KR101278118B1 - Triazolylcoumarin derivatives having fluorous alkyl group and process for preparation and use thereof - Google Patents

Abstract

An object of the present invention is a triazolylcoumarin derivative having a novel polyfluorinated alkyl group represented by the following Chemical Formula 1, which combines immobilization, immobilization monitoring, and adhesion of an organic low molecular weight compound, a preparation method thereof, and an immobilization process for preparing a chemical array thereof. It is about use as a labeling system for verification.
[Formula 1]

In Formula 1, A, B, and D are alkylene groups having 1 to 6 carbon atoms; a is an integer from 5 to 9; b is an integer of 2-5.

Description

Triazolylcoumarin derivatives having a polyfluoroalkyl group, a method for preparing the same, and use thereof {Triazolylcoumarin derivatives having fluorous alkyl group and process for preparation and use}

The present invention provides a triazolylcoumarin derivative having a polyfluorinated alkyl group having immobilization, immobilization monitoring, and adhesion of organic low molecular weight compounds, a method for preparing the same, and a labeling system for preparing a chemical array thereof. It is about the use as a tag system.

One of the main tasks of the postgenomic era is to understand the function in the biological system of gene products, proteins [Curr. Opin. Chem. Biol. 2009, 13, 549-555], bioactive small organic compounds are chemical tools or biotechnology as an effective means for the exploration of biological phenomena and drug discovery. Also called a probe (bioprobe). In particular, securing organic low molecular weight compounds that can regulate specific functions of proteins as a means of identifying unknown life phenomena enables the understanding of living organisms and the study of novel therapeutics [Nature 2004]. , 432, 846-854].

As an effective means for exploring biological phenomena and drug discovery, the effective securement of chemical tools, along with securing small organic compound libraries with various backbones and substituents, As a result, a high-throughput screening technique for efficiently detecting a binding pair of organic low molecular weight compounds to a protein or protein group to be studied is required.

A chemical array is defined as a solid surface in which organic small molecule compounds are spatially addressable and high density integrated, chemical microarrays, and small molecule microarrays. Molecular microarrays (SMMs), chemical chips, ligand chips, etc. [Protein Targeting with Small Molecules, Wiley, 2009. Chapter 4. Recent Developments and Advances in Chemical Arrays, pp 57-80 ; Chem. Soc. Rev. 2008, 37, 1385-1394; Drug Discov. Today 2006, 11, 661-668; Mol. Biosyst. 2006, 2, 58-68]. For example, when a chemical array is processed with a protein to be studied, a specific fluorescence signal from a reporter group of fluorescent dyes used as a label of the protein may be used. The presence of binding ligands can be identified primarily, and a secondary multidimensional assay of the ligands and their analogs can be used to discover chemical tools that can control the function of the proteins. Currently, high-precision equipment, which can generate more than 1,000 microspots per square centimeter, is commercialized, allowing thousands to tens of thousands of binding assays to be performed simultaneously with a small sample volume. It can be considered as a future alternative technology for high-density well plate systems that are expected to be limited [Exploiting Chemical Diversity for Drug Discovery, RSC Biomolecular Sciences, 2006, Chapter 9. High-Density Plates, Microarrays, Microfluidics, pp 203-232].

Chemical array technology consists of elements such as a) organic low molecular compound library design and synthesis / b) immobilization of library molecules / c) organic low molecular compound-protein binding detection / d) data analysis. A) and d) are widely applied in the field of chip-based and solution-phase high-throughput screening, while b) immobilization of library molecules and c) organic low molecular weight. Compound-protein binding detection techniques are unique to chemical arrays (Protein Targeting with Small Molecules, Wiley, 2009. Chapter 4. Recent Developments and Advances in Chemical Arrays, pp 57-80).

The present invention is a b) immobilization technology of the library molecule of the above-described element technology [Top. Curr. Chem. 2007, 278, 311-342], in particular, seeks alternative techniques for the verification method of the immobilization process. Validation of the immobilization process for the manufacture of existing chemical arrays involves the immobilization of organic low molecular weight compounds (typically biotin) or fluorescent dyes with known binding proteins, followed by labeled binding proteins (avidin or strep for biotin). This can be achieved by the binding of strabividin or by detecting the fluorescence of the fluorescent dye itself, and in the chemical array manufacturing step, organic low molecular weight compounds or fluorescent dyes with known binding proteins are generated as reference spots [J]. . Am. Chem. Soc. 1999, 121, 7967-7968; J. Am. Chem. Soc. 2003, 125, 8420-8421; Angew. Chem. Int. Ed. 2003, 42, 2376-2379; J. Comb. Chem. 2004, 6, 862-868.

