KR20140135858A - Novel 2,3-QA, 2,3-QP Compounds, Agent Selecting Zinc Ion Using The Same, Detecting Method And Detecting Device Thereof - Google Patents

Abstract

The present invention relates to a novel 2-((pyridine-2-ylmethyl)amino)-N-(quinolin-8-yl)acetamide or 3-((pyridine-2-ylmethyl)(pyridine-3-ylmetyl)amino)-N-(quinolin-8-yl)propanamide compound, a detecting agent of zinc ion (Zn2+) using the same, a detection method, and a detection device.

Description

[0001] The present invention relates to a novel 2,3-QA, 2,3-QP compound, a zinc ion detecting agent using the same, a detecting method, Detecting Method And Detecting Device Thereof}

The present invention relates to novel 2 - ((pyridin-2-ylmethyl) (pyridin-3- ylmethyl) amino) -N- (quinolin- detection method using (pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) detection agent, which is typically selective to specific propanamide compound, zinc ions (Zn ^{+ 2)} containing the compound this And a detection device.

When a molecule absorbs ultraviolet light or visible light, the electrons in the ground state are excited to a high energy state. Thereafter, the electrons in the excited state return to the stable bottom state and emit light at the same time. Fluorescence is a phenomenon in which a phosphor absorbs energy and then releases energy into light. A phosphor is a molecule that absorbs energy from the outside and emits light. Compounds having fluorescent properties can be applied to a variety of applications such as chemical sensors, electroluminescent display devices, and the like.

A range of zinc concentrations in vivo is nanomolar concentrations at micromolar concentrations, and small molecule sensors have been developed to detect zinc concentrations in vivo. While many types of zinc sensors can bind to zinc to detect zinc at low concentrations at the nanomolar level, the ability of the sensor to bind weakly to zinc is also important to detect small changes in zinc concentration.

For this purpose, research on zinc sensors has been continued. Zinc sensors can be divided into a chelating group that zinc can bind and an aromatic group that produces fluorescence when zinc is combined. Therefore, zinc sensors having chelating groups or aromatic groups in molecules have been actively studied, but effective sensors have not been developed so far.

Roger G. Harrison Inorganica Chimica Acta 394 (2013) 542-551 Xu, Z .; Baek, K.-H .; Kim, H. N .; Cui, J .; Qian, X .; Spring, D. R .; Shin, I .; Yoon, J. J. Am. Chem. Soc. 2010, 132, 601. Jianjun Du, Jiangli Fan, XiaojunPeng, Honglin Li, ShiguoSunSensors and Actuators B 144 (2010) 337-341 Mathieu Soibinet, Isabelle Dechamps-Olivier, Aminou Mohamadou, Michel Aplincourt InorganicaChemica Acta 394 (2013) 542-551

It is an object of the present invention to provide a zinc ion detecting agent, a detecting method and a detecting apparatus using the same to produce a compound which selectively recognizes only zinc ions (Zn ^{2 +} ) among various metal ions and exhibit fluorescence.

In order to achieve the above object,

2 - ((pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) acetamide of formula Methyl) (pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) propanamide compound.

[Chemical Formula 1]

(2)

The present invention also provides a process for preparing the compound of formula (1), wherein 2-chloro-N- (quinol-8-yl) acetamide and 2,3-dipycolylamine are reacted.

The present invention also provides a process for preparing the compound of formula (2), wherein 3-chloro-N- (quinol-8-yl) propanamide and 2,3-dipycolylamine are reacted.

The present invention also provides the zinc ions (Zn ^{+ 2)} detecting comprising a compound or compounds of formula 2 of the formula (1).

The present invention also provides a method for detecting zinc ion (Zn ^{2 +} ) comprising the compound of Formula 1 or the compound of Formula 2.

The present invention also provides a zinc ion (Zn ^{+ 2)} detecting apparatus including the zinc ions (Zn ²⁺⁾ detection agent.

It is an advantage that compounds of formulas 1 and 2 of the present invention is due to the difference in carbon numbers between the binding site (binding site) and the phosphor to detect the optionally zinc ions (Zn ^{2 +)} may be used as a fluorescent chemical sensor , And can be usefully used as a detecting agent and a detecting apparatus for detecting zinc ion (Zn ^{2 +} ) using the same. In addition, the compound of formula (I) can effectively detect zinc ions (Zn ^< ^{2 + &} gt ^; ) in vivo.

