KR20120130906A - Coumarine derivative for detecting thiol compounds - Google Patents

Abstract

PURPOSE: A novel coumarin derivative for detecting compounds with thiol groups is provided to be used as a thiol-detecting probe. CONSTITUTION: A coumarin derivative for detecting thiol compounds is denoted by chemical formula 1. The compound of chemical formula 1 is (E)-7-(diethyl amino)-3-(3-oxobut-1-enyl)-2H-chromene-2-one, 3-((7-(diethyl amino)-2-oxo-2H-chromene-3-yl)methylene)pentane-2,4-dion, (E)-3-(7-(diethyl amino)-2-oxo-2H-chromene-3-yl)acrylaldehyde, (E)-7-(diethyl amino)-3-(3-(2-hydroxyphenyl)-3-oxoprop-1-enyl)-2H-chromene-2-one, (E)-7-(diethylamino)-3-(3-oxo-3-phenyl-1-prop-1-enyl)-2H-chromene-2-one, (E)-7-(diethyl amino)-3-(3-(2-hydroxyphenyl)cryloyl)-2H-chromene-2-one, 3-cinnamoyl-7-(diethyl amino)-2H-chromene-2-one, and (E)-7-(diethyl amino)-3-(4-(3-(2-hydroxyphenyl)-3-oxoprop-1-enyl)phenyl)-2H-chromene-2-one.

Description

Coumarin derivatives for detecting thiol compounds

The present invention relates to novel compounds useful for thiol compound probes and, more particularly, to novel coumarin derivatives useful for the detection of compounds having thiol groups ex vivo and / or in vivo. will be.

There are a variety of substances that contain thiol groups in vivo. Representative materials containing a thiol group include cysteine, homocysteine, gamma-glutamylcysteinylglycine (GSH), and the structures thereof are as follows.

Compounds containing the thiol group play a myriad of roles in the activity of cells involved in life support. For example, redox homeostasis (Redox Homeostasis; TP Dalton, HG Shertzer and A. Puga, Annu . Rev. Pharmacol . Toxicol ., 1999, 39 , 67), cellular growth (ZA Wood, E. Schroeder, JR Harris and LB Poole, TransBiochem . Sci ., 2003, 28 , 32), and they are also known to be involved in cancer and AIDS (DM Townsend, KD Tew and H. Tapiero, Biomed . Pharmacother ., 2003, 57 , 145).

Therefore, if such a thiol group can be selectively detected, it is expected that it can contribute a lot to discover the activity of cells at the molecular level.

The inventors have designed a novel compound that can effectively detect thiol group-containing biomaterial compounds not only in vivo but also in vivo, specifically in cells. In particular, considering the nucleophilic nature of the thiol, Michael acceptor groups were introduced into coumarin, and new derivatives were designed so that the thiol acts as a Michael donor to selectively react. .

Accordingly, it is an object of the present invention to provide novel compounds capable of detecting thiol group-containing materials by selectively reacting with thiol group-containing materials, in particular thiol group-containing bio-derived materials.

According to one aspect of the invention, there is provided a compound of formula 1 useful as a probe for the detection of a thiol group-containing material:

≪ Formula 1 >

은 상기 화학식 1의 화합물에 결합되는 결합(bond)를 나타낸다).Wherein Y is a group of formula (2), a group of formula (3), or a group of formula (4) (in groups of formulas 2-4, R ₁ is hydrogen or acetyl; R ₂ is hydrogen, C ₁ to C ₄ Is alkyl, or phenyl optionally substituted with a hydroxy group of R ₃ is hydrogen or hydroxy:

Represents a bond to the compound of Formula 1).

<Formula 2>

<Formula 3>

&Lt; Formula 4 >

The compound of formula 1 according to the present invention is useful as a thiol-detecting probe by selectively reacting coumarin derivatives with a compound having a thiol in vitro or in vivo. Can be used.