However, since organic compounds have different properties and properties according to their structure, individual compounds are likely to show differences in the efficiency of the immobilization step, thus improving the quality of chemical arrays in which thousands of tens of thousands of organic molecules are accumulated. For quality control, it is necessary to monitor the immobilization process of the library molecules individually for each micropoint, and the present invention provides a fluorescent labeling system for confirming the immobilization state of the library molecules for each micropoint in the chemical array manufacturing step. (fluorescent tag system), its manufacturing method, and its use.

It is an object of the present invention to provide a triazolylcoumarin derivative having a polyfluoroalkyl group which combines immobilization, immobilization monitoring, and adhesion function of the organic low molecular weight compound.

It is another object of the present invention to provide a method for producing a triazolylcoumarin derivative having the polyfluoroalkyl group.

Still another object of the present invention is to provide a use of the triazolylcoumarin derivative having a polyfluoroalkyl group as a labeling system for verifying an immobilization process for qualitative improvement of chemical array preparation.

The present invention provides a triazolylcoumarin derivative having a polyfluoroalkyl group represented by the following general formula (1) as a novel compound having immobilization, immobilization monitoring, and adhesion function of an organic small molecule compound:

[Formula 1]

In Formula 1, A, B, and D are alkylene groups having 1 to 6 carbon atoms; a is an integer from 5 to 9; b is an integer of 2-5.

In the present invention, the "alkylene group having 1 to 6 carbon atoms" is an aliphatic hydrocarbon group having 1 to 6 carbon atoms including methylene, ethylene, propylene, butylene and the like.

Preferably A, B, and D are alkylene groups having 1 to 3 carbon atoms; a is an integer from 5 to 9; b is an integer of 3 to 5;

The triazolylcoumarin derivative compounds according to the present invention may be more specifically exemplified as the following compounds, and the present invention is not limited by the following compounds.

The triazolylcoumarin derivative having a polyfluorinated alkyl group of the formula 1 according to the present invention is prepared by, for example, the following Scheme 1. However, the method of preparing the compound of Formula 1 according to the present invention is not limited to the following preparation method, and various modifications of the following preparation method will be possible to those skilled in the art. Unless stated otherwise, the definitions of substituents in the following schemes are the same as in Formula 1.

The preparation method for preparing a triazolylcoumarin derivative having a polyfluorinated alkyl group of the present invention is azide compound having a methyl 7-ethynylcoumarin-4-acetate compound (2) and a polyfluorinated alkyl group, as shown in Scheme 1 below. Reacting 3) to prepare 7-triazolylcoumarin-4-acetate compound (4) having a polyfluoroalkyl group; A second step of preparing a 7-triazolylcoumarin-4-acetic acid compound (5) having a polyfluoroalkyl group from the prepared 7-triazolylcoumarin-4-acetate compound (4) having a polyfluoroalkyl group; 7-triazolylcoumarin-4-acetic acid having a polyfluoroalkyl group is reacted with 7-triazolylcoumarin-4-acetic acid compound (5) prepared above and a diamine compound (6) having a protecting group (PG). A third step of preparing acetamide compound (7); And deprotecting the 7-triazolylcoumarin-4-acetamide compound (7) having the polyfluoroalkyl group prepared above, to obtain a triazolylcoumarin compound (1) having a polyfluoroalkyl group of the formula (1) according to the present invention. Fourth step.

[Reaction Scheme 1]

In the above scheme, A, B, D, a and b are the same as defined in the formula (1).

In detail step by step, the first step of the production method is a polyfluorinated alkyl group of Formula 3 under the conditions of a conventional copper-catalyzed alkyne-azide cycloaddition reaction (copper-catalyzed alkyne-azide cycloaddition reaction) It is carried out using an azide compound having a. For the compound of Formula 3, it is preferable to use 1.0 to 1.2 molar equivalents, and more preferably 1.0 to 1.1 molar equivalents, relative to the compound of Formula 2. At this time, the reaction temperature is good in the boiling point range of 0 ℃ to the solvent, more preferably in the range of 0 ℃ to room temperature. The reaction time is 1 hour to 12 hours, preferably 1 hour to 5 hours.

