KR20160038129A - Compound based on coumarin hemicyanine scaffold for selectively detect peroxynitrite - Google Patents

Abstract

The present invention relates to a compound based on coumarin hemicyanine structure for selectively detecting peroxynitrite. According to the present invention, the compound based on a coumarin hemicyanine structure maintains the structure in environment inside cells and has a changed fluorescence or absorption spectrum by selectively reacting with exogenous or endogenous peroxynitrite. Therefore, the compound of the present invention is useful to detect peroxynitrite in cells.

Description

[0001] The present invention relates to a compound based on coumarin hemicyanine scaffold for selectively detecting peroxynitrite,

The present invention relates to compounds based on cumarine hemicyanide for selectively detecting peroxynitrite.

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) play a very important role in physiology and pathology (Non-Patent Document 1). In particular, peroxynitrite (ONOO ^- ) among the types of ROS and RNS is a very important factor involved in various cell functions in vivo due to its strong oxidizing property and nucleophilic property.

More specifically, peroxynitrite reacts with carbon dioxide to form nitrosoperoxycarbonate (ONOOCO ₂ ^- ) as an intermediate, and the resulting nitrosopentyloxycarbonate is rapidly decomposed to form carbonate radicals (CO ₃ ^- ) Is generated. The carbonate radicals generated here cause cell damage such as cell apoptosis or necrotic cell death. In addition, peroxynitrite having a strong oxidizing ability can directly react with an electron-rich moiety of a biomolecule such as DNA (deoxyribonucleic acid) or protein to interfere with the arrangement of the biomolecule, and as a result, , Cancer, diabetes, inflammatory diseases, neuropathic diseases and the like (Non-Patent Document 2). On the other hand, it is also known that peroxynitrite, preferably controlled and generated in vivo, plays a role as a signaling element for inducing normal function of cells. Therefore, in order to accurately understand the relevance of peroxynitrite to in vivo cell function, a method for selectively detecting peroxynitrite is needed.

Accordingly, various fluorescent probes have been developed for detecting peroxynitrite in vivo. Specifically, Non-Patent Document 3 describes a probe based on a direct oxidation reaction of boronate. In addition, a probe based on a reaction with active ketones (Non-Patent Document 4), a probe based on metal complex formation (Non-Patent Document 5), a probe using a nonmetal element tellurium (Non-patent document 6) and the like are known. However, not only is the half life (<20 ms) of peroxynitrite very short in certain physiological conditions, but also the method of detecting peroxynitrite selectively for various kinds of ROS such as ClO ^- , H ₂ O ₂ There is a limit to use (Non-Patent Document 7). In addition, there is a problem that it is difficult to selectively detect peroxynitrite using a probe according to the above method due to the reducing ability of biomolecules present in vivo.

Accordingly, the present inventors have conducted studies to develop probes for selectively detecting peroxynitrite in vivo, and found that the compounds based on the coumarin hemi-cyan structure according to the present invention are useful as peroxynitrite The present inventors have confirmed that fluorescence or light absorption changes due to selective reaction with light and that the detection can be easily performed, and the present invention has been completed.

Chen, Tian, Shin and Yoon 2011; Dickinson and Chang 2011; Pacher, Beckman and Liaudet 2007; Winterbourn 2008; Xu, Lee, Lee, Lee, and Yoon 2013; Yang, Seidlits, Adams, Lynch, Schmidt, Anslyn and Shear 2010. Kryston, Georgiev, Pissis and Georgakilas 2011; Soon, Donnelly, Turner, Hung, Crouch, Sherratt, Tan, Lim, Lam, Bica, Lim, Hickey, Morizzi, Powell, Finkelstein, Culvenor, Masters, Duce, White, Barnham and Li 2011. Chen et al. 2013b; Sikora, Zielonka, Lopez, Joseph and Kalyanaraman 2009; Sun, Xu, Kim, Flower, Lowe, Yoon, Fossey, Qian, Bull and James. Peng and Yang 2010; Sun, Wang, Liu, Chen, Tam and Yang 2009; Yang et al. 2006. Rausaria, Kamadulski, Rath, Bryant, Chen, Salvemini and Neumann 2011. Koide, Kawaguchi, Urano, Hanaoka, Komatsu, Abo, Terai and Nagano 2012; Yu, Li, Wang and Han 2013. Chen, Ren, Wright and Ai 2013b; Oushiki, Kojima, Terai, Arita, Hanaoka, Urano and Nagano 2010; Yu, Song, Li, Wang and Han 2012.

It is an object of the present invention to provide a coumarin hemi-cyan structure-based compound.

Another object of the present invention is to provide a process for producing the above compound.

A further object of the present invention are peroxy nitrite (ONOO ^-) containing the compound to provide a sensor for detecting chemicals.