1 is a result of analyzing the ex vivo binding characteristics of the compound according to the present invention and the thiol-containing compound (2-mercaptoethanol) by 200MHz NMR. In Figure 1 A is a 200 MHz NMR chart of the compound according to the invention, B is a 200 MHz NMR chart of the product obtained after the addition of 2-mercaptoethanol to the compound, respectively.
Figure 2 shows the results of analyzing the selective binding properties of the compound according to the invention and the thiol-containing amino acid (homocysteine) by the relative fluorescence response (Ratiometric fluorescence responses) through the fluorescence spectrum.
Figure 3 shows the results of analyzing the selective binding properties of the compound according to the invention and the thiol-containing material (glutathione) in the cell via confocal laser scanning microscopy imaging.

The present invention introduces a Michael acceptor group to coumarin, specifically, an α, β-unsaturated carbonyl group, and the thiol acts as a Michael donor to selectively react. As a novel coumarin derivative designed to allow this to occur, a novel coumarin derivative capable of selectively reacting with a compound having a thiol group in vitro as well as in vivo is provided.

That is, the present invention provides a compound of formula 1 useful as a probe for the detection of a thiol group-containing substance:

&Lt; Formula 1 >

은 상기 화학식 1의 화합물에 결합되는 결합(bond)를 나타낸다).Wherein Y is a group of formula (2), a group of formula (3), or a group of formula (4) (in groups of formulas 2-4, R ₁ is hydrogen or acetyl; R ₂ is hydrogen, C ₁ to C ₄ Is alkyl, or phenyl optionally substituted with a hydroxy group of R ₃ is hydrogen or hydroxy:

Represents a bond to the compound of Formula 1).

<Formula 2>

<Formula 3>

&Lt; Formula 4 >

More preferred compounds of Formula 1 are as follows:

(E) -7- (diethylamino) -3- (3-oxobut-1-enyl) -2H-chromen-2-one;

3-((7- (diethylamino) -2-oxo-2H-chromen-3-yl) methylene) pentane-2,4-dione;

(E) -3- (7- (diethylamino) -2-oxo-2H-chromen-3-yl) acrylaldehyde;

(E) -7- (diethylamino) -3- (3- (2-hydroxyphenyl) -3-oxoprop-1-enyl) -2H-chromen-2-one;

(E) -7- (diethylamino) -3- (3-oxo-3-phenyl-1-prop-1-enyl) -2H-chromen-2-one;

(E) -7- (diethylamino) -3- (3- (2-hydroxyphenyl) acryloyl) -2H-chromen-2-one;

3-cinnamoyl-7- (diethylamino) -2H-chromen-2-one;

(E) -7- (diethylamino) -3- (4- (3- (2-hydroxyphenyl) -3-oxoprop-1-enyl) phenyl) -2H-chromen-2-one.

Among these compounds, particularly preferred compounds are (E) -7- (diethylamino) -3- (3- (2-hydroxyphenyl) -3-oxoprop-1-enyl) -2H-chromen-2 It may be on.

The compound of formula 1 according to the present invention may exist in the form of racemics or stereoisomers (eg, diastereomeric forms), and the present invention includes both the racemates and stereoisomers.

In the compound of Formula 1, the compound of Formula 1a, wherein Y is a group of Formula 2, may be an aldol reaction using 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde or It can be prepared using the Wittig reaction.

<Formula 1a>

In the compound of Formula 1a, R ₁ and R ₂ are as defined above.

The starting material 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde is prepared by a known method (e.g., Repub.Korean Kongkae Taeho Kongbo, 2010112792, 20 Oct 2010). Can be prepared.

The aldol reaction is a reactant such as 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde and acetone, acetylacetone, 2-hydroxy-acetophenone, or acetophenone as starting materials. Can be carried out by reacting in the presence of a catalyst. As the catalyst, pyrrolidine or piperidine can be used, and the amount of the catalyst is used per mole of starting material 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde. It may be used in the range of 0.005 mL to 0.01 mL, and typically about 0.01 mL may be used. In addition, an alcohol such as ethanol, dichloromethane, or a mixed solvent of alcohol and dichloromethane may be used as the solvent, and may be performed under reflux conditions for about 6 to 12 hours.

The Witig reaction uses 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde, triphenylphosphoranylidine acetaldehyde and triethylamine as starting materials. Can be performed. Triphenylphosphoranilidine acetaldehyde may use about 1.2 molar equivalents relative to the starting material and triethylamine may use about 1 molar equivalents.

