHS was present or absent, pH effects were investigated for H ₂ S detection of Compound 1 (FIG. 25). In the presence of a 20 equivalent (equiv.) Of NaHS (H ₂ S donor), Compound 1 showed a clear fluorescence increase at 480 nm between pH values 5 and 8, which are widely observed under physiological conditions. In contrast, compound 1 showed a negligible fluorescence change between pH values 1 and 7. However, at pH values 8 and 11 Compound 1 showed a slight increase in fluorescence due to the sensitivity of disulfide bonds bound to the TPE moiety. As a result, it was found that Compound 1 can provide an increase in fluorescence capable of detecting H ₂ S at most physiological pH values.

Experimental Example  9: hydrogen sulfide gas detection Kit  Manufacture and Detection of Hydrogen Sulfide Gas Using the Same

Silica gel TLC plates were coated with compound 1 to detect H ₂ S gas. As shown in FIG. 26A, the silica gel TLC plate with glass on the back was cut into 2 cm x 2 cm pieces, soaked in DCM solution of compound 1 (0.5 mM), and dried. Each Compound 1 coated plate was placed on a vial containing excess amount of volatile solution for 30 seconds (FIG. 26B). Glass plates include H ₂ S, H ₂ O ₂ , acetaldehyde, ethyl acetate (EA), acetone, diethyl ether, chloroform (CH ₂ Cl ₂ ) , Hydrochloric acid (HCl), trifluoroacetic acid (TFA), sulfuric acid (H ₂ SO ₄ ), acetic acid (AcOH), hydrazine (N ₂ H ₄ ), Various vapors have been exposed, including methylamine (CH ₃ NH ₂ ), ammonia (NH ₃ ) and formaldehyde. A small UV lamp was used to observe the change in fluorescence color under excitation conditions of 365 nm. When exposed to H ₂ S a fairly bright blue fluorescence appeared, but when exposed to other vapors this reaction did not occur (Figure 26c). In addition, as shown in FIG. 26D, as the concentration of the NaHS solution increased, the weak blue fluorescence of the plate coated with Compound 1 gradually increased.

Compound 1 easily generates AIEgen of the H ₂ S-selective dimer, resulting in the formation of Compound 2, which is a TPE dimer, on a silica gel TLC plate with glass on the back. Silica gel TLC plates coated with compounds 1, 2 and 3 were prepared (FIG. 27). Light blue fluorescence was observed in Compound 1 coated plates after exposure to H ₂ S vapor, and fluorescence was also observed in Compound 2 coated plates, but not in Compound 3 coated plates. As a result, shown that the compounds (1) coated on a glass silica gel TLC plate may be selectively detected by the H ₂ S gas, it may be used as the sensor kit for H ₂ S gas detection of toxicity in a working environment.

In addition, the present invention can provide a naphthalimide derivative represented by the following formula (4) or a pharmaceutically acceptable salt thereof.

[Formula 4]

In Formula 4, at least one of R ₁ to R ₃ is halogen and the other is hydrogen, and R ₄ may be any one selected from the group consisting of phenyl, -COOH, -OH, and (C1 to C6) alkyl. .

More specifically, in Formula 4, at least one of R ₁ to R ₃ is fluoro, the remainder is hydrogen, and R ₄ is (C 1 to C 6) alkyl, or a pharmaceutically acceptable derivative thereof. It may be a salt.

The present invention can provide a composition for detecting water, comprising a naphthalimide derivative represented by the following formula (4) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 4]

In Formula 4, at least one of R ₁ to R ₃ is halogen and the other is hydrogen, and R ₄ may be any one selected from the group consisting of phenyl, -COOH, -OH, and (C1 to C6) alkyl. .

More specifically, in Formula 4, at least one of R ₁ to R ₃ is fluoro, the remainder is hydrogen, and R ₄ is (C 1 to C 6) alkyl, or a pharmaceutically acceptable derivative thereof. It may be a salt.

The naphthalimide derivative represented by Chemical Formula 4 or a pharmaceutically acceptable salt thereof may exhibit a double emission wavelength in the presence of water in a sample, and may change the fluorescence intensity according to the water content contained therein.

According to an embodiment of the present invention, as a result of confirming the absorbance and fluorescence spectrum of the water of the compounds 1 to 4 represented by the formula (4), as shown in Figure 28a Compound 1 has an absorption band at about 350 nm, at 452 and 558 nm Two strong emission bands appeared. The stoke shift (Δλ) of the double emission was 102 and 208 nm, respectively, and the pale yellow solution of compound 1 showed brilliant white fluorescence.