Another object of the present invention is to provide a method for detecting peroxynitrite (ONOO ^- ) using the above chemical sensor.

In order to achieve the above object, the present invention provides a compound represented by the following general formula (1).

[Chemical Formula 1]

In Formula 1,

R ¹ , R ² , R ³ , R ⁴ and R ⁵ are independently -H, -OH, halogen, C _{1 -10} linear or branched alkyl, or C _{1 -10} linear or branched alkoxy.

The present invention also relates to a process for producing a compound represented by the formula (1)

Reacting a compound represented by the formula (2) with a compound represented by the formula (3) to prepare a compound represented by the formula (4) (step 1);

(Step 2) of preparing a compound represented by the formula (5) by substituting the compound represented by the formula (4) obtained in the step 1 with a formaldehyde group; And

Reacting a compound represented by the formula (5) and a compound represented by the formula (6) obtained in the above step 2 to prepare a compound represented by the formula (1) (step 3) to provide.

[Reaction Scheme 1]

In the above Reaction Scheme 1,

R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined in the above formula (1).

Furthermore, the present invention provides a chemical sensor for detection of peroxynitrite (ONOO ^- ) containing the above compound.

The present invention also provides a method for detecting peroxynitrite (ONOO ^- ) using the chemical sensor.

Compounds based on the coumarin hemicaine structure according to the present invention maintain their structure in the intracellular environment and selectively react with exogenous or endogenous peroxynitrite to change fluorescence or absorbance spectrum. Therefore, peroxynitrite Can be used effectively.

1 is an image showing the reaction mechanism of the compound (CHCN) prepared in Example 1 and peroxynitrite,
FIG. 2 is an image obtained by observing a change in fluorescence spectrum when the compound prepared in Example 1 reacts with peroxynitrite (0-20 μM), and the illustration shows an image obtained before and after the addition of peroxynitrite (F _{515 nm} / F _{635 nm} ) of fluorescence intensity as a function of concentration and fluorescence change,
FIG. 3 is an image showing a change in absorption spectrum when the compound prepared in Example 1 reacts with peroxynitrite (0-20 μM)
FIG. 4 is an image showing fluorescence spectra observed when the ROS, RNS, and biothiol were respectively reacted with the compound prepared in Example 1, and the illustration shows that the respective ROS, RNS, and biothiol were reacted with the compound prepared in Example 1 (F _{515 nm} / F _{635 nm} ) before and after the reaction,
FIG. 5 is an image showing the absorption spectrum observed when the ROS, RNS, and biothiol were respectively reacted with the compound prepared in Example 1,
FIG. 6 is an image obtained by slowly changing the peak of ¹ H NMR of peroxynitrite in the compound prepared in Example 1,
Fig. 7 is an image obtained by slowly changing the peak observed in the mass spectrometry by slowly titrating peroxynitrite into the compound prepared in Example 1,
8 is a fluorescence image observed by using a confocal microscope after sequential treatment of the compound prepared in Example 1 and extraneous peroxynitrite in vivo cells,
9 is a fluorescence image obtained by inducing peroxynitrite to be generated in living cells, treating the compound prepared in Example 1 and observing using a confocal microscope,
10 is an image showing the change of the fluorescence intensity ratio (F _{515 nm} / F _{635 nm} ) according to various pH ranges,
11 is an image showing a change in the ratio of fluorescence intensity (F _{515 nm} / F _{635 nm} ) to various NaCl ion concentrations,
12 is an image for evaluating the emission intensity of the compound prepared in Example 1. Fig.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound represented by the following formula (1).

[Chemical Formula 1]

In Formula 1,

R ¹ , R ² , R ³ , R ⁴ and R ⁵ are independently -H, -OH, halogen, C _{1 -10} linear or branched alkyl, or C _{1 -10} linear or branched alkoxy.

Preferably,

R ¹ , R ² , R ³ , R ⁴ and R ⁵ are independently -H, -OH, C _{1 -5} straight or branched chain alkyl, or C _{1 -5} straight or branched alkoxy.

More preferably,

R ¹ and R ² are independently ethyl,

R ³ , R ⁴ and R ⁵ are independently methyl.

Among these active oxygen species, ONOO ^- , NO, O ₂ , H ₂ O ₂ , OCl ^- , OH, t - BuOO, and the like, only the exogenous or endogenous peroxynitrite (ONOO ^- ) Can be selectively used.

The present invention also relates to a process for producing a compound represented by the formula (1)

Reacting a compound represented by the formula (2) with a compound represented by the formula (3) to prepare a compound represented by the formula (4) (step 1);

(Step 2) of preparing a compound represented by the formula (5) by substituting the compound represented by the formula (4) obtained in the step 1 with a formaldehyde group; And

Reacting a compound represented by the formula (5) and a compound represented by the formula (6) obtained in the above step 2 to prepare a compound represented by the formula (1) (step 3) to provide.