1 is an X-ray crystal structure of 2,3-QA-Zn (NO ₃ ) ₂ , which is a complex of 2,3-QA compound and zinc nitrate synthesized in Example 1.
FIG. 2 is a graph showing fluorescence intensity measured when various metals are added to the 2,3-QA compound. FIG.
FIG. 3 is a graph showing the intensity of fluorescence while gradually increasing the concentration of zinc ion (Zn ^{2 +} ) in a 2,3-QA compound.
4 is a graph showing fluorescence of 2,3-QA compound according to pH in the presence of zinc ion (Zn ^{2 +} ).
FIG. 5 is a graph showing fluorescence intensities of 2,3-QA compounds and zinc ions (Zn ^{2 +} ) when various metal ions are present.
FIG. 6 is a graph showing the absorbance of a 2,3-QA compound while gradually increasing the concentration of zinc ion (Zn ^{2 +} ).
FIG. 7 is a graph showing fluorescence intensities measured when various metals are added to a 2,3-QP compound. FIG.
FIG. 8 is a graph showing the intensity of fluorescence while gradually increasing the concentration of zinc ion (Zn ^{2 +} ) in a 2,3-QP compound.
9 is a graph showing fluorescence of 2,3-QP compound according to pH in the presence of zinc ion (Zn ^{2 +} ).
10 is a graph showing fluorescence intensities of 2,3-QP compounds and zinc ions (Zn ^{2 +} ) when various metal ions are present.
11 is a graph showing the absorbance of a 2,3-QP compound while gradually increasing the concentration of zinc ion (Zn ^< ^{2 + &} gt ^; ).
FIG. 12 is a spectrum showing ¹ H NMR changes of a 2,3-QA compound due to an increasing amount of Zn (NO ₃ ) ₂ in a CD ₃ OD solvent.
13 is a spectrum obtained by measuring ¹ H NMR changes of a 2,3-QP compound resulting from increasing the amount of Zn (NO ₃ ) ₂ under a CD ₃ OD solvent.
FIG. 14 is a graph showing fluorescence changes of Zn (NO ₃ ) ₂ with respect to the mole fraction of 2,3-QP compound in Job's plot.
15 is an image obtained by measuring the fluorescence ability of a 2,3-QA compound in a cell in the presence of zinc ion (Zn ^{2 +} ).

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

The invention 2 of the following general formula (1) has a selectivity for zinc ions (Zn ^{+ 2)} - ((pyridin-2-ylmethyl) (pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) acetamide Amide or 3 - ((pyridin-2-ylmethyl) (pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) propanamide compound of formula 2.

[Chemical Formula 1]

(2)

The compound of formula (1) is then reacted with a 2,3-QA compound of formula (1), a 2 - ( 3- ((pyridin-2-ylmethyl) (pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) propanamide compound is referred to as a 2,3-QP compound.

The 2,3-QA compound of formula (1) has one methylene group between 2,3-dipicolylamine (2,3-DPA) and quinoline as a phosphor (Non-patent document 3) -QP compound has two methylene groups between 2,3-dipicolylamine (2,3-DPA) and the quinoline phosphor. The 2,3-QA compound of Formula 1 can simultaneously detect zinc ion (Zn ^{2 +} ) and cadmium ion (Cd ^{2 +} ), and the 2,3-QP compound of Formula 2 can detect zinc ion (Zn ^{2 +} Can be detected.

The present invention provides a process for preparing 2,3-QA compounds of formula (1), wherein 2-chloro-N- (quinol-8-yl) acetamide and 2,3-dipycolylamine are reacted . 2-chloro-N- (quinol-8-yl) acetamide can be prepared by reacting 8-aminoquinoline with 2-chloroacetyl chloride, and 2,3-dipicolylamine ) Can be added and reacted to prepare the 2,3-QA compound of formula (1).