Compound 2 showed absorption at 365 nm, emission at 550 nm (Δλ = 180 nm) and 435 nm (Δλ = 70 nm) as shown in FIG. 28B, and a pale yellow solution showed a brilliant yellow fluorescent color.

In addition, toluene, dichloromethane (DCM), tetrahydrofuran (THF), CHCl ₃ , acetone (acetone), acetonitrile (ACN), N, N-dimethylformamide (N Absorption and fluorescence spectra of compounds 1, 2, 3 and 4 in various organic solvents such as N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol (methanol; MeOH) and ethanol (EtOH) As a result, as shown in Table 1, Compound 1 showed two absorption bands at 350 and 460 nm with dual emission of about 440 and 560 nm in DMF and DMSO, and showed yellow fluorescence.

In addition, absorption bands were observed at about 350 nm for toluene, DCM, THF, CHCl ₃ , acetone, MeOH and EtOH solutions, and the highest single emission band was found in the 419-447 nm range.

Toluene, DCM, THF, CHCl ₃ , acetone and ACN solutions of compound 1 showed a dark blue fluorescence, while bright blue fluorescence was observed in MeOH and EtOH solutions.

The bright white fluorescence of Compound 1 that appeared in water in the above results was not seen in the experimental organic solvent.

From the above results, it was confirmed that Compound 1 exhibits a unique absorption and dual release to water, which are distinguished from various organic solvents, in ethanol and methanol, which are magnetic solvents having the same polarity as water. It is confirmed that the ratio change can be provided through the dual emission, and the accurate detection and real time monitoring can be easily visualized by providing the change of the fluorescent color.

Accordingly, the present invention can provide a fluorescent probe for moisture detection comprising a naphthalimide derivative represented by the following formula (4) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 4]

In Formula 4, at least one of R ₁ to R ₃ is halogen and the other is hydrogen, and R ₄ may be any one selected from the group consisting of phenyl, -COOH, -OH, and (C1 to C6) alkyl. .

The present invention can provide a strip for detecting moisture, comprising a naphthalimide derivative represented by the following formula (4) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 4]

In Formula 4, at least one of R ₁ to R ₃ is halogen and the other is hydrogen, and R ₄ may be any one selected from the group consisting of phenyl, -COOH, -OH, and (C1 to C6) alkyl. .

According to another embodiment of the present invention, for simple moisture detection using Compound 1, a paper strip impregnated with Compound 1 was prepared by immersing filter paper in a solution of Compound 1 (1.0 mM) in CHCl ₃ and drying in an oven for one day. And, repeatedly immersed in an aqueous solution of EtOH and dried to confirm the reversible fluorescence reaction of the paper strip in which Compound 1 is infiltrated against water, as shown in Figure 32b when the paper strip is immersed in an aqueous solution of EtOH containing 10% moisture, The color changed rapidly from blue to white and the strip returned to blue fluorescence again by simple drying of the solvent.

As it is confirmed from the above results that the paper strip can be repeatedly used for moisture detection, the paper strip can be used very effectively for moisture detection by quickly and simply visualizing fluorescence color change as well as reusability.

In addition, the present invention comprises the steps of reacting by adding a sample to a composition comprising the naphthalimide derivative or a pharmaceutically acceptable salt thereof; And measuring one or more indicators selected from the group consisting of absorbance, fluorescence intensity, and optical change of the reacted reactant.

The sample may be an organic solvent.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

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

Example  13: 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 solutions were used as analytical or HPLC grade, HR-ESI-MS data obtained from Liquid Chromatography Mass Spectrometer (LC / MS) of the Korea Basic Science Institute (Seoul), NMR Spectra Bruker (500 MHz) instrument Was recorded.

Example  14: UV / Vis  Absorption and Fluorescence Spectroscopy

All solutions used for spectroscopic studies were used in HPLC grade.

Toluene, dichloromethane (DCM), tetrahydrofuran (THF), chloroform, acetone, acetone, acetonitrile (ACN), N, N-dimethylformamide (N Compound stock solution was prepared using N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol, methanol and ethanol.

All UV / Vis absorbance and fluorescence spectra were recorded on UV-2600 (Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) and RF-6000 (Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) spectrometers, respectively.