[Reaction Scheme 1]

In the above Reaction Scheme 1,

R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined in the above formula (1).

Hereinafter, the process for preparing the compound represented by the formula (1) according to the present invention will be described in detail.

In the process for preparing a compound represented by the formula (1) according to the present invention, the step (1) is a step of reacting a compound represented by the formula (2) with a compound represented by the formula (3) Is a step of dissolving a compound represented by the formula (2), a compound represented by the formula (3) and piperidine in a solvent, heating under reflux, and then adding an acid to obtain a compound represented by the formula (4).

At this time, as the reaction solvent, tetrahydrofuran; Dioxane; Ether solvents including ethyl ether, 1,2-dimethoxyethane and the like; Lower alcohols including methanol, ethanol, propanol and butanol; But are not limited to, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonitrile, acetonazenesulfonate, toluene sulfonate, chlorobenzene sulfonate, Sulfonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate , Mandelate and the like can be used, and it is preferable to use ethanol.

As the acid, hydrochloric acid, acetic acid, etc. may be used alone or in combination.

Further, the reaction is preferably carried out at a temperature of 0 ° C between the boiling point of the solvent, and the reaction time is not particularly limited, but preferably 0.5-10 hours.

In the process for preparing a compound represented by the formula (1) according to the present invention, the step (2) is a step for preparing a compound represented by the formula (5) by substituting a formaldehyde group for the compound represented by the formula (4) Specifically, the compound represented by the formula (4) is dissolved in a solvent, and phosphorous chloride is added and stirred to prepare a compound represented by the formula (5).

At this time, as the reaction solvent, tetrahydrofuran; Dioxane; Ether solvents including ethyl ether, 1,2-dimethoxyethane and the like; Lower alcohols including methanol, ethanol, propanol and butanol; But are not limited to, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonitrile, acetonazenesulfonate, toluene sulfonate, chlorobenzene sulfonate, Sulfonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate , Mandelate and the like can be used, and it is preferable to use dimethylformamide (DMF).

In addition, the reaction is preferably carried out at a temperature of 0 ° C between the boiling point of the solvent, and the reaction time is not particularly limited, but preferably 0.5-10 hours.

In the process for preparing a compound represented by the formula (1) according to the present invention, the above step 3 is a step of reacting the compound represented by the formula (5) obtained in the above step 2 with the compound represented by the formula (6) More specifically, the compound represented by formula (5) and the compound represented by formula (6) are dissolved in a solvent, heated under reflux and purified to prepare a compound represented by formula (1).

At this time, as the reaction solvent, tetrahydrofuran; Dioxane; Ether solvents including ethyl ether, 1,2-dimethoxyethane and the like; Lower alcohols including methanol, ethanol, propanol and butanol; But are not limited to, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonitrile, acetonazenesulfonate, toluene sulfonate, chlorobenzene sulfonate, Sulfonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate , Mandelate and the like can be used, and it is preferable to use ethanol.

In addition, the reaction is preferably carried out at a temperature of 0 ° C between the boiling point of the solvent, and the reaction time is not particularly limited, but preferably 0.5-10 hours.

Further, the present invention provides a chemical sensor for detecting peroxynitrite (ONOO ^- ) containing the compound represented by the above formula (1). Here, the chemical sensor is characterized by detecting peroxynitrite (ONOO ^- ) in a biological sample.

The present invention also provides a method for detecting peroxynitrite (ONOO ^- ) using the above chemical sensor. Here, the detection method is characterized in that peroxynitrite (ONOO ^- ) is detected in a biological sample.

Specifically, as shown in FIG. 1, a double bond in a pentagonal ring of the compound represented by the formula (1) occurs due to a nucleophilic attack of an oxygen atom having an anion of peroxynitrite (ONOO ^- ) Peroxynitrite (ONOO ^- ) can be detected by solely or in combination with changes in fluorescence or light absorption characteristics resulting from the change of the--conjugation system.

1 is an image showing the reaction mechanism of the compound (CHCN) prepared in Example 1 and peroxynitrite.

Compounds based on the coumarin hemicyanide structure according to the present invention are expected to maintain their structure in the intracellular environment and selectively react with exogenous or endogenous peroxynitrite to change the fluorescence or the absorption spectrum. The following series of experiments were performed.

First, experiments were carried out to evaluate the change of fluorescence and absorption spectrum according to the concentration of peroxynitrite (ONOO ^- ). As a result, the compound prepared in Example 1 reacted with peroxynitrite and fluorescence and absorption spectra (See Figs. 2 and 3 of Experimental Example 1).