[Production method of 2,3-QA compound]

The present invention also relates to a process for the preparation of the 2,3-QP compound of Formula 2, wherein 3-chloro-N- (quinol-8-yl) propanamide and 2,3-dipicolylamine are reacted to provide. (Quinol-8-yl) propanamide can be prepared by reacting 8-aminoquinoline with 3-chloropropionyl chloride, and 2,3-dipicolylamine (2,3- DPA) may be added and reacted to prepare the 2,3-QP compound of formula (2).

[Production method of 2,3-QP compound]

The present invention relates to a zinc ion (Zn ^{2 +} ) detecting agent comprising the 2,3-QA compound of the formula (1) or the 2,3-QP compound of the formula ( ² ) May include a 2,3-QA compound or a 2,3-QP compound and a solvent, and the solvent is preferably methanol, but is not limited thereto.

The 2,3-QA compounds are various metal ions ^{(e.g., Mn 2 +, Fe 2 +} , Co 2 +, Ni 2 +, Cu 2+, Zn 2 +, Cd 2 +, Hg 2 +, Li +, Na ^{+, K +, Mg 2 +} , Ca 2 +, Ag +, may be combined with Cr ^{3 +),} in combination with other metal ions other than zinc ions but the cadmium ion (Cd ^{2 +)} and the weak fluorescence increase , And the fluorescence intensity is more than twice that of zinc ion (Zn ^{2 +} ). That is, the 2,3-QA compounds can be highly selective for the excellent zinc ion (Zn ^{2 +).} In addition, the 2,3-QA compound showed an increase in fluorescence intensity when zinc ion (Zn ^{2 +} ) was added in a cell experiment using fibroblast. That is, the 2,3-QA compound can detect zinc ions (Zn ^{2 +} ) in cells.

The compound 2,3-QP also various metal ions ^{(e.g., Mn 2 +, Fe 2 +} , Co 2 +, Ni 2 +, Cu 2+, Zn 2 +, Cd 2 +, Hg 2 +, Li +, Na ^+, K ^+, Mg ^{2 +,} Ca ^{2 +,} binding, but little increase in the fluorescence intensity, zinc ions and Ag ^+, Cr ^{3 +)} and may be bonded to the other metal ions other than zinc ions (Zn ^{2 +} ), The intensity of fluorescence increases gradually and shows strong fluorescence intensity. That is, the 2,3-QP compounds can be highly selective for the excellent zinc ion (Zn ^{2 +).}

The present invention provides a method for detecting zinc ions (Zn ^< ^{2 + &} gt ^; ) comprising the 2,3-QA compound of formula (1) or the 2,3-QP compound of formula ( ² ). More particularly, the present invention provides a method for detecting zinc ions (Zn ^< ^{2 + &} gt ^; ) using the 2,3-QA compound or 2,3-QP compound.

The present invention also provides a zinc ion (Zn ^{+ 2)} detecting apparatus including the zinc ions (Zn ²⁺⁾ detection agent. The detection agent may be a fluorescence chemosensor, and preferably a probe.

The zinc fluorescent probe of the detection device exhibits sensitivity mainly at a pH of 5 to 11, preferably at a pH of 6 to 10, in the case of a compound. If the pH is out of the above range, the fluorescence intensity of the 2,3-QA compound and the 2,3-QP compound is weakened. If the pH is less than 5, protonation of the hydrogen cation may occur.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are provided for illustrating the present invention, and the scope of the present invention is not limited to Examples and Experimental Examples.

Example  1. 2,3- QA  [2 - ((Pyridin-2- Yl methyl ) (Pyridin-3- Yl methyl ) Amino) -N- (quinolin-8-yl) Acetamide ] Preparation of compound

1.1 2- Chloro -N- ( Quinol -8-yl) Acetamide  Manufacturing method

2-chloroacetyl chloride (461 mg, 2.4 mmol) was dissolved in 5 mL of chloroform in an ice bath. To the solution was added 8-aminoquinoline (288 mg, 2.0 mmol) And pyridine (222 mg, 2.8 mmol) in 10 mL of chloroform was added slowly and stirred for 1 hour. The mixed solution was then warmed to room temperature and further stirred for two hours. The mixture was cooled at room temperature, the solvent was removed, and the resulting white oil was separated by column chromatography under dichloromethane eluent.