The second step of the process is carried out under conventional ester hydrolysis reaction conditions. At this time, the reaction temperature is good in the boiling point range of 0 ℃ to the solvent, more preferably in the range of 0 ℃ to room temperature. The reaction time is 1 hour to 12 hours, preferably 1 hour to 5 hours.

The third step of the preparation method is carried out under conventional 'amide bond forming reaction' conditions using carboxylic acid and amine. The compound of Formula 6 has a conventional amino group protecting group (PG) including t-butoxycarbonyl (hereinafter referred to as 'Boc'). With respect to the compound of Formula 5, the compound of Formula 6 may preferably use 1.0 to 1.2 molar equivalents, more preferably 1.0 to 1.1 molar equivalents. At this time, the reaction temperature is good in the boiling point range of 0 ℃ to the solvent, more preferably in the range of 0 ℃ to room temperature. The reaction time is 1 hour to 12 hours, preferably 1 hour to 5 hours.

The fourth step of the production process is carried out under reaction conditions for removing conventional amino group protecting groups. When the amino group protecting group is Boc, it is carried out in a conventional organic solvent such as dichloromethane (hereinafter referred to as 'DCM') using a conventional organic acid such as trifluoroacetic acid (hereinafter referred to as 'TFA') and the like. do. At this time, the reaction temperature is good in the boiling point range of 0 ℃ to the solvent, more preferably in the range of 0 ℃ to room temperature. The reaction time is reacted for 1 minute to 5 hours, preferably 1 minute to 30 minutes.

In order to confirm whether the compounds of Formulas 1, 4, 5 and 7 were produced according to the preparation method of the present invention, the structure was analyzed by NMR and Mass spectra.

Since the triazolyl coumarin derivative having the polyfluorinated alkyl group of Formula 1 has the function of immobilization, immobilization monitoring, and adhesion of the organic low molecular weight compound, the triazolyl coumarin derivative may be used as a labeling system for verifying the immobilization process for preparing a chemical array. An example thereof may be provided in FIG. 4, the first step of attaching an organic low molecular weight compound to a compound of Formula 1 to prepare a compound of Formula 8; A second step of immobilizing the compound of formula 8 on a solid surface having a polyfluorinated alkyl group; The third step is to monitor the immobilization process of the compound of Formula 8 by detecting fluorescence generated from the compound of Formula 8. The following examples do not limit the use of the compound of formula 1 according to the present invention.

In FIG. 4, solid support includes a resin or the like used in conventional 'solid-phase organic synthesis'; X is a conventional organic functional group; A fluorously modified solid surface with a polyfluoroalkyl group is a solid surface that has been surface treated with a polyfluoroalkyl group including a fluorously modified glass slide with a polyfluoroalkyl group.

In detail, the use step by step, by reacting the compound of Formula 1 with the organic low-molecular compound or a solution phase of the organic low-molecular compound attached to the solid support in the step of attaching the organic low-molecular compound The organic low molecular weight compound is labeled with the compound of formula 1 by forming a chemical bond in the liver.

The second step of this application immobilizes the compound of formula 8 via polyfluoroalkyl interaction on a solid surface with polyfluoroalkyl groups, for example a glass slide with polyfluoroalkyl groups.

The third step of this application monitors the immobilization process of the compound of formula 8 via fluorescence detection resulting from the triazolylcoumarin backbone of immobilized compound 8.

The present invention relates to a triazolylcoumarin derivative having a novel polyfluoroalkyl group, which combines immobilization, immobilization monitoring, and adhesion of organic low molecular weight compounds, and is a labeling system for verifying an immobilization process for improving the quality of chemical array preparation. Can be used as

1 illustrates a reference point-based immobilization verification and a label system-based immobilization verification method to be invented.
2 shows a schematic configuration of a fluorescent labeling system.
3 shows a schematic configuration of a fluorescent labeling system having a polyfluoroalkyl group.
4 shows the use of the compound of formula 1 as a labeling system.

Hereinafter, the present invention will be described in detail with reference to examples.