In addition, experiments were carried out to evaluate the selectivity of the coumarin hemicyan structure-based compound according to the present invention to peroxynitrite. As a result, it was found that the compound prepared in Example 1 selectively reacted with peroxynitrite, Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS), Cysteine, Homocysteine, and Glutathione have been reported to have different spectra No change in fluorescence and absorption spectrum was observed, indicating no selectivity (see FIGS. 4 and 5 of Experimental Example 2).

Furthermore, in order to observe the reaction mechanism of the peroxynitrite and the coumarin hemicyan structure-based compound according to the present invention, experiments using ¹ H NMR and mass spectroscopy showed that peroxynitrite and the The reaction mechanism of the compound was shown to have the mechanism behavior shown in FIG. 1 (FIG. 6 and FIG. 7 of Experimental Example 3).

Experiments were conducted using W138 VA13 cells (normal human lung cells) and RAW 264.7 cells (macrophage cells) in order to evaluate the detection ability of intracellular extracellular or endogenous peroxynitrite. As a result, One compound selectively reacts with an exogenous or endogenous peroxynitrite to change the fluorescence and absorption spectrum (see FIGS. 8 and 9 of Experimental Example 4).

Furthermore, experiments were conducted to evaluate the stability of the coumarin hemicyanide-based compounds according to the present invention in various pH or ion concentration environments. As a result, it was found that the compound prepared in Example 1 had a structure Of the total population. In addition, the compound prepared in Example 1 showed that the structure was stably maintained even at an ion concentration of 0.0 to 2.0 mM, and it was confirmed that the chemical structure could be stably maintained in the cells (see FIGS. 10 and 10 of Experimental Example 5) 11).

Further, as a result of conducting an experiment to evaluate the optical stability of a coumarin hemicyan structure-based compound according to the present invention, the compound prepared in Example 1 showed excellent optical stability (see FIG. 12 of Experimental Example 6) .

Further, as a result of conducting an experiment to evaluate the biocompatibility of a coumarin hemicyan structure-based compound according to the present invention, the compound prepared in Example 1 showed excellent biocompatibility (see Experimental Example 7).

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the present invention is not limited thereto.

< Example  1 (E) -2- (2- (7- ( Diethylamino ) -2-oxo-2H- Kromen Yl) vinyl) -1,3,3-trimethyl-3H- Of indole  Produce

Step 1: 7- ( Diethylamino ) -2H- Kromen -2-one

4-Diethylaminosalicylaldehyde (1.93 g, 10 mmol), malonic acid diethyl (3.2 g, 20 mmol) and piperidine (1 mL) were dissolved in ethanol (30 mL) and heated to reflux for 6 hours. The mixture was cooled to room temperature, and ethanol was removed under reduced pressure. To the product was added concentrated hydrochloric acid (20 mL) and glacial acetic acid (20 mL), stirred for 6 hours, and then hydrolyzed. The solution was cooled to room temperature and 100 mL ice water was added. NaOH solution (40%) was added in small amounts to adjust the pH to 5. After stirring for 30 minutes, the mixture was filtered using water, and recrystallized with toluene to obtain the target compound (1.74 g, yield: 80.1%).

¹ H-NMR (CDCl ₃ )? 7.55 (d, IH), 7.23 (d, IH), 6.59 (d, ), 1.21 (t, 6H).

Step 2: 7- ( Diethylamino ) -2-oxo-2H- Kromen -3- Of carbaldehyde  Produce

Phosphorus chloride (2 mL) in argon was added dropwise to anhydrous DMF (2 mL) to give a red solution. The solution was mixed with the compound obtained in step 1 (1.50 g, 6.91 mmol) dissolved in anhydrous DMF (10 mL) and stirred for 30 minutes. The mixture was heated at reflux for 12 hours at 60 &lt; 0 &gt; C and 100 mL of ice water was added. The pH was adjusted to 5 using NaOH solution (20%). The mixture was filtered and recrystallized to obtain the desired compound (1.20 g, yield: 70.8%).

_{^{1 H-NMR (CDCl 3)}} δ 10.13 (s, 1H), 8.26 (s, 1H), 7.43 (d, 1H), 6.67 (d, 1H), 6.50 (s, 1H), 3.49 (m, 4H) , &Lt; / RTI &gt; 1.25 (t, 6H).

Step 3: (E) -2- (2- (7- ( Diethylamino ) -2-oxo-2H- Kromen Yl) vinyl) -1,3,3-trimethyl-3H- Of indole  Produce

2-oxo-2H-chromen-3-carbaldehyde (50 mg, 2 mmol) and 1,2,3,3-tetramethyl-3H- indolium (60 mg, ) Was dissolved in 20 mL of ethanol and heated under reflux for 10 hours. After cooling to room temperature, the ethanol was removed under reduced pressure. The resulting solid was purified by column chromatography using silica gel and mobile phase using DCM / methanol (20/1, v: v) to obtain the title compound as a dark purple solid (0.312 g, yield: 68%).