Yield: 353 mg (79.9%),

_{^{1 H-NMR (CDCl 3,}} 400MHz): δ = 10.92 (s, 1H), 8.82 (d, 1H), 8.76 (d, 1H), 8.20 (d, 1H), 7.59 (m, 2H), 7.50 ( m, 1 H), 4.32 (s, 2 H) ppm.

1.2 2,3- Of dipicolylamine (2,3-DPA)  Manufacturing method

Pyridine (0.52 mL, 5.1 mmol) and 2-pyridylcarboxaldehyde (0.48 mL, 5 mmol) were dissolved in 10 mL of methanol and stirred for 5 minutes. Sodium borohydride (0.21 g, 5.5 mmol) was slowly added to the reaction solution for 5 minutes, and the red mixed solution was stirred overnight. Then, about 10% hydrochloric acid (HCl) at a high concentration of 35% was slowly added. The solvent of the mixed solution was removed, concentrated, and dissolved in 50 mL of water. Then, in the process of extracting with chloroform using a separating funnel, impurities were removed with a saturated sodium carbonate solution. The product collected with chloroform was removed with sodium sulfate and the solvent was removed. The resulting oil phase product was separated by column chromatography under an eluent condition of 10: 1 by volume ratio of chloroform to methanol.

Yield: 0.52 g (52.2%).

^{1 H NMR (400 MHz, DMSO} -d 6, 25 ℃): δ = 8.53 (s, 1H), 8.48 (d, 1H), 8.43 (d, 1H), 7.74 (m, 2H), 7.45 (d, 1H), 7.33 (t, 1H), 7.23 (s, 1H), 3.78 (s, 2H), 3.73 (s, 2H), 2.86

1.3 2 - ((Pyridin-2- Yl methyl ) (Pyridin-3- Yl methyl ) Amino) -N- (quinolin-8-yl) Acetamide (2,3- QA )

(0.46 g, 2.1 mmol) of Example 1.1, 2,3-dipicolylamine (0.40 g, 2 mmol) of Example 1.2, N, N < RTI ID = -Diisopropylethylamine (0.39 mL, 2.2 mmol) and potassium iodide (KI) (0.37 g, 2.2 mmol) were dissolved in 30 mL of acetonitrile and stirred under reflux in a nitrogen atmosphere for 24 hours Respectively. The mixed solution was cooled at room temperature and extracted three times with dichloromethane. The solvent was then removed and the resulting oil was separated by column chromatography under the conditions of an eluent of 30: 1 by volume of chloroform to methanol.

Yield: 0.35 g (46.1%).

^{1 H NMR (400 MHz, DMSO} -d 6, 25 ℃): δ = 11.44 (s, 1H), 9.10 (d, 1H), 8.78 (s, 1H), 8.61 (d, 1H), 8.49 (d, 1H), 7.35 (t, 1H), 7.25 (t, IH), 8.44 (m, 2H) ), 3.89 (s, 2H), 3.84 (s, 2H), 3.46 (s, 2H) ppm.

^{13 C NMR (400 MHz, DMSO} -d 6, 25 ℃): δ = 168.9, 157.8, 150.4, 149.1, 149.0, 148.7, 137.8, 136.8, 136.7, 133.9, 133.3, 127.8, 127.1, 123.5, 123.2, 122.6, 122.4, 121.8, 115.3, 59.8, 55.9 ppm.

HRMS (ESI): [M + H < ⁺ & gt ^; ]; calcd, 384.18, found, 384.18.

_{Anal.Calcd for C 23 H 21 N 5O} (383.45): C 72.04, H 5.52, N 18.26;

Found: C 71.73, H 5.36, N 18.16.

Example  2. 2,3-QP [3 - ((Pyridin-2- Yl methyl ) (Pyridin-3- Yl methyl ) Amino) -N- (quinolin-8-yl) Propanamide ] Preparation of compound

2.1 3- Chloro -N- (quinolin-8-yl) propanamide

3-Chloropropionyl chloride (2.00 mL, 20.9 mmol) was dissolved in chloroform (10 mL) under ice bath. To the solution was added 8-aminoquinoline (0.740 g, 5.13 mmol) And pyridine (0.43 mL, 5.2 mmol) in 10 mL of chloroform was slowly added and stirred for 1 hour. The mixed solution was then warmed to room temperature and further stirred for two hours. After removal of the solvent, the resulting powdery product was washed with dichloromethane and diethyl ether.