The following examples are merely illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

[Example 1] N- (2- (2- (ethoxy) ethyl 2-aminoethoxy)) -2- (7- (1- (4,4,5,5,6,6,7, 7 , 8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3- triazol - 4-yl) -2-oxo-2H -chromen -4-yl) Preparation of Acetamide

(a) step: methyl {7- [1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluor Preparation of Undecyl) -1H- [1,2,3] triazol-4-yl] -2-oxo-2H-chromen-4-yl} acetate

2.94 g (12.1 mmol) of methyl 7-ethynylcoumarin-4-acetate was added to a mixed solution of 60 mL of acetonitrile and 120 mL of water at room temperature, followed by 4,4,5,5,6,6,7,7,8 6.10 g (12.1 mmol) of 8,9,9,10,10,11,11,11-heptadecafluoroundecyl azide, 1.80 mL (12.8 mmol) of triethylamine, 580 mg (3.03) of copper iodide (I) mmol) was added. The reaction mixture was stirred at the same temperature for 3 hours, then water was added and extracted four times with dichloromethane. The organic layers were combined, dried over magnesium sulfate, evaporated to remove the solvent, and the residue was purified by silica gel column chromatography (n-hexane: ethyl acetate 1: 1 v / v) to give 6.52 g of the target compound (yield: 72%). )

¹ H NMR (500 MHz, Acetone-d ₆ ) δ 2.34-2.39 (m, 2H), 2.43-2.50 (m, 2H), 3.71 (s, 3H), 4.02 (s, 2H), 4.71 (t, J = 6.9 Hz, 2H), 6.46 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.83 (sd, J = 1.5 Hz, 1H), 7.91 (dd, J = 1.6, 8.3 Hz, 1H ), 8.65 (s, 1 H); MS (ESI) m / z 746 ([M + H] ⁺ ).

(b) step: {7- [1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluorounde Preparation of ac) -1H- [1,2,3] triazol-4-yl] -2-oxo-2H-chromen-4-yl} acetic acid

Methyl {7- [1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H 1.80 g (2.42 mmol) of [[1,2,3] triazol-4-yl] -2-oxo-2H-chromen-4-yl} acetate are mixed with 150 mL of tetrahydrofuran and 150 mL of water at room temperature Lithium hydroxide monohydrate (1.01 g, 24.2 mmol) was added to the solution. The reaction mixture was stirred at the same temperature for 3 hours, adjusted to pH 3-4 with 1N aqueous hydrochloric acid solution, water was added, and extracted four times with ethyl acetate. The organic layers were combined, dried over magnesium sulfate, and evaporated under reduced pressure to obtain 1.77 g (yield: 99%) of the target compound.

¹ H NMR (500 MHz, Acetone-d ₆ ) δ 2.33-2.39 (m, 2H), 2.43-2.51 (m, 2H), 3.98 (s, 2H), 4.71 (t, J = 6.9 Hz, 2H), 6.47 (s, 1H), 7.83 (d, J = 1.5 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.92 (dd, J = 1.5, 8.2 Hz, 1H), 8.65 (s, 1H ); MS (ESI) m / z 732 ([M + H] ⁺ ).

step (c): t-butyl 2- (2- (2- (2- (7- (1- (4,4,5,5,6,6,7,7,8,8,9,9, 10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) acetamido) Preparation of ethoxy) ethoxy) ethyl carbamate

At room temperature (7- [1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl)- 1.77 g (2.42 mmol) of 1H- [1,2,3] triazol-4-yl] -2-oxo-2H-chromen-4-yl} acetic acid and N-Boc-2,2 '-(ethylenedi 660 mg (2.66 mmol) of oxy) diethylamine were added to 100 mL of dichloromethane, followed by 835 mg (4.35 mmol) of ethyl diisopropyl carbodiimide hydrogen chloride. The reaction mixture was stirred at the same temperature for 2 hours and then water was added and extracted with dichloromethane. The organic layer was dried over magnesium sulfate, dark evaporated to remove the solvent, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain 1.71 g (yield: 73%) of the target compound.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1.35 (s, 9H), 2.21-2.15 (m, 2H), 2.39-2.32 (m, 2H), 3.05 (q, J = 5.8 Hz, 2H), 3.24 (q, J = 5.6 Hz, 2H), 3.36 (q, J = 6.5 Hz, 2H), 3.43 (t, J = 5.6 Hz, 2H) 3.49 (s, 4H), 3.76 (s, 2H), 4.57 (t, J = 7.0 Hz, 2H), 6.43 (s, 1H), 6.76 (t, J = 5.4 Hz, 1H), 7.83 (s, 1H), 7.85 (s, 2H), 8.37 (t, J = 5.4 Hz, 1 H), 8.83 (s, 1 H); MS (ESI) m / z 962 ([M + H] ⁺ ).