^{1 H NMR (300 MHz, CDCl} 3 -d 3) δ 10.05 (s, 1H), 8.60 (d, J = 16.2 Hz, 1H), 8.13 (d, J = 9.3 Hz, 1H), 8.10 (d, J 1H), 4.31 (s, 3H), 3.54 (q, J = 7.1 Hz, 1H), 6.72 (d, J = , 4H), 1.85 (s, 6H), 1.34 (t, J = 7.1 Hz, 6H).

¹³ C NMR (75 MHz, MeOD-d ₃ )? 158.1, 154.7, 150.3, 143.1, 140.3, 132.3, 128.7, 122.7, 113.8, 112.4, 110.0, 96.4, 78.1, 51.7, 45.1, 32.6, 25.4, 11.4. MS: [M ⁺ ] &^lt; / RTI ^&gt;

< Experimental Example  1 > peroxynitrite ( ONOO ^- ) Fluorescence and Absorbance  Evaluation of spectrum change

<1-1> Evaluation of Fluorescence Spectrum Change

The following experiment was carried out in order to observe the change of the fluorescence spectrum when the compound prepared in Example 1 was reacted with peroxynitrite.

The fluorescence spectrum changed by addition of peroxynitrite to the compound prepared in Example 1 at a concentration of 0-20 μM in PBS (Phosphate buffered saline) buffer (pH 7.4) was measured with RF-5301 / PC Shimada) fluorescence spectrophotometer (1 cm quartz cell), and the results are shown in Fig.

FIG. 2 is an image obtained by observing a change in fluorescence spectrum when the compound prepared in Example 1 reacts with peroxynitrite (0-20 μM), and the illustration shows an image obtained before and after the addition of peroxynitrite (F _{515 nm} / F _{635 nm} ) of fluorescence intensity as a function of fluorescence intensity and fluorescence change.

As shown in FIG. 2, the fluorescence intensity (A: red) intensity near 635 nm in the fluorescence spectrum decreased and the fluorescence intensity (B: green) intensity around 515 nm increased as the concentration of peroxynitrite was increased . This demonstrates that the fluorescence intensity shifts to a shorter wavelength as the compound of Example 1 reacts with peroxynitrite through a mechanism such as that shown in FIG. 1 and a change of the--conjugation system occurs. Further, as shown in the illustration of FIG. 2, it was confirmed that the ratio of the fluorescence intensity (F _{515 nm} / F _{635 nm} ) linearly increased with respect to the concentration of peroxynitrite. Further, the Y-intercept value (0.025) of the regression line showing the standard deviation of the response (SD), the slope of the calibration curve (S, 1.66) and the slope The lower limit of detection concentration value was found to be 49.7 nM through the equation expressed by the following equation (1).

In the above equation (1)

SD is the response standard deviation,

S is the slope.

&Lt; 1-2 > Evaluation of change in absorption spectrum

In order to observe the change of the absorption spectrum when the compound prepared in Example 1 and the peroxynitrite were reacted, the same procedure as in Experimental Example 1-1 was carried out. The results are shown in Fig.

FIG. 3 is an image showing a change in absorption spectrum when the compound prepared in Example 1 and peroxynitrite (0-20 μM) were reacted. FIG.

As shown in FIG. 3, the absorption (A: blue) intensity near 300 nm and 568 nm of the absorption spectrum decreases as the concentration of peroxynitrite is increased, and the intensity of light absorption (B: yellow) near 422 nm increases Respectively. This demonstrates that the compound of Example 1 reacts with peroxynitrite through a mechanism such as in FIG. 1, resulting in the change of the--conjugation system.

Therefore, the compound based on the coumarin hemicaine structure according to the present invention reacts with peroxynitrite to change its fluorescence and absorption spectrum, so that it can be usefully used for detecting peroxynitrite.

< Experimental Example  2 > peroxynitrite ( ONOO ^- ) Selectivity evaluation

The following experiment was conducted to evaluate the selectivity of the compound prepared in Example 1 for peroxynitrite.

<2-1> Evaluation of Selectivity by Fluorescence Spectrum Change

Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS), and the like were used instead of 0-20 μM of peroxynitrite to evaluate the selectivity of the compound prepared in Example 1, , Or biothiols were used. More specifically, AHP (2,2'-azobisisobutyronitrile), which is 2,2'-azobis (2-amidinopropane) dihydrochloride, azobis (2-amidinopropane) obtaining a peroxyl radical from the ^{_{dihydrochloride)), · O 2 -}} , t-BuOO ·, H 2 O 2, ClO - , respectively 30μM, cysteine (cysteine, Cys), homocysteine (homocysteine, Hcy), Was performed in the same manner as in Experimental Example < 1-1 > except that glutathione (GSH) was used at a concentration of 100 mu M, respectively. The results are shown in Fig.