Yield: 1.00 g (75.3%),

^{1 H-NMR (DMSO-d} 6, 400MHz): δ = 10.32 (NHCO, s, 1H), 8.94 (d, J = 4.0 Hz, 1H), 8.66 (d, J = 4.0 Hz, 1H), 8.41 ( 8.0 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.58 _{2 -, t, J = 8.0Hz} , 2H), 3.14 (-CH 2 -, t, J = 8.0 Hz, 2H).

_{^{13 C NMR (DMSO-d 6}} , 300MHz): δ = 170.9, 159.0, 150.7, 149.7, 140.8, 137.3, 123.5, 122.9, 109.8, 60.4, 58.8.

IR (KBr): 3334 (NH), 1688 (C = O), 1529,1484,1424,1386,1345,1324,1261,1236,1186,1163,951,828,796,756,745,696,588 cm ^-1 .

FAB MS m / z (M ⁺ ): calcd, 259.06, found, 259.27.

2.2 3 - ((Pyridin-2- Yl methyl ) (Pyridin-3- Yl methyl ) Amino) -N- (quinolin-8-yl) Propanamide ( 2,3-QP compound)  Manufacturing method

(0.49 g, 2.1 mmol) of Example 2.1, 2,3-dipicolylamine (0.40 g, 2 mmol) of Example 1.2, N, N < RTI ID = - diisopropyl ethyl amine was dissolved (0.39 mL, 2.2 mmol) and potassium iodide (KI) (0.37 g, 2.2 mmol) in acetonitrile 30 mL, under reflux in a nitrogen atmosphere and stirred for 24 hours. The mixed solution was cooled at room temperature and extracted three times with dichloromethane. The solvent was then removed and the resulting oil phase product was separated by column chromatography under the conditions of an eluent of 30: 1 by volume of chloroform to methanol.

Yield: 0.34 g (43.0%).

^{1 H-NMR (DMSO-d} 6, 400 MHz, 25 ℃): δ = 10.58 (s, 1H), 8.87 (d, 1H), 8.67 (d, 1H), 8.61 (s, 1H), 8.45 (d 2H), 3.78 (d, 4H), 2.86 (m, 4H), 7.80 (m, 2H).

^{13 C-NMR (400 MHz,} DMSO-d 6, 25 ℃): δ = 170.9, 158.8, 150.2, 148.7, 148.7, 148.2, 136.6, 136.6, 136.3, 134.9, 134.0, 127.9, 127.0, 123.2, 122.9, 122.1 , 121.7, 116.7, 58.8, 54.5, 50.0, 34.9 ppm.

HRMS (ESI): [M + H < ⁺ & gt ^; ]; calcd, 398.20, found, 398.19.

Anal.Calcd for C ₂₄ H ₂₃ N ₅ O (397.47): C 72.52, H 5.83, N 17.62;

Found: C 72.48, H 5.78, N 17.68.

Experimental Example  One. Example  1 2,3- QA  Compound and zinc ion ( Zn ^{2 +} ) Complex  Crystal structure analysis

(38.3 mg, 0.1 mmol) synthesized in Example 1 and zinc nitrate (Zn (NO ₃ ) ₂ ) (14.6 mg, 0.05 mmol) were dissolved in 1 mL of methanol and mixed with each other. The mixed solution was allowed to stand at room temperature for one day to produce crystals. The crystal structure of the complex was analyzed by X-ray analysis, and the results are shown in Fig. It can be seen that the 2,3-QA compound and the zinc ion bind to each other in a one-to-one manner. Two nitrogen atoms in the 2,3-DPA moiety and two nitrogen atoms in the 8-aminoquinoline moiety are combined with zinc ion (Zn ^{2 +} I could confirm that it was for boats.