(d) step: N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -2- (7- (1- (4,4,5,5,6,6,7,7) , 8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen Preparation of -4-yl) acetamide

t-butyl 2- (2- (2- (2- (7- (1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11) , 11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) acetamido) ethoxy) ethoxy 1.70 g (1.77 mmol) of ethyl carbamate were added to dichloromethane (48 mL) at 0 ° C., followed by addition of 12 mL of trifluoroacetic acid. The reaction mixture was stirred at the same temperature for 10 minutes and then evaporated to remove the solvent, and then the residue was purified by silica gel column chromatography (methanol: triethylamine: dichloromethane 7: 1: 92 v / v), 1.05 g (yield: 69%) of the title compound were obtained.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 2.21-2.15 (m, 2H), 2.41-2.32 (m, 2H), 2.64 (t, J = 5.7 Hz, 2H), 3.45-3.23 (m, 8H ), 3.50 (m, 4H), 3.76 (s, 2H) 4.56 (t, J = 7.0 Hz, 2H), 6.43 (s, 1H), 7.83 (s, 1H), 7.85 (s, 2H), 8.40 ( t, J = 5.5 Hz, 1H), 8.83 (s, 1H); MS (ESI) m / z 862 ([M + H] ⁺ ).

Example 2 N- (3- (2- (2- (3- aminopropoxy ) ethoxy ) ethoxy ) propyl) -2- (7- (1- (4,4,5,5,6) , 6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3- triazol - 4-yl) -2- Preparation of oxo-2H -chromen -4-yl) acetamide

(a) step: t-butyl 1- (7- (1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11 -Heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) -2-oxo-7,10,13-trioxa Preparation of 3-Azahexadecane-16-yl carbamate

In the same manner as in Example 1 (c), except that obtained in Examples 1 (a) and 1 (b) {7- [1- (4,4,5,5,6,6,7,7, 8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H- [1,2,3] triazol-4-yl] -2-oxo-2H-chromen 2.38 g (3.25 mmol) of -4-yl} acetic acid and 1.15 g (3.58 mmol) of N-Boc-4,7,10-trioxa-1,13-tridecanediamine were added to 150 mL of dichloromethane at room temperature, followed by ethyl 1.13 g (5.86 mmol) of diisopropyl carbodiimide hydrogen chloride were added. The reaction mixture was stirred at the same temperature for 2 hours, then water was added and extracted with dichloromethane. The organic layer was dried over magnesium sulfate, dark evaporated to remove the solvent, and the residue was purified by silica gel column chromatography (ethyl acetate) to obtain 2.53 g (yield: 75%) of the target compound.

¹ H NMR (500 MHz, CDCl ₃ ) δ 1.41 (s, 9H), 1.77-1.69 (m, 4H), 2.27-2.16 (m, 2H), 2.37-2.31 (m, 2H), 3.18 (q, J = 6.2 Hz, 2H), 3.39 (q, J = 6.2 Hz, 2H), 3.48 (t, J = 5.9 Hz, 2H) 3.57-3.53 (m, 6H), 3.63-3.61 (m, 4H), 3.69 ( s, 2H), 4.55 (t, J = 6.9 Hz, 2H), 4.92 (s, 1H), 6.42 (s, 1H), 7.02 (s, 1H), 7.71 (d, J = 1.1 Hz, 1H), 7.82-7.78 (m, 2 H), 7.93 (s, 1 H); MS (ESI) m / z 1034 ([M + H] ⁺ ).