In the compound of Formula 1, Y is a group of Formula 3, wherein the compound of Formula 1b is prepared by using an aldol reaction using 3-acetyl-7- (diethylamino) -2H-chromen-2-one It can manufacture.

<Formula 1b>

In the compound of Formula 1b, R ₃ is as defined above.

The starting material 3-acetyl-7- (diethylamino) -2H-chromen-2-one can be prepared by known methods (e.g. J. Am . Chem . Soc . 2004 , 126, 2282-2283). Can be prepared by The aldol reaction is carried out by reacting a reactant such as 2-hydroxy benzaldehyde, benzaldehyde, etc. in the presence of a catalyst, using 3-acetyl-7- (diethylamino) -2H-chromen-2-one as a starting material. Can be. Piperidine can be preferably used as the catalyst. The amount of catalyst used may be in the range of 0.005 mL to 0.01 mL with respect to 1 mol of the starting material 3-acetyl-7- (diethylamino) -2H-chromen-2-one, typically about 0.01 mL Can be used. In addition, an alcohol such as ethanol may be preferably used as the solvent, and may be performed under reflux conditions for about 20 hours.

In the compound of Formula 1, Y is a group represented by Formula 4, and the compound of Formula 1c is a Suzuki coupling reaction using 3-bromo-7- (diethylamino) -2H-chromen-2-one. ), Followed by an aldol reaction.

<Formula 1c>

In the compound of Formula 1c, R ₃ is as defined above.

The starting material 3-bromo-7- (diethylamino) -2H-chromen-2-one is known in the art (e.g. J. Am . Chem . Soc . 2004 , 126, 2282-2283) It can be prepared by. The Suzuki coupling reaction was carried out using 3-bromo-7- (diethylamino) -2H-chromen-2-one as a starting material, 4-formyl phenyl boronic acid, and the like. It can be carried out by reacting the reactant with in the presence of a catalyst. As the catalyst, a palladium-based catalyst such as dichlorobis (triphenylphosphine) palladium (II) can be preferably used. The catalyst may be used in an amount of about 10 mol% based on 1 mol of the starting material 3-bromo-7- (diethylamino) -2H-chromen-2-one. As a solvent, an aprotic solvent such as tetrahydrofuran may be preferably used, and may be performed at about 60 ° C. for about 12 hours. The following aldol reaction can be carried out using a 2-hydroxy acetophenone and a pyrrolidine catalyst. The amount of the catalyst may be used in the range of 0.005 mL to 0.01 mL per mole of Suzuki coupling reaction product, and typically about 0.01 mL may be used. In addition, an alcohol such as ethanol may be preferably used as the solvent, and may be performed at room temperature for about 12 hours.

Hereinafter, the present invention will be described in more detail through Examples and Test Examples. However, the following examples and test examples are intended to illustrate the invention, not to limit the invention.

Example  1. (E) -7- ( Diethylamino ) -3- (3- Oxo Boot -1-enyl) -2H- Kromen -2-one

0.15 g of 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde was added to a round bottom flask, and 1.8 ml of ethanol was added to dissolve it. 0.07 mL of acetone and 0.01 mL of piperidine were added to the solution and refluxed for 6 hours. When the color of the solution turned red, it was cooled to room temperature and concentrated under reduced pressure when the starting material disappeared by TLC. The resulting concentrate was purified by column chromatography (eluent: dichloromethane: ethyl acetate: n-hexane = 1: 2: 7, v / v) to give the title compound.

Yield 10%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.77 (s, 1H, -C CH C (CO ₂ )-), 7.46 (d, 1H, J = 16 Hz, -C CH CH-), 7.31 (d, 1H, J = 9.2Hz, -C (NEt ₂ ) CH CH C-), 7.13 (d, 1H, J = 16Hz, -CCH CH- ), 6.61 (dd, 1H, J = 2.4, 8.8Hz, -C (NEt ₂ ) CH CHC-), 6.48 (d, 1H, J = 2.4 Hz, -C (NEt ₂ ) CH C-), 3.44 (q, 4H, J = 7.2 Hz, -N CH ₂ CH ₃ ), 2.35 (s, 3H, -CO CH ₃ ), 1.23 (t, 6H, J = 7.2 Hz, -NCH ₂ CH ₃ ).