Experimental Example  2. Example  1 2,3- QA  Fluorescence of compounds and Absorption analysis

Various metal ions, a 2,3-QA compound prepared in Example ^{1 (Zn 2 +, Cd 2} +, Na +, K +, Mg 2 +, Ca 2 +, Al 3 +, Pb 2 +, Hg 2 ^{+, Cr 3 +, Mn 2} +, Fe 3 +, Co 2 +, Ni 2 +, Cu 2 +) and the reaction was observed by fluorescence properties. The 2,3-QA compound was dissolved in methanol to make 10 μM. 3 μL of the solution was added to 3 mL of 10 mM Bis-tris buffer, and 200 μM of a solution containing various metal ions dissolved in methanol was added to the solution Afterwards, the fluorescence intensities were measured using a fluorescence instrument (λex = 350 nm). At this time, the metal ion solution was prepared using nitrate salt.

The results of fluorescence analysis for each metal ion are shown in FIG. As a result, ^{Na +, K +, Mg 2} +, Ca 2 +, Al 3 +, Pb 2 +, Hg 2 +, Cr 3 +, Mn 2 +, Fe 3 +, Co 2 +, Ni 2 +, Cu 2 ⁺ And cadmium ions (Cd ^{2 +} ) showed weak fluorescence increase. However, zinc ion (Zn ^{2 +} ) showed fluorescence intensity more than twice that of cadmium ion (Cd ^{2 +} ) (FIG. 2). Thus, it was found that the 2,3-QA compounds of the invention selectively detect zinc ions (Zn ^{+ 2).}

The fluorescence intensity was measured while gradually increasing the concentration of zinc ion (Zn ^{2 +} ) to 0 to 5 equivalents to 10 μM of the 2,3-QA compound prepared in Example 1. The intensity of fluorescence increased to 3 equivalents, and the intensity of fluorescence increased slightly from 3.5 equivalents, and the maximum value was obtained at 5 equivalents. The results are shown in FIG.

The results of the influence of pH on the fluorescence of the 2,3-QA compound in the presence of zinc ion (Zn ^{2 +} ) are shown in FIG. 10 μM of zinc ion (Zn ^{2 +} ) was added to 10 μM of the 2,3-QA compound, and the solutions of pH 2 to 11 were measured with a fluorescence instrument. Mainly exhibited sensitivity at pH 5 to 11, and excellent sensitivity at pH 6 to 11. When the pH range was out of the above range, the fluorescence intensity of the 2,3-QA compound was weak, and protonation occurred at a pH below the above range.

The effect of other metals on the fluorescence intensity of complexes of 2,3-QA compounds and zinc ions (Zn ^{2 +} ) is shown in FIG. 2,3-QA other metal ions in the compound ^{10 μM (Zn 2 +, K} +, Ca 2 +, Na +, Mg 2 +, Al 3 +, Cr 3 +, Mn 2 +, Fe 2 +, Co 2 + ^{, Ni 2 +, Cu 2 +} , Cd 2 +, Pd 2 +, Hg 2+) were dissolved respectively by 10μM, 20 μM, 50 μM, were then put into a zinc ion (Zn ^{+ 2)} 10μM measuring the fluorescence. ^{Zn 2 +, K +, Ca} 2 +, Na +, Mg 2 +, Al 3 +, Cr 3 +, Mn 2 +, Fe 2 +, Ni 2 +, Cd 2 +, Pd 2 + metal ions such as are The fluorescence intensities of 2,3-QA compounds and zinc ions (Zn ^{2 +} ) were not inhibited. However, the metal ions such as Co ^{2 +} , Cu ^{2 +} , and Hg ^{2 +} QA compound and zinc ion (Zn ^< ^{2 + &} gt ^; ) (Fig. 5).

The concentration of zinc ion (Zn ^{2 +} ) was adjusted to 2, 4, 6, 8, 10, and 20 μM in 20 μM of a 2,3-QA compound using a UV-vis instrument (Perkin-Elmer model Lambda 2S UV / vis spectrometer) Absorbance was measured while gradually increasing to 12, 14, 16, 18, 20, 24, 28, 32, 40, and 60 μM. As a result, the absorbance decreased at 310 nm and the absorbance increased at 360 nm. An isosbestic point was found at 338 nm and a 2,3-QA-Zn ^{2 +} complex was formed at this point (FIG. 6).