(b) step: N- (3- (2- (2- (3-aminopropoxy) ethoxy) ethoxy) propyl) -2- (7- (1- (4,4,5,5,6) , 6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2- Preparation of oxo-2H-chromen-4-yl) acetamide

In the same manner as in Example 1 (d), except that t-butyl 1- (7- (1- (4,4,5,5,6,6,7,7,8,8,9,9,10 , 10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) -2-oxo- 2.52 g (2.44 mmol) of 7,10,13-trioxa-3-azahexadecane-16-yl carbamate was added to 72 mL of dichloromethane at 0 ° C., followed by addition of 18 mL of trifluoroacetic acid. After stirring for 10 minutes at the same temperature, the solvent was evaporated to remove the solvent, and then the residue was purified by silica gel column chromatography (methanol: triethylamine: dichloromethane 7: 1: 92 v / v) to obtain the target compound 1.77. g (yield: 78%) was obtained.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1.57-1.52 (m, 2H), 1.66-1.61 (m, 2H), 2.20-2.14 (m, 2H), 2.43-2.32 (m, 2H), 2.57 (t, J = 6.8 Hz, 2H), 3.12 (q, J = 6.0 Hz, 2H), 3.48-3.35 (m, 14H), 3.73 (s, 2H), 4.56 (t, J = 7.1 Hz, 2H) , 6.42 (s, 1 H), 7.82 (s, 1 H), 7.85 (s, 2 H), 8.30 (t, J = 5.5 Hz, 1 H), 8.83 (s, 1 H); MS (ESI) m / z 934 ([M + H] ⁺ ).

[Experimental Example 1] Experiment for confirming the possibility of use as a labeling system for a chemical array

(a) step: preparation of labeled biotin

Experimental Example a-1 From N-hydroxysuccinimide Ester of Biotin, N- (2- (2- (2- (2- (2- (7- (1- (4,4,5,5,6,6) , 7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazolyl-4-yl) -2-oxo- 2H-chromen-4-yl) acetamido) ethoxy) ethoxy) ethyl) -5- (2-oxoexohydro-1H-cyino [3,4-d] imidazol-4-yl) pentane Preparation of Amides

17 mg (0.050 mmol) of N-hydroxysuccinimide ester of Biotin (Compound 9) and N- (2- (2- (2-aminoethoxy) ethoxy prepared in Example 1 above ) Ethyl) -2- (7- (1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluor Undecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) acetamide (Compound 1a) 50 mg (0.060 mmol) was diluted with dichloromethane at room temperature. 1 mL of N, N-dimethylformamide and 14 μl of triethylamine (0.10 mmol) were added thereto. The reaction mixture was stirred at the same temperature for 6 hours, followed by evaporation to remove the solvent, and then the residue was purified by silica gel column chromatography (dichloromethane: methanol 9: 1 v / v) to obtain the target compound of Chemical Formula 8a. mg (yield: 58%) were obtained.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1.32-1.22 (m, 2H), 1.47-1.34 (m, 4H), 1.59-1.55 (m, 1H), 2.05 (t, J = 7.1 Hz, 2H ), 2.19-2.17 (m, 2H), 2.37-2.33 (m, 2H), 2.56 (d, J = 12.5 Hz 1H), 2.79 (dd, J = 4.7, 12.4 Hz, 1H), 3.17 (sd, J = 5.5 Hz, 2H), 3.24 (d, J = 5.3 Hz, 2H), 3.42 (d, J = 5.4 Hz, 4H), 3.50 (s, 4H), 3.76 (s, 2H), 4.11 (s, 1H ), 4.29 (t, J = 6.1 Hz, 1H), 4.56 (t, J = 6.6 Hz, 2H), 6.35 (s, 1H), 6.42 (d, J = 11.9 Hz, 2H), 7.84 (d, J = 10.4 Hz, 4H), 8,38 (s, 1 H), 8.83 (s, 1 H); MS (ESI) m / z 1088 ([M + H] ⁺ ).