¹³ C NMR (100 MHz, CDCl ₃ ): δ 188.7, 160.5, 156.6, 151.7, 144.1, 144.1, 137.8, 129.9, 127.0, 114.3, 109.5, 108.7, 96.9, 45.0, 28.3, 12.4.

HRMS (FAB ⁺ , m-NBA): m / z obs'd 286.1440 ([M + H] &lt; + &gt;,cal'd 286.1443 for C ₁₇ H ₂₀ NO ₃ ).

Example  2. 3-((7- ( Diethylamino ) -2-oxo-2H- Kromen -3-yl) methylene) pentane-2,4- Dion's  Produce

0.15 g of 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde was added to a round bottom flask, and 1.8 ml of ethanol was added to dissolve it. 0.09 mL of acetylacetone and 0.01 mL of piperidine were added to the solution and refluxed for 12 hours. When the color of the solution turned red, it was cooled to room temperature and concentrated under reduced pressure when the starting material disappeared by TLC. The resulting concentrate was purified by column chromatography (eluent: dichloromethane: ethyl acetate: n-hexane = 1: 2: 7, v / v) to give the title compound.

Yield 48%.

¹ H NMR (200 MHz, CDCl ₃ ): δ 7.78 (s, 1H, -C CH C (CO ₂ )-), 7.59 (s, 1H, -C CH C (COCH ₃ ) _2- ), 7.28 (d , 1H, J = 9 Hz, -C (NEt ₂ ) CH CH C-), 6.60 (dd, 1H, J = 2.6, 9 Hz, -C (NEt ₂ ) CH CHC-), 6.46 (d, 1H, J = 2.6 Hz, -C (NEt ₂ ) CH C-), 3.45 (q, 4H, J = 7 Hz, -N CH ₂ CH ₃ ), 2.44 (s, 3H, -CO CH ₃ ), 2.36 (s, 3H, -CO CH ₃ ), 1.23 (t, 6H, J = 7.2 Hz, -NCH ₂ CH ₃ ).

¹³ C NMR (50 MHz, CDCl ₃ ): δ 205.5, 197.2, 161.1, 157.0, 152.1, 143.8, 141.7, 133.9, 130.6, 112.5, 109.6, 108.4, 96.9, 45.0, 31.4, 26.1, 12.4.

HRMS (FAB ⁺ , m-NBA): m / z obs'd 328.1544 ([M + H] &lt; + &gt;,cal'd 286.1443 for C ₁₉ H ₂₂ NO ₄ ).

Example  3. (E) -3- (7- ( Diethylamino ) -2-oxo-2H- Kromen -3 days) Acrylic aldehyde Manufacture of de

0.2 g of 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde was added to a round bottom flask, and 10 ml of acetonitrile was added and dissolved. 0.3225 g of triphenylphosphoranylidine acetaldehyde and 0.12 mL of triethylamine were added to the solution and refluxed for 6 hours. When the color of the solution turned red, it was cooled to room temperature and concentrated under reduced pressure when the starting material disappeared by TLC. The obtained concentrate was recrystallized by adding 2 mL of ethanol, and filtered to prepare the title compound.

Yield: 80%.

¹ H NMR (200 MHz, CDCl ₃ ): δ 9.58 (d, 1H, J = 7.8 Hz, -CO H ), 8.34 (s, 1H, -C CH C (CO ₂ )-), 7.55 (d, 1H , J = 15.6 Hz, -C CH CH-), 7.49 (d, 1H, J = 8.6 Hz, -C (NEt ₂ ) CH CH C-), 6.93 (dd, 1H, J = 7.8, 15.6 Hz,- CCH CH COH-), 6.78 (dd, 1H, J = 2.4, 8.6 Hz, -C (NEt ₂ ) CH CHC-), 6.58 (d, 1H, J = 2.4 Hz, -C (NEt ₂ ) CH C- ), 3.48 (q, 4H, J = 7.2 Hz, -N CH ₂ CH ₃ ), 1.13 (t, 6H, J = 7.2 Hz, -NCH ₂ CH ₃ ).