FIG. 4 is an image showing fluorescence spectra observed when the ROS, RNS, and biothiol were respectively reacted with the compound prepared in Example 1, and the illustration shows that the respective ROS, RNS, and biothiol were reacted with the compound prepared in Example 1 (F _{515 nm} / F _{635 nm} ) before and after the reaction with the fluorescent material.

As shown in Fig. 4, it was confirmed that the compound prepared in Example 1 exhibited a high fluorescence intensity around 515 nm of the fluorescence spectrum when it was reacted with peroxynitrite only. ROS, RNS and biothiol, except peroxynitrite, were found to exhibit high fluorescence intensity near 635 nm of the fluorescence spectrum, as in the untreated group. 4, the ratio of the fluorescence intensity (F _{515 nm} / F _{635 nm} ) was higher when using peroxynitrite than when using ROS, RNS and biothiol except peroxynitrite It was confirmed that the fluorescence intensity was about 474 times higher. This demonstrates that the compound of Example 1 is only selective for peroxynitrite.

<2-2> Absorbance  Evaluation of selectivity through spectrum change

The selectivity of the compound prepared in Example 1 was evaluated in the same manner as in Experimental Example <2-1>, and the results are shown in FIG.

FIG. 5 is an image showing the absorption spectrum observed when the ROS, RNS, and biothiol were respectively reacted with the compound prepared in Example 1. FIG.

As shown in FIG. 5, it was confirmed that the compound prepared in Example 1 exhibited a low absorption intensity around 568 nm of the absorption spectrum when it was reacted with only peroxynitrite. It was confirmed that ROS, RNS and biothiol except peroxynitrite exhibited a high absorption intensity at around 568 nm of the absorption spectrum as in the untreated group. This demonstrates that the compound of Example 1 is only selective for peroxynitrite.

Therefore, the compound based on the coumarin hemicyan structure according to the present invention selectively reacts with peroxynitrite to change its fluorescence and absorption spectrum, so that it can be usefully used for detecting peroxynitrite.

< Experimental Example  3> Observation of reaction mechanism

Peaks were observed using ¹ H NMR and mass spectroscopy to observe the reaction mechanism of the peroxynitrite with the compound prepared in Example 1. At this time, ¹ H NMR spectrum was measured on a CDCl ₃ solution containing tetramethylsilane (TMS) as an internal standard using a Bruker AM-300 spectrometer. In addition, the mass spectrum was measured using a JMS-HX 110A / 110A Tandem mass spectrometer (JEOL).

<3-1> ^One H NMR  Measurement through appropriate titration ( titration ) Experiment

Peroxynitrite was slowly titrated to ^{0 -} 20 μM against 1 μM of the compound prepared in Example 1, and the change in the peak observed by ¹ H NMR was observed. The results are shown in Fig.

FIG. 6 is an image obtained by slowly titrating peroxynitrite into the compound prepared in Example 1 and observing the change in peak appearing in ¹ H NMR.

As shown in Fig. 6, before addition of peroxynitrite, a peak (8.03 ppm, 7.98 ppm) corresponding to the double bond hydrogen between hemicyanine and coumarin of the compound prepared in Example 1 It was observed that the peaks gradually disappear as peroxynitrite was titrated.

<3-2> Titration through mass spectrometry ( titration ) Experiment

Peroxynitrite was gradually titrated to 1-20 [mu] M against 1 [mu] M of the compound prepared in Example 1, and the change in the peak appearing in the mass spectrum was observed. The results are shown in Fig.

FIG. 7 is an image obtained by slowly changing the peak of the compound produced in Example 1 and observing the change of the peak in the mass spectrometry.

As shown in Fig. 7, when the peroxynitrite was reacted with the compound (CHCN) prepared in Example 1 by the same amount, not only a peak near 401.2 m / z corresponding to the compound of Example 1 was observed, each "CHCN-O", "CHCN -O 2 H", "CHCN-NO 2" 417.1 m / z, 435.0 m / z, 446.2 m / z peak was observed corresponding to.

Further, when an excessive amount of peroxynitrite was reacted with the compound (CHCN) prepared in Example 1, the reaction product, 1,3,3-trimethyloxiindole, and cumarin-3 176.2 m / z, 246.3 m / z peak corresponding to coumarin-3-aldehyde was observed.

Therefore, it can be seen that the compound based on the coumarin hemicyan structure according to the present invention has a mechanism behavior as shown in FIG. 1 when reacted with peroxynitrite.