Experimental Example a-2 From N-hydroxysuccinimide Ester of Biotin, N- (1- (7- (1- (4,4,5,5,6,6,7,7,8,8, 9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) -2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl) -5- (2-oxohexahydro-1H-cyino [3,4-d] imidazol-4- (1) Preparation of pentanamide

N-hydroxysuccinimide ester of Biotin (Compound 9) (35 mg, 0.10 mmol) and N- (3- (2- (2- (3-aminopro) prepared in Example 2, above. Foxy) ethoxy) ethoxy) propyl) -2- (7- (1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10,11, 11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) acetamide (Compound 1b) 115 mg (0.120) mmol) was added to 2 mL of dichloromethane at room temperature, and then 0.2 mL of N, N-dimethylformamide and 28 μl (0.20 mmol) of triethylamine were added thereto. The reaction mixture was stirred at the same temperature for 9 hours, then evaporated to remove the solvent, and then the residue was purified by silica gel column chromatography (dichloromethane: methanol 9: 1 v / v) to obtain the target compound of Chemical Formula 8b. mg (96%) was obtained.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1.30-1.22 (m, 4H), 1.50-1.43 (m, 4H), 1.64-1.56 (m, 5H), 2.03 (t, J = 7.4 Hz, 2H ), 2.19-2.16 (m, 2H), 2.37-2.33 (m, 2H), 2.56 (d, J = 12.4 Hz 1H), 2.80 (dd, J = 5.1, 12.4 Hz, 1H), 3.09-3.03 (m , 3H), 3.12 (q, J = 6.5 Hz, 2H), 3.49-3.43 (m, 9H), 3.73 (s, 2H), 4.12-4.10 (m, 1H), 4.29-4.28 (m, 1H), 4.56 (t, J = 7.1 Hz, 2H), 6.35 (s, 1H), 6.42 (d, J = 6.5 Hz, 2H), 7.74 (t, J = 5.4 Hz, 1H), 7.83 (s, 1H), 7.85 (d, J = 2.2 Hz, 2H), 8,25 (t, J = 5.5 Hz, 1H), 8.83 (s, 1H); MS (ESI) m / z 1160 ([M + H] ⁺ ).

Experimental Example a-3 N- (2- (2- (2- (2- (7- (1- (4,4,5)) from biotin attached to a Kenner's safety-catch linker. , 5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazolyl-4-yl ) -2-oxo-2H-chromen-4-yl) acetamido) ethoxy) ethoxy) ethyl) -5- (2-oxoexohydro-1H-cyino [3,4-d] imidazole Preparation of -4-yl) pentanamide

100 mg (0.076 mmol) of the compound of Chemical Formula 10 above from aminomethyl polystyrene (PS) resin (1.2 mmol / g, purchased from Novabiochem) and N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -2- (7- (1- (4,4,5,5,6,6,7,7,8,8,9,9,10,10, 11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) acetamide (Compound 1a) 65 mg (0.076 mmol) was added to 3 mL of N-methylpyrrolidinone at room temperature, followed by stirring at 55 ° C. for 10 hours. After cooling to room temperature, the mixture was filtered, washed with tetrahydrofuran, and the filtered solution was evaporated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane: methanol 9: 1 v / v) to give 69 mg (yield: 84%) of the target compound of Chemical Formula 8a.

Experimental Example a-4 From Biotin Attached to Kenner's Safety-catch Linker, N- (1- (7- (1- (4,4,5,5,6,6,7, 7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chrome Men-4-yl) -2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl) -5- (2-oxohexahydro-1H-cyino [3,4-d ] Imidazol-4-yl) pentanamide Preparation

100 mg (0.076 mmol) of the compound of Formula 10 and N- (3- (2- prepared in Example 2) from an aminomethyl polystyrene (PS) resin (1.2 mmol / g, purchased from Novabiochem) (2- (3-aminopropoxy) ethoxy) ethoxy) propyl) -2- (7- (1- (4,4,5,5,6,6,7,7,8,8,9, 9,10,10,11,11,11-heptadecafluoroundecyl) -1H-1,2,3-triazol-4-yl) -2-oxo-2H-chromen-4-yl) acetamide (Compound 1b) 71 mg (0.076 mmol) was added to 3 mL of N-methylpyrrolidinone at room temperature, followed by stirring at 55 ° C. for 10 hours. After cooling to room temperature, the mixture was filtered, washed with tetrahydrofuran, and the filtered solution was evaporated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane: methanol 9: 1 v / v) to give 78 mg (yield: 89%) of the target compound of Chemical Formula 8b.