Example  4. (E) -7- ( Diethylamino ) -3- (3- (2- Hydroxyphenyl ) -3- Oxoprop -1-enyl) -2H- Kromen -2-one

0.123 g of 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde was added to a round bottom flask, and 1.8 ml of ethanol was added to dissolve it. 0.11 mL of 2-hydroxy-acetophenone and 0.01 mL of pyrrolidine were added to the solution, followed by stirring for 12 hours. When the color of the solution passed through the red color to produce a solid, the solid was filtered using 5 times 1mL of ethanol, and dried to prepare the title compound.

Yield: 60%.

¹ H NMR (DMSO-d ₆ , 200 MHz): δ 12.63 (s, 1H), 8.51 (s, 1H), 8.12 (d, 3J = 15.0 Hz, 1H), 8.01 (m, 1H), 7.76 (d , 3J = 15.0Hz, 1H), 7.52 (m, 2H), 7.01 (m, 2H), 6.80 (dd, 3J = 9.0Hz, 4J = 2.4Hz, 1H), 6.60 (d, 4J = 9.0Hz, 1H ), 3.59 (q, 3J = 7.0 Hz, 4H), 1.15 (t, 3J = 7.0 Hz, 6H).

¹³ C NMR (CDCl ₃ , 75 MHz): δ 194.44, 163.50, 160.19, 156.70, 152.15, 147.10, 140.74, 136.14, 130.25, 130.21, 121.29, 120.29, 118.85, 118.27, 114.56, 109.67, 108.94, 96.87, 45. .

HRMS (FAB ⁺ , m-NBA): m / z obs'd 364.1552 ([M + H] &lt; + &gt;,calc'd 364.1549 for C ₂₂ H ₂₂ NO ₄ ).

Example  5. (E) -7- ( Diethylamino ) -3- (3-oxo-3- Phenyl -One- Prof -1-enyl) -2H- Big Preparation of Romen-2-one

To the round bottom flask, add 0.0246 g of 7- (diethylamino) -2-oxo-2H-chromen-3-carbaldehyde, add 1 ml of a mixed solvent of ethanol and dichloromethane (4: 1, v / v) Dissolved. 0.022 g of acetophenone and 0.01 mL of pyrrolidine were added to the solution, followed by stirring for 12 hours. When the color of the solution passed through the red color to produce a solid, the solid was filtered using 5 times 1mL of ethanol, and dried to prepare the title compound.

Yield: 70%.

¹ H NMR (CDCl ₃ , 300 MHz): δ 8.27 (d, 3J = 15.0 Hz, 1H), 8.12 (d, 3J = 7.1 Hz, 2H), 7.80 (s, 1H), 7.68 (d, 3J = 15.0 Hz , 1H), 7.58-7.48 (m, 3H), 7.36 (d, 3J = 9.0Hz, 1H), 6.65 (dd, 3J = 9.0Hz, 4J = 2.4Hz, 1H), 6.52 (d, 4J = 2.4Hz , 1H), 3.50 (q, 3J = 7.0 Hz, 4H), 1.28 (t, 3J = 7.0 Hz, 6H).

¹³ C NMR (CDCl ₃ , 75 MHz): δ 190.8, 160.3, 156.6, 151.9, 146.3, 139.9, 138.4, 132. 7, 130.0, 128.7, 128.5, 123.0, 115.0, 109.5, 108.9, 96.9, 45.1, 12.5 (18 carbon peaks).

HRMS (FAB ⁺ , m-NBA): m / z obs'd 348.1604 ([M + H] ⁺ , calc'd 348.1600 for C ₂₂ H ₂₂ NO ₃ ).

Example  6. (E) -7- ( Diethylamino ) -3- (3- (2- Hydroxyphenyl ) Acryloyl) -2H- Kromen -2-one

0.2 g of 3-acetyl-7- (diethylamino) -2H-chromen-2-one was added to a round bottom flask, and 15 ml of ethanol was added to dissolve it. 0.28 mL of 2-hydroxy benzaldehyde and 0.03 mL of piperidine were added to the solution and refluxed for 20 hours. When the color of the solution turned red, it was cooled to room temperature and concentrated under reduced pressure when the starting material disappeared by TLC. The resulting concentrate was purified by column chromatography (eluent: dichloromethane: ethyl acetate: n-hexane = 1: 2: 7, v / v) to give the title compound.