Synthesis Example  1: Synthesis of Compounds 1-4

Compounds 3 and 4 were prepared by methods previously reported (Ghorbanian, S, et al .; J Chem Technol Biotechnol 2000, 75, 1127-1134 and Lee, MH et al .; J. Am. Chem. Soc. 2012, 134, 1316-1322.), And compound 1 and 2 were synthesized using compound 4 as a starting material.

Scheme 1

1. Compound 1 Synthesis

Scheme 2

Dimethylformamide (DMF; 0.1 mL) was slowly added to a mixture of difluoroacetic acid (0.13 mL, 2.06 mmol) and oxalyl chloride (0.26 mL; 3.07 mmol) dissolved in dichloromethane (DCM, 50 mL).

The reaction mixture was stirred overnight at room temperature under N ₂ gas and confirmed by thin layer chromatography (TLC).

After evaporating difluoroacetic acid in a TLC plate the reaction mixture was immediately used for the subsequent reaction. A DCM solution containing Compound 4 was added to the reaction mixture containing difluoroacetyl chloride and refluxed at 60 ° C. under N ₂ gas for 3 hours.

The reaction mixture was quenched in aqueous sodium bicarbonate solution, diluted with DCM (50 mL) and washed with water (100 mL).

The organic layer was collected, dried over anhydrous Na ₂ SO ₄ , and after solution removal, silica gel column chromatography was performed using ethyl acetate / hexane as eluent to obtain a yellowish white solid compound 1 (0.66 g, 92%).

HR-ESI-MS m / z [M] ⁺ calc. 346.1439, [M + H] ⁺ obs. 347.1513. ¹ H NMR (DMSO- d 6, 500 MHz): δ 0.94 (t, J = 7.5 Hz, 3H); 1.32-1.40 (m, 2 H); 1.59-1.65 (m, 2 H); 4.03 (t, J = 7.5 Hz, 2H); 6.65 (t, J = 53.5 Hz, 1 H); 7.90 (t, J = 7.8 Hz, 1H); 8.11 (d, J = 8.0 Hz, 1H); 8.48-8.54 (m, 3H) ppm. ¹³ C NMR (DMSO-d ₆ , 125 MHz): δ 163.2, 162.8, 162.0, 161.8, 161.6, 137.9, 131.0, 131.0, 129.1, 128.1, 126.9, 125.2, 122.3, 122.0, 119.6, 110.2, 108.3, 106.3, 39.4, 29.6, 19.8, 13.7 ppm.

2. Compound 2 Synthesis

Scheme 3

Trifluoroacetic anhydride (0.14 g, 0.53 mmol) and sodium carbonate (0.03 g, 0.26 mmol) in a mixture of 1,4-dioxane (5 mL) dissolved in trifluoroacetic anhydride ( trifluoroacetic anhydride (0.11 mL, 0.79 mmol) was added slowly and the reaction mixture was stirred for 2 h at N ₂ gas room temperature.

The mixture was diluted with ethyl acetate (50 mL), washed with water and the organic layer was collected and dried over anhydrous Na ₂ SO ₄ .

After removal of the solvent, the crude product was purified by silica gel column chromatography using ethyl acetate / hexane (v / v, 1: 2) as eluent to give a yellowish white solid Compound 2 (0.16 g, 82%). . HR-ESI-MS m / z [M] ⁺ calc. 364.1342, [M + H] ⁺ obs. 365.1415. ¹ H NMR (DMSO-d ₆ , 500 MHz): δ 0.93 (t, J = 7.5 Hz, 3H); 1.31-1.42 (m, 2 H); 1.58-1.67 (m, 2 H); 4.05 (t, J = 7.5 Hz, 2H); 7.92-7.95 (m, 1 H); 7.96 (d, J = 8.0 Hz, 1H); 8.38-8.9 (m, 1 H); 8.53-8.55 (m, 2 H); 11.9 (s, 1 H) ppm. ¹³ C NMR (DMSO-d ₆ , 125 MHz): δ 163.1, 162.7, 156.4, 156.1, 155.8, 155.5, 136.7, 131.1, 130.6, 129.3, 128.1, 127.3, 126.3, 124.4, 122.4, 121.0, 119.3, 117.0, 114.7, 112.4, 39.3, 29.5, 19.7, 13.6 ppm.