< Experimental Example  4> Evaluation of detection ability of exogenous and endogenous peroxynitrite in living cells

The following experiment was conducted to evaluate the detection ability of the compound prepared in Example 1 for exogenous and endogenous peroxynitrite in vivo cells.

First, W138 VA13 cells (normal human lung cells) and RAW 264.7 cells (macrophage cell line) is obtained from the American Type Culture Collection (Manassas, VA), under 37 ℃ 5% CO ₂ environment 10 × culture medium [1% Protea OSU Were cultured on a glass slide surface in a protease peptone, 0.2% glucose, 0.1% yeast extract, 0.003% ethylenediaminetetraacetic acid (EDTA) ferric sodium salt. Specifically, cells were scraped and adhered into 6-well plates according to the manufacturer's instructions.

<4-1> Evaluation of detection ability of exogenous peroxynitrite in vivo

W138 VA13 cells were treated with 5 μM of the compound (CHCN) prepared in Example 1 for 30 minutes and then washed with Dulbecco's Phosphate Buffered Saline (DPBS). Subsequently, exogenous peroxynitrite was treated at concentrations of 0.1 mM and 0.3 mM, respectively, and imaged using a confocal microscope. The results are shown in Fig.

Fig. 8 is a fluorescence image observed by using a confocal microscope after sequential treatment of the compound prepared in Example 1 and extrinsic peroxynitrite in a living body cell.

As shown in Fig. 8, when the peroxynitrite was not treated, red fluorescence (red fluorescence channel, 575-675 nm) by the compound (CHCN) prepared in Example 1 was observed, indicating that CHCN It can be confirmed that it is maintained in a perfect form. Then, peroxynitrite, which acts as an oxidizing agent, is oxidized and CHCN is oxidized to weaken the red fluorescence and enhance the green fluorescence (green fluorescence channel, 490-540 nm).

In addition, it can be confirmed from the image merged with the green fluorescence channel and the red fluorescence channel that is confirmed in yellow that the ratio ratiometric reaction of the CHCN with the change in the concentration of peroxynitrite in the biocell is excellent .

<4-2> Evaluation of detection ability of endogenous peroxynitrite in living cells

In order to produce endogenous peroxynitrite, lipopolysaccharide (LPS) was added to RAW 264.7 cells having the characteristics of generating Reactive Oxygen Species (ROS) or Reactive Nitrogen Species (RNS) Treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 30 min, followed by incubation for 1 hour at 37 ° C for 1 hour, Led to the production of Roxynitrite. Then, 100 μM of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) or aminoguanidine (AG) as a nitric oxide synthesis inhibitor was further treated with 1 mM of a peroxide capturing agent. The cells were treated with 5 μM of the compound (CHCN) prepared in Example 1 for 30 minutes, washed three times with Dulbecco's Phosphate Buffered Saline (DPBS) and imaged using a confocal microscope. The results are shown in Fig.

FIG. 9 is a fluorescence image obtained by inducing peroxynitrite to generate biocells, treating the compound prepared in Example 1, and observing using a confocal microscope.

As shown in FIG. 9, when the compound (CHCN) prepared in Example 1 was treated without inducing the production of peroxynitrite by RAW 264.7 cells, red fluorescence by CHCN (red fluorescence channel, 575-675 nm ) Was observed, indicating that the CHCN was maintained intact in the cytoplasm of the living cell. On the other hand, when RAW 264.7 cells were treated with LPS, interferon-γ, and PMA in order to induce peroxynitrite generation, the oxidative nature of endogenous peroxynitrite caused by RAW 264.7 cells caused CHCN The red fluorescence is weakened and the green fluorescence (green fluorescence channel, 490-540 nm) is strengthened. RAW 264.7 cells were treated with LPS, interferon-γ, and PMA sequentially, and then treated with CHCN and then treated with AG or TEMPO to inhibit the generation of endogenous peroxynitrite, resulting in stronger red fluorescence than green fluorescence Can be confirmed.

In addition, it can be confirmed from the image merged with the green fluorescence channel and the red fluorescence channel that is confirmed in yellow that the ratio ratiometric reaction of the CHCN with the change in the concentration of peroxynitrite in the biocell is excellent .

Therefore, the compound based on the coumarin hemicaine structure according to the present invention maintains its structure in a cell and selectively reacts with exogenous or endogenous peroxynitrite to change the fluorescence and the absorption spectrum. Therefore, the peroxynitrite Can be used effectively.

< Experimental Example  5> pH  Or stability of ion concentration environment

In order to evaluate the stability of the compound prepared in Example 1 in various pH ranges or ion concentrations, the pH and the NaCl ion concentration were varied, and the ratio of fluorescence intensity (F _{515 nm} / F _{635 nm} ). The results are shown in Fig. 10 and Fig.