Yield 40%.

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.21 (s, 1H, OH), 8.56 (s, 1H, -CCHC (CO ₂ )-), 7.99 (d, 1H, J = 16 Hz, -COCHCH 7.94 (d, 1H, J = 16 Hz, -COCHCH-), 7.67 (d, 1H, J = 8.8 Hz, -C (NEt ₂ ) CHCHC-), 7.61 (d, 1H, J = 6.8 Hz) , 7.25 (dt, 1H, J = 1.6, 8.4 Hz), 6.92 (d, 1H, J = 8 Hz), 6.86 (t, 1H, J = 7.6 Hz), 6.78 (dd, 1H, J = 2.4, 8.8 Hz , -C (NEt ₂ ) CHCHC-), 6.58 (d, 1H, J = 2.4 Hz, -C (NEt ₂ ) CHC-), 3.48 (q, 4H, J = 7.2 Hz, -NCH ₂ CH ₃ ), 1.14 (t, 6H, J = 7.2 Hz, -NCH ₂ CH ₃ ).

¹³ C NMR (75 MHz, DMSO-d ₆ ): δ 186.2, 160.3, 158.6, 157.6, 153.3, 148.7, 138.0, 132.2, 128.8, 124.6, 122.0, 119.9, 116.6, 116.1, 110.5, 108.3, 96.3, 44.8, 12.8.

HRMS (FAB +, m-NBA): m / z obs'd 364.1544 ([M + H] &lt; + &gt;,cal'd 364.1549 for C ₂₂ H ₂₂ NO ₄ ).

Example  7. 3- Cinnamo -7- ( Diethylamino ) -2H- Kromen -2-one

0.2 g of 3-acetyl-7- (diethylamino) -2H-chromen-2-one was added to a round bottom flask, and 15 ml of ethanol was added to dissolve it. 0.25 mL of benzaldehyde and 0.01 mL of piperidine were added to the solution and refluxed for 20 hours. When the color of the solution turned red, it was cooled to room temperature and concentrated under reduced pressure when the starting material disappeared by TLC. The resulting concentrate was purified by column chromatography (eluent: dichloromethane: ethyl acetate: n-hexane = 1: 2: 7, v / v) to give the title compound.

Yield: 30%.

¹ H NMR (200 MHz, DMSO-d ₆ ): δ 8.61 (s, 1H), 7.98 (d, 1H, J = 16 Hz), 7.72 (m, 4H), 7.45 (m, 3H), 6.82 (d, 1H, J = 9.0Hz), 6.62 (s, 1H), 3.48 (q, 4H, J = 6.8Hz, -N CH ₂ CH ₃ ), 1.15 (t, 6H, J = 6.8Hz, -NCH ₂ CH ₃ ).

Example  8. (E) -7- ( Diethylamino ) -3- (4- (3- (2- Hydroxyphenyl ) -3- Oxoprop -1-enil) Phenyl ) -2H- Kromen -2-one

(1) 4- (7- ( Diethylamino ) -2-oxo-2H- Kromen -3 days) Of benzaldehyde  Produce

0.296 g of 3-bromo-7- (diethylamino) -2H-chromen-2-one was put into a round bottom flask, and 8 ml of tetrahydrofuran (THF) was added and dissolved. 300 mg of 4-formyl phenyl boronic acid and 36 mg of dichlorobis (triphenylphosphine) palladium (II) were added to the solution, followed by slowly dropwise adding an aqueous potassium carbonate solution (2M, 8 mL). The reaction mixture was stirred at 60 ° C. for 12 h. Methanol was added to the reaction mixture for crystallization, followed by filtration and drying to give the title compound.

Yield: 63.5%.

¹ H NMR (200 MHz, CDCl ₃ ): δ 10.06 (s, 1H), 7.94 (s, 4H), 7.84 (s, 1H), 7.40 (d, J = 8.8 Hz, 1H), 6.69 (dd, J = 2.6 Hz 1H), 6.57 (d, J = 2.6 Hz, 1H), 3.53 (q, J = 7 Hz, 4H), 1.26 (t, J = 7 Hz, 6H).