Experimental Example  10: Characterization of moisture sensitive compounds

The absorbance and fluorescence spectrum of water of Compounds 1 to 4 synthesized by the above method were confirmed.

As a result, as shown in FIG. 28A, Compound 1 showed an absorption band at about 350 nm and two strong emission bands at 452 and 558 nm. The stoke shift (Δλ) of the double emission was 102 and 208 nm, respectively, and this large stoke shift was due to the efficient intramolecular charge transfer of the naphthalimide moiety. In addition, the pale yellow solution of compound 1 showed brilliant white fluorescence.

Compound 2 showed absorption at 365 nm, emission at 550 nm (Δλ = 180 nm) and 435 nm (Δλ = 70 nm) as shown in FIG. 28B, and a pale yellow solution showed a brilliant yellow fluorescent color.

On the other hand, in contrast to Compounds 1 and 2 under the same conditions as in FIG. 28C, Compound 3 was absent at 350 nm due to the absence of acetamide groups to which fluorine was added, and a single emission was observed at 477 nm (Δλ = 127 nm). The colorless solution of compound 3 showed light blue fluorescence.

In addition, Compound 4 having an amine group instead of acetamide group added with fluorine showed absorption at 435 nm as shown in FIG. 28D, and a single emission was observed at 545 nm (Δλ = 110 nm). The yellow solution of compound 4 showed a greenish yellow fluorescence.

From the above results, it was confirmed that the acetamide groups to which the fluoros of Compounds 1 and 2 played an important role in dual fluorescence emission and large stoke shift based on ICT progression.

On the other hand, in order to understand the double emission of compounds 1 and 2 for water, emission spectra were identified at two emission wavelengths.

As a result, the excitation spectrum of Compound 1 recorded at 452 and 558 nm appeared as one excitation band at 350 nm, respectively, and the result was confirmed to be the same as the absorption of Compound 1.

From the above results, it was confirmed that two excited species are formed after the excitation of compound 1 in water.

In the case of compound 2, the excitation spectrum recorded at 435 nm was confirmed as a single excitation band at 365 nm identically to the absorption of compound 2, whereas the excitation spectrum at 550 nm appeared as an excitation band at 380 nm, The results are red shifted compared to the uptake of compound 2, which may be due to a significant increase in the ICT properties of naphthalimide.

In addition, toluene, dichloromethane (DCM), tetrahydrofuran (THF), CHCl ₃ , acetone (acetone), acetonitrile (ACN), N, N-dimethylformamide (N Absorption and fluorescence spectra of compounds 1, 2, 3 and 4 in various organic solvents such as N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol (methanol; MeOH) and ethanol (EtOH) It was confirmed.

As a result, the peaks of the absorption and emission bands of Compounds 1, 2, 3 and 4 were identified as in Table 1.

Compound 1 identified two absorption bands at 350 and 460 nm with dual emission of about 440 and 560 nm in DMF and DMSO and showed yellow fluorescence.

In addition, absorption bands were observed at about 350 nm for toluene, DCM, THF, CHCl ₃ , acetone, MeOH and EtOH solutions, and the highest single emission band was found in the 419-447 nm range.

Toluene, DCM, THF, CHCl ₃ , acetone and ACN solutions of compound 1 showed a dark blue fluorescence, while bright blue fluorescence was observed in MeOH and EtOH solutions.

The bright white fluorescence of Compound 1 that appeared in water in the above results was not seen in the experimental organic solvent.

From the above results, it was confirmed that Compound 1 exhibits a unique absorption and double release of water, which are distinguished from various organic solvents, in ethanol and methanol, which are magnetic solvents of the same polarity as water.

Therefore, Compound 1 may provide a change in ratio through the double release of the water contained in the organic solvent, it is possible to easily visualize the change in the fluorescent color to enable accurate detection and real-time monitoring.