Fig. 10 is an image showing the change of the fluorescence intensity ratio (F _{515 nm} / F _{635 nm} ) according to various pH ranges.

11 is an image showing a change in the ratio of fluorescence intensity (F _{515 nm} / F _{635 nm} ) to various NaCl ion concentrations.

As shown in FIG. 10, the compound prepared in Example 1 showed a stable structure at pH 4-9, and when the pH was over 9, the structure was decomposed to change the ratio of fluorescence intensity .

Also, as shown in Fig. 11, it was confirmed that the compound prepared in Example 1 was stably maintained in the ion concentration environment of 0.0 to 2.0 mM.

Therefore, the compound based on the coumarin hemicaine structure according to the present invention maintains its structure in a cell and selectively reacts with exogenous or endogenous peroxynitrite to change the fluorescence and the absorption spectrum. Therefore, the peroxynitrite Can be used effectively.

< Experimental Example  6> Evaluation of light stability

In order to evaluate the light stability of the compound prepared in Example 1, the emission intensity was measured using a UV-vis spectra (Scinco 3000 spectrophotometer, 1 cm quartz cell) at 25 ° C. The results are shown in FIG. 12 .

12 is an image for evaluating the emission intensity of the compound prepared in Example 1. Fig.

As shown in FIG. 12, the compound prepared in Example 1 showed that the structure was stably maintained for 24 hours, indicating that the radiation intensity was excellent.

Therefore, the compound based on the coumarin hemicaine structure according to the present invention maintains its structure in a cell and selectively reacts with exogenous or endogenous peroxynitrite to change the fluorescence and the absorption spectrum. Therefore, the peroxynitrite Can be used effectively.

< Experimental Example  7> Assessment of cytotoxicity

In order to evaluate the cytotoxicity of the compounds prepared in Example 1, the compounds were added to W138 VA13 cells (normal human lung cells) and RAW 264.7 cells (macrophage cells) used in Experimental Example 4, respectively.

As a result, all the cells died after 24 hours of incubation of the compound prepared in Example 1 and the above cells. However, since it is confirmed that the cells remain intact for 2 hours, it can be seen that the compound of the present invention has sufficient cytotoxicity to detect intracellular peroxynitrite using the compound based on the cumarine hemicyan structure.

Claims (10)

A compound represented by the following formula (1):
[Chemical Formula 1]

(In the formula 1,
^{R 1, R 2, R 3} , R 4 and R ⁵ are independently -H, -OH, halogen, C _{1 -10} linear or branched alkyl, alkoxy or linear or branched C _{1 -10} in).
The method according to claim 1,
Wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are independently -H, -OH, C _{1 -5} straight or branched chain alkyl, or C _{1 -5} straight or branched alkoxy.
The method according to claim 1,
R ¹ and R ² are independently ethyl,
R ³ , R ⁴ and R ⁵ are independently methyl.
As shown in Scheme 1 below,
Reacting a compound represented by the formula (2) with a compound represented by the formula (3) to prepare a compound represented by the formula (4) (step 1);
(Step 2) of preparing a compound represented by the formula (5) by substituting the compound represented by the formula (4) obtained in the step 1 with a formaldehyde group; And
(3), which comprises reacting the compound represented by the formula (5) and the compound represented by the formula (6) obtained in the step 2 to prepare a compound represented by the formula (1) Way:
[Reaction Scheme 1]

(In the above Reaction Scheme 1,
R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same as defined in the formula (1).
A chemical sensor for detecting peroxynitrite (ONOO ^- ) comprising the compound of claim 1.
6. The method of claim 5,
Wherein the chemical sensor detects peroxynitrite (ONOO ^- ) in a biological sample.
6. The method of claim 5,
It said chemical sensor is in the five-membered ring of the double bonds in the compound represented by formula (1) of claim 1, peroxy nitrite (ONOO ^-) л- arising from nucleophilic attack of the oxygen atom stands for the anion of (nucleophilic attack) Characterized in that the change in fluorescence or absorption characteristics due to the change of the conjugated system is measured singly or in combination.
A method for detecting peroxynitrite (ONOO ^- ) using the chemical sensor of claim 5.
9. The method of claim 8,
Wherein the detection method detects peroxynitrite (ONOO ^- ) from a biological sample.
9. The method of claim 8,
The above-mentioned detection method is a method for detecting the presence of a--amino group in the double bond in the pentagonal ring of the compound represented by the general formula (1), which is caused by a nucleophilic attack of an oxygen atom having anion of peroxynitrite (ONOO ^- ), Characterized in that a change in fluorescence or light absorption characteristics caused by a change in a conjugated system is measured singly or in combination.

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