(2) (E) -7- ( Diethylamino ) -3- (4- (3- (2- Hydroxyphenyl ) -3- Oxoprop -1-enil) Phenyl ) -2H- Kromen -2-one

0.128 g of the compound obtained in the above (1) was added to a round bottom flask, and 5 ml of ethanol was added to dissolve it. 0.096 ml of 2-hydroxyacetophenone and 0.01 ml of pyrrolidine were added to the solution, followed by stirring at room temperature for 12 hours. 5 mL of 1 mL of ethanol was added to the reaction mixture for crystallization, followed by filtration and drying to prepare the title compound.

Yield 72%.

¹ H NMR (300 MHz, CDCl ₃ ): δ 12.90 (s, 1H), 7.95 (m, 2H), 7.81 (d, J = 8.25 Hz, 2H), 7.79 (s, 1H), 7.70 (d, J = 8.25 Hz, 2H), 7.67 (d, J = 15.0 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 8.82 Hz, 1H), 7.03 (d, J = 8.34 Hz, 1H), 6.98 (t, J = 7.2 Hz, 1H), 6.63 (m, 1H), 6.53 (m, 1H), 3.45 (q, J = 7.2 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H).

¹³ C NMR (CDCl ₃ , 75 MHz): δ 193.7, 163.6, 161.4, 156.4, 150.9, 145.1, 141.0, 138.6, 136.4, 133.7, 129.7, 129.3, 128.7, 128.6, 120.1, 119.8, 119.3, 118.9, 118.6, 24 carbon peaks (109.1, 109.0, 97.0, 44.9, 12.5).

HRMS (FAB +, Glycerol): m / z obs'd 440.1861 ([M + H] &lt; + &gt;,cal'd 440.1862 for C ₂₈ H ₂₆ NO ₄ ).

Test Example  One. In vitro ( ex vivo ) Thiol  Binding Properties with Compounds

3.63 mg of the compound obtained in Example 4 was dissolved in DMSO-d ₆ 0.5 mL was used to dissolve in the NMR tube. 0.005 mL of 2-mercaptoethanol was added to the solution, and 10 minutes later, the structure of the material before and after the reaction was confirmed through 200 MHz NMR, and the results are shown in FIG. 1. In FIG. 1, A is a 200 MHz NMR chart for the compound obtained in Example 4, and B is a 200 MHz NMR chart of the product obtained after addition of 2-mercaptoethanol to the compound obtained in Example 4. FIG. From the results of FIG. 1, it can be seen that thiol was added to β-site from the ketone site of the compound obtained in Example 4.

In addition, ¹ H NMR (DMSO-d ₆ , 200 MHz) and HRMS analysis of the compound produced after the reaction are as follows. Thus, the compound obtained in Example 4 was prepared through 7- (diethylamino) -3- (1- (2-hydroxyethylthio) -3- (2) through a thiol compound and Michael reaction. It can be seen that -hydroxyphenyl) -3-oxopropyl) -2H-chromen-2-one was formed.

¹ H NMR (DMSO-d ₆ , 200 MHz): δ 11.63 (s, 1H), 7.92 (m, 1H), 7.90 (s, 1H), 7.52 (m, 1H), 7.39 (d, 3J = 8.8 Hz, 1H), 6.96 (m, 2H), 6.68 (dd, 3J = 9.0Hz, 4J = 2.4Hz, 1H), 6.52 (d, 4J = 2.4Hz, 1H), 4.76 (t, 3J = 5.4Hz, 1H) , 4.48 (t, 3J = 7.4Hz, 1H), 3.78 (d, 3J = 7.4Hz, 2H), 3.45 (m, 6H), 2.56 (m, 2H), 1.15 (t, 3J = 7.0Hz, 6H) .

HRMS (FAB ⁺ , m-NBA): m / z obs'd 442.1680 ([M + H] ⁺ , cal'd 442.1688 for C ₂₄ H ₂₈ NO ₅ S).