Solvent	One		2		3		4
	λ abs (nm)	λ em (nm)	λ abs (nm)	λ em (nm)	λ abs (nm)	λ em (nm)	λ abs (nm)	λ em (nm)
Toluene	350	419	345	414	345	434	405	480
DCM a	350	419	345	414	345	439	405	490
THF	350	428	344	414	344	444	420	502
CHCl 3	350	423	350	414	352	442	405	495
Acetone	350	427	350	418	358	448	420	508
			445	534
ACN b	350	427	350	418,537	352	450	415	514
	445	461	432	553
DMF c	350	435,560	350	424,537	363	457	430	520
	460	444	443	537
DMSO d	350	444,560	350	424,537	365	463	435	530
	460	446	445	537
MeOH	350	447	350	434,550	352	467	430	535
			430	542
EtOH	350	444	350	434,550	363	463	430	530
			430	550
H 2 O	350	452,558	365	435,550	350	477	435	545

^a Dichloromethane, ^b Acetonitrile, ^c N, N -Dimethylformamide, ^d Dimethyl sufoxide

In addition, Compound 2 was found to exhibit two absorption wavelengths at 350 and 440 nm with two emission wavelengths at about 430 and 550 nm in several polar solvents such as ACN, MeOH, EtOH, DMF, and DMSO. The solutions showed a variety of fluorescent colors, from sky blue to yellow green.

From the above results, it was confirmed that Compound 2 significantly increased the ICT process of the naphthalimide moiety in the polar solvent, and this result was due to the strong electron withdrawing property of Compound 2 and compared with that of Compound 1 having a fluoroacetyl group. By ICT of 4-amino naphthalimide residues which are very sensitive to solvents.

However, in Toluene, DCM, THF and CHCl3 solvents, Compound 2 showed absorption of about 350 nm and single emission at about 420 nm with dark blue fluorescence.

In contrast to compounds 1 and 2, compounds 3 and 4 without fluorinated acetamide groups showed only a simple solvation color change and a single absorption and emission band depending on the polarity of the experimental solvent as shown in Table 1.

Based on the above results, using a compound 1 as a representative moisture sensitive fluorescence probe that can easily visualize fluorescence color change in various nonpolar / polar organic solvents and detect water through ratiometric fluorescence, water of different ratios ([Water] ]%, v / v) was confirmed the effect of moisture detection in EtOH, MeOH and ACN solution.

As a result, as shown in FIG. 29, the fluorescence change of Compound 1 in the EtOH solution containing water of different ratios was confirmed. Referring to FIG. 29A, in anhydrous EtOH solution, Compound 1 showed a central emission band of 442 nm and gradually decreased, and a new emission band centered at 558 nm appeared and increased within 50% of [Water], and 526 nm. Clear isoelectric point is shown at.

However, when the [Water] increased further from 50% to 90%, the emission of 442 nm was slightly increased to 452 nm by the maximum wavelength shift, while the emission of 558 nm was reduced to the isoabsorption point of 515 nm.

In addition, it is suggested that Compound 1 can easily visualize the change in the fluorescence color according to the moisture ratio contained in the EtOH solution, and in fact, a parallel experiment was performed using MeOH and ACN solutions containing various ratios of water. Could be seen.

The ratio of fluorescence intensity versus [Water] (%) was obtained from the ratio quantitative fluorescence change of compound 1 to moisture in various organic solvents such as ACN, MeOH, and EtOH, as shown in FIG. The linear correlation between the fluorescence intensity ratio and the moisture ratio of Compound 1 in the EtOH solvent is shown to enable accurate moisture sensing.

In the linear correlation of FIG. 30B, the moisture in the ACN was 0-70%, and as shown in FIGS. 30C and 30D, MeOH was found to be 0-30% and 50-90% of moisture, and in FIGS. 30E and 30F. 0-15% and 60-90% moisture was found in EtOH.

From the results, it was confirmed that Compound 1 was able to confirm a wide range of moisture content contained in the sample under various analytical conditions.

In addition, the moisture detection limits using Compound 1 in ACN, MeOH and EtOH solvents were 0.78, 0.26 and 0.024% (v / v), respectively.

From the above results, it was confirmed that Compound 1 can be used as a ratiometric fluorescent moisture sensitive probe that enables quantitative analysis of moisture as well as simple visual analysis.

In order to confirm the detection mechanism of Compound 1 with respect to moisture, other portions of DMSO-d ₆ and CDCl ₃ were identified in the ¹ H NMR spectrum of Compound 1 as shown in FIG. 31B.

As a result, DMSO-d ₆ was used instead of D 2 O to avoid proton-deuterium exchange in the difluoroacetamide group of Compound 1.

In addition, referring to FIG. 31A, the protons Ha and Hb for the difluoroacetamide moiety in the ¹ H NMR spectrum of CDCl ₃ were identified at 8.6 and 6.2 ppm, respectively. However, as the DMSO-d ₆ ratio increased, as shown in FIGS. 31B and 31C, Ha and Hb moved to 11.3 and 6.7 ppm, respectively.