Experimental Example  3. Example 2  2,3- QP  Fluorescence of compounds and Absorption analysis

Various metal ions using a 2,3-QP compound prepared in Example ^{2 (Mn 2 +, Fe 2} +, Co 2 +, Ni 2 +, Cu 2 +, Zn 2 +, Cd 2 +, Hg 2 ^+, Na ^+, K ^+, Mg ^{+ 2,} Ca ^{+ 2,} Al ^{+ 3,} Cr ^{+ 3,} by Pb ²⁺⁾ and the reaction was observed fluorescence properties. The 2,3-QP compound was dissolved in methanol to make 10 μM. 3 μL of the solution was added to 3 mL of 10 mM Bis-tris buffer, and 200 μM of a solution containing various metal ions dissolved in methanol was added to the solution The fluorescence intensity was then measured using a fluorescence instrument (λex = 350 nm). At this time, the metal ion solution was prepared using nitrate salt.

The results of fluorescence analysis for each metal ion are shown in Fig. As a result, ^{Mn 2+, Fe 2 +, Co} 2 +, Ni 2 +, Cu 2 +, Cd 2 +, Hg 2 +, Na +, K +, Mg 2 +, Ca 2 +, Al 3 +, Cr 3 ⁺ , And Pb ^{2 +} showed almost no increase in fluorescence intensity, whereas zinc ion (Zn ^{2 +} ) showed strong fluorescence intensity (FIG. 7). Therefore, it was found that the 2,3-QP compound of the present invention reacts very selectively with respect to zinc ions (Zn ^{2 +} ).

The fluorescence intensity was measured while gradually increasing the concentration of zinc ion (Zn ^{2 +} ) to 0 to 5 equivalents to 10 μM of the 2,3-QP compound prepared in Example 2. The intensity of fluorescence was continuously increased and showed a maximum value at 5 equivalents. The results are shown in FIG.

The results of the influence of pH on the fluorescence of 2,3-QP compound in the presence of zinc ion (Zn ^{2 +} ) are shown in FIG. 10 μM of zinc ion (Zn ^{2 +} ) was added to 10 μM of 2,3-QP compound, and each solution was measured with a fluorescence instrument from pH 2 to 11. Mainly exhibited sensitivity at pH 5 to 11, and excellent sensitivity at pH 6 to 10. When the pH range is out of the above range, the fluorescence intensity of the 2,3-QP compound is weak, and protonation may occur at a pH lower than the above range.

In addition, the influence of other metals on the fluorescence intensity of the 2,3-QP compound and the zinc ion (Zn ^{2 +} ) complex is shown in FIG. The compound 2,3-QP other metal ions to ^{5 μM (Zn 2 +, K} +, Ca 2 +, Na +, Mg 2 +, Al 3 +, Cr 3 +, Mn 2 +, Fe 2 +, Co 2 ^{+, Ni 2 +, Cu 2} +, Cd 2 +, Pb 2 +, Hg 2 +) of 50 μM, 100 μM, into each by 250 μM, and then insert the zinc ion (Zn ^{2 +)} 50 μM fluorescent intensity Were measured. ^{K +, Ca 2 +, Na} +, Mg 2 + metal ions such as they did not interfere with the fluorescence intensity of the 2,3-QP compound and zinc ions ^{(Zn 2 +), Cr 3} +, Mn 2 +, Fe 2 ^{+, Co 2 +, Ni 2} +, Cu 2 +, Cd 2 +, Hg 2+, Pb 2 +, Al 3 + , such as the metal ions, the amount of metal to be much more 2,3-QP compound and zinc ions (Zn ^< ^{2 + &} gt ^; ) (Fig. 10).

In addition, UV-vis instrument (Perkin-Elmer model Lambda 2S UV / vis spectrometer) to the compound 2,3-QP to 20 μM zinc ions (Zn ^{+ 2)} concentrations for 2, 4, 6, 8, 10 using, Absorbance was measured while gradually increasing to 12, 14, 16, 18, 20, 24, 28, 32, 40, and 60 μM. As a result, the absorbance decreased at 310 nm and the absorbance increased at 360 nm. An isosbestic point was found at 324 nm and a 2,3-QP-Zn ^{2 +} complex was formed at this point (FIG. 11).