Test Example  2. In vitro ( ex vivo ) Thiol  Selective binding properties with group-containing amino acids

The compound obtained in Example 4 was dissolved in 9 cuvettes at a concentration of 10 uM using a mixture of 100 mM HEPES buffer (pH = 7.4) and DMSO 1: 4 ratio. Thereafter, 500 equivalents of blank, Val, Phe, Pro, His, Ser, Lys, Asn, and Asp were added to each cuvette, and 1 hour afterward, the relative fluorescence responses were measured using the fluorescence spectrum. After that, 500 equivalents of homocysteine was added, and after 1 hour, the relative fluorescence reaction was measured using the fluorescence spectrum. The result is shown in FIG.

From the results of FIG. 2, the reaction did not occur with an amino acid that does not have a thiol group, but it can be seen that the reaction occurs selectively when homocysteine is added. Through selective reactions, it can be usefully used as a probe capable of detecting thiol compounds.

Test Example  3. Intracellular Thiol  Binding Properties with Compounds

HeLa cells in DMEM medium containing 100 units / mL penicillin, 100 mg / mL streptomycin, 10% heat-inactivated fetal bovine serum (HeLa ATCC Catalog No. CCL-2; human papilloma virus 18 (HPV-18)) Was incubated. The cell culture was inoculated in each of three culture plates of 35mm diameter HeLa cells at a concentration of 1X10 ⁵ cells, and the compound prepared in Example 4 was incubated at a concentration of 2.5 uM. One of the culture dishes was treated with α-lipoic acid (LPA, 500 uM) to increase the concentration of intracellular glutathione (GSH) (group 2), and the other N -ethylmaleimide (NEM, 100 mM). Treatment reduced the concentration of intracellular glutathione (GSH) (group 3). Cells of group 1 (control group), group 2, and group 3 were incubated at 37 ° C. for 24 hours and then visualized using confocal laser scanning microscopy. Confocal laser scanning microscopy imaging was activated using three channels and each excitation wavelength (λ _ex ) was 405 nm, 488 nm, and 555 nm. The results are shown in FIG.

From the results of FIG. 3, when only the compound prepared in Example 4 was added, blue fluorescence was detected at 405 nm, and the intensity of 405 nm was added when α-lipoic acid (LPA) capable of separating disulfide bonds was added. It can be seen that is counted. On the other hand, when N -ethylmaleimide capable of introducing a protecting group was added to the thiol group, the intensity disappeared at 405 nm and the strength increased at 488 nm. Therefore, the compound according to the present invention can be usefully used as a probe capable of detecting a compound containing a thiol group in vitro as well as in vitro.

Claims (2)

A compound of formula
&Lt; Formula 1 >

Wherein Y is a group of formula (2), a group of formula (3), or a group of formula (4) (in groups of formulas 2-4, R ₁ is hydrogen or acetyl; R ₂ is hydrogen, C ₁ to C ₄ Is alkyl, or phenyl optionally substituted with a hydroxy group of R ₃ is hydrogen or hydroxy:

Represents a bond to the compound of Formula 1).
(2)

(3)

&Lt; Formula 4 >

The method of claim 1,
(E) -7- (diethylamino) -3- (3-oxobut-1-enyl) -2H-chromen-2-one;
3-((7- (diethylamino) -2-oxo-2H-chromen-3-yl) methylene) pentane-2,4-dione;
(E) -3- (7- (diethylamino) -2-oxo-2H-chromen-3-yl) acrylaldehyde;
(E) -7- (diethylamino) -3- (3- (2-hydroxyphenyl) -3-oxoprop-1-enyl) -2H-chromen-2-one;
(E) -7- (diethylamino) -3- (3-oxo-3-phenyl-1-prop-1-enyl) -2H-chromen-2-one;
(E) -7- (diethylamino) -3- (3- (2-hydroxyphenyl) acryloyl) -2H-chromen-2-one;
3-cinnamoyl-7- (diethylamino) -2H-chromen-2-one; And
(E) -7- (diethylamino) -3- (4- (3- (2-hydroxyphenyl) -3-oxoprop-1-enyl) phenyl) -2H-chromen-2-one
Compound of formula 1, characterized in that selected from the group consisting of.

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