The downfield shift is due to the intermolecular hydrogen bond between the difluoroacetamide proton of compound 1 and the sulfonyl oxygen atom of the DMSO-d ₆ solvent.

Hydrogen bonds played an important role in significantly increasing the electron-pulling effect on the ICT of naphthaleneimide with a fluorescence color change from blue to yellow.

This effect can be demonstrated by a clear upfield shift of the ¹ H NMR signal from the directional proton of Compound 1 in DMSO-d ₆ , and similar Compound ¹ H NMR spectrum with increasing DMSO-d ₆ ratio of CDCl ₃ in Compound 2 Change was confirmed.

Experimental Example  11: Preparation and identification of strips for detecting moisture in organic solvents

For simplicity, the water detection using the compound 1, the compound 1 (1.0 mM) of the filter paper was immersed in the CHCl ₃ solution was dried in an oven for one day to prepare a paper strip impregnated with a compound 1.

33, the strip was immersed in EtOH, MeOH and ACN solution containing moisture (0, 5, 10, 20 and 30%, v / v) at various ratios and 365 nm using a portable UV lamp. Fluorescence color changes were visualized with excitation light.

As a result, it was confirmed that the paper strip in which Compound 1 was infiltrated showed dark blue fluorescence as shown in FIG. 32A.

The paper strips immersed in pure EtOH, MeOH, and ACN solvents showed blue fluorescence, and the characteristic fluorescence changes rapidly as the moisture ratio increases, and the color change is observed similarly to FIG. 29.

In addition, it was repeatedly immersed in an aqueous solution of EtOH and dried to confirm the reversible fluorescence of the paper strip in which Compound 1 was infiltrated with respect to moisture.

As a result, as shown in FIG. 32B, when the paper strip was immersed in an aqueous solution of EtOH containing 10% moisture, the fluorescent color rapidly changed from blue to white, and the strip returned to blue fluorescence by simple solvent drying.

As it is confirmed from the above results that the paper strip can be repeatedly used for moisture detection, the paper strip can be used very effectively for moisture detection by quickly and simply visualizing fluorescence color change as well as reusability.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

The scope of the present invention is represented by the following claims, and it should be interpreted that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.

Claims (15)

1. A compound represented by formula (1) or a pharmaceutically acceptable salt thereof:

   [Formula 1]

   

   Wherein X is a mitochondrial target moiety.
2. According to claim 1, wherein the mitochondrial target moiety is triphenylphosphonium cation (triphenylphosphonium cation), rhodamine (rhodamine) and hemiyanine (hemicyanine), characterized in that the compound or a pharmaceutically acceptable Possible salts.
3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound or a pharmaceutically acceptable salt thereof is represented by the following Chemical Formula 2:

   [Formula 2]

   
4. 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]

   

   Wherein X is a mitochondrial target moiety.
5. 5. The mitochondrial NAD (P) H according to claim 4, wherein the mitochondrial target moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine. Detection composition.
6. A fluorescent chemical sensor 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]

   

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

   [Formula 1]

   

   Wherein X is a mitochondrial target moiety.
9. 9. The mitochondrial NAD (P) H according to claim 8, wherein the mitochondrial target moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine, and hemicyanine. Detection kit.
10. 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]

    

    Wherein X is a mitochondrial target moiety.
11. The composition of claim 10, wherein the mitochondrial target moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine, and hemiyanine.
12. 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]

    

    Wherein X is a mitochondrial target moiety.
13. 13. The kit for detecting cancer cells according to claim 12, wherein the mitochondrial target moiety is selected from the group consisting of triphenylphosphonium cation, rhodamine and hemicyanine.
14. (a) treating a compound of formula 1 according to claim 1 or a pharmaceutically acceptable salt by reacting with a cell; And

    (b) measuring at least one indicator selected from the group consisting of fluorescence wavelength, fluorescence intensity, and optical change of the reacted reactant.
15. The method of claim 14, wherein the indicator is UV-Vis (Ultraviolet-visible) spectrophotometer, fluorescence photometer, electrospray ionization mass spectroscopy and confocal microscope characterized in that any one or more selected from the group consisting of using a method Method for detecting NAD (P) H in the mitochondria.

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