Experimental Example  4. Example  1 2,3- QA Compound and zinc  ion( Zn ^{2 +} )

¹ H NMR titration experiments were performed on CD ₃ OD to determine the binding state of 2,3-QA compound and zinc ion (Zn ^{2 +} ). 0.2, 0.4, 0.6, 0.8, 1.0 equivalent of zinc ion (Zn ^{2 +} ) was added to the 2,3-QA compound and ¹ H NMR was measured. When zinc ions (Zn ^{2 +} ) are added, the methylene hydrogen cations of 2,3-DPA become diastereotopic and split into doublet of doublets, (Fig. 12). In addition, the methylene hydrogen cations between amines and amide carbons of 2,3-DPA were also observed. It was also found that the hydrogen cation in the directional ring structure existing in most of the upfield moves to the down field. All these changes are complete when 1.0 equivalent of zinc ion (Zn ^{2 +} ) is added, indicating that the 2,3-QA compound and the zinc ion (Zn ^{2 +} ) are bound in a one-to-one ratio. In addition, the crystal structure of FIG. 1 also demonstrates that the 2,3-QA compound and the zinc ion (Zn ^{2 +} ) bind at a ratio of 1: 1.

Experimental Example 5 . Example  2, 2,3- QP Compound and zinc  ion( Zn ^{2 +} )

In order to investigate the binding state of 2,3-QP compound and zinc ion (Zn ^{2 +} ), ¹ H NMR titration experiments were performed on CD ₃ OD. 0.2, 0.4, 0.6, 0.8, 1.0 equivalent of zinc ion (Zn ^{2 +} ) was added to the 2,3-QP compound and ¹ H NMR was measured. The 2,3-QP compound showed a broad NMR signal when 1.0 equivalent of zinc ion (Zn ^{2 +} ) was added. Thus, binding analysis could not be defined with NMR (Fig. 13). Of 2,3-QP compound and zinc ions (Zn ^{+ 2)} Job's plot (Fig. 14) the result of the experiment, 2,3-QP compound and zinc ions (Zn ^{+ 2)} to determine the binding of a 1: 1 .

Experimental Example  6. Example  1 2,3- QA  Compound and zinc ion ( Zn ^{2 +} ) In the cell

Intracellular experiments were conducted to examine the fluorescence ability of 2,3-QA compounds in the presence of zinc ion (Zn ^{2 +} ), and the results are shown in FIG. Each 0 (A and E) human skin fibroblasts (dermal fibroblast), 1 (B and F), 10 (C and G), and 100 (D and H) μM of zinc ions (Zn ^{2 +)} Yes after exposure for a time, to remove the phosphate-buffered saline (phosphate buffered saline, PBS) remaining zinc ion in solution (Zn ^{2 +).} Then, after exposure to 10 μM of the 2,3-QA compound for 30 minutes, the 2,3-QA compound remaining in the phosphate buffered saline solution was removed and fluorescence images were observed (FIG. 15).

Human skin fibroblasts are zinc ions (Zn ^{+ 2)} is not present, there was no fluorescence when there is no 2,3-QA compound. Intracellular fluorescence intensity increased with increasing concentration of zinc ion (Zn ^{2 +} ). Thus, a 2,3-QA compound prepared in example 1 can detect zinc ions (Zn ^{+ 2)} in the cells by fluorescence.

Claims (7)

2 - ((pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) acetamide of formula Methyl) (pyridin-3-ylmethyl) amino) -N- (quinolin-8-yl) propanamide.
[Chemical Formula 1]

(2)

2-chloro-N- (quinol-8-yl) acetamide and 2,3-dipycolylamine. 3-chloro-N- (quinol-8-yl) propanamide and 2,3-dipycolylamine. A zinc ion (Zn ^< ^{2 + &} gt ^; ) detecting agent comprising a compound of the formula (1) or a compound of the formula ( ² ). A method for detecting zinc ion (Zn ^< ^{2 + &} gt ^; ) using the compound of formula (1) or the compound of formula ( ² ). Zinc ions (Zn ²⁺⁾ zinc ion containing the detection agent of claim 4 (Zn ^{+ 2)} detecting device. According to claim 6, wherein said detection device is a probe (probe), and zinc ions, characterized in that of sensitivity at pH 5 to 11 (Zn ^{+ 2)} detecting device.

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