Step (b): Labeled Biotin Immobilization and Immobilization Monitoring

N, N-dimethylformamide solution of the compounds of Formulas 8a and 8b prepared in step (a) was prepared at 5 mM, 2.5 mM, 1 mM and 0.5 mM concentrations, followed by 384-well plates. 40 μl each). A 384-well plated sample prepared using a commercially available microarrayer (purchased from Qarray mini, Genetix) was spotted on a glass slide with a fluoroalkyl group (purified from Fluorous). It was left for time. As shown in Figure 5a, a commercial slide scanner (slide scanner, ArrayWoRx, purchased from Applied Precision) was used to detect the fluorescence of the labeling system with the A350 channel (absorbed at 360 nm, emitted at 460 nm). It was confirmed that the compounds of Formulas 8a and 8b prepared in step a) were immobilized on the glass slide. For comparison, the compounds of Formulas 4, 5, 7a, 7b, 1a and 1b were also immobilized in the same manner on the same glass slide.

(c) step: detection of binding of streptavidin and biotin

1 μg / mL sample obtained by diluting streptavidin-Cy3 (streptavidin-Cy3, 1 mg / mL in 0.01 M phosphate buffered saline, pH 7.4, containing 1% BSA and 15 mM sodium azide, Sigma) The glass slide prepared in step b) was treated for 30 minutes, washed with phosphate buffered saline (0.01 M, pH 7.4, containing 1% BSA, purchased from Sigma) for 10 minutes, and then washed with distilled water for 5 minutes. Repeat three times. After drying the washed glass slides, fluorescence of Cy3 with CY3 channel (absorbed at 540 nm, emitted at 595 nm) using one commercial slide scanner (slide scanner, ArrayWoRx, purchased from Applied Precision), as shown in Figure 5b below It was confirmed that the compounds of formulas 8a and 8b prepared in the above step (a) immobilized on the glass slide specifically bind to streptavidin.

Experimental results confirming that the compound of Formula 1 can be utilized as a labeling system for chemical arrays are shown in FIG. 4.

The triazolylcoumarin derivative having a novel polyfluorinated alkyl group prepared in an embodiment of the present invention is a polyfluorinated alkyl group for immobilization, a triazolylcoumarin skeleton as a fluorophore moiety for immobilization monitoring, and an attachment for an organic small molecule compound. It is composed of a linker, it can be seen that it can be used as a fluorescent labeling system having a polyfluorine group to verify the immobilization process for the qualitative improvement of chemical array manufacturing.

Claims (6)

Triazolylcoumarin derivative represented by the following formula (1):
[Formula 1]

In Chemical Formula 1,
A, B, and D are alkylene groups having 1 to 6 carbon atoms;
a is an integer from 5 to 9;
b is an integer of 2-5.
The method of claim 1,
A, B, and D are alkylene groups having 1 to 3 carbon atoms;
a is an integer from 5 to 9;
b is a triazolylcoumarin derivative, characterized in that an integer of 3 to 5.
The method of claim 1,
Triazolylcoumarin derivatives selected from the following compounds.

(a) 7-triazolylcoumarin-4-acetate having a polyfluorinated alkyl group of formula 4 by reacting methyl 7-ethynylcoumarin-4-acetate of formula 2 with an azide having a polyfluoroalkyl group of formula 3 Preparing a;
(b) preparing a polyfluorinated alkyl group-substituted 7-triazolylcoumarin-4-acetic acid of formula 5 by ester hydrolysis of the compound of formula 4 prepared in step (a);
(c) reacting the compound of formula 5 prepared by step (b) with a diamine having a protecting group (PG) of formula (6) to a polyfluoroalkyl group-substituted 7-triazolylcoumarin-4-acetamide of formula (7) Preparing a; And
(d) deprotecting the compound of formula (7) prepared by step (c) to obtain triazolylcoumarin of formula (1);
Method for preparing a triazolylcoumarin derivative having a polyfluoroalkyl group comprising:
[Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

(7)

In the above formulas,
A, B, and D are alkylene groups having 1 to 6 carbon atoms;
a is an integer from 5 to 9;
b is an integer of 2-5.
A fluorescent label device comprising the triazolylcoumarin derivative of the formula (1) according to any one of claims 1 to 3.
A labeling system comprising a process of preparing a target compound by attaching an organic low molecular weight compound to the triazolylcoumarin derivative of Formula 1 according to any one of claims 1 to 3 and monitoring the immobilization of the target compound through fluorescence detection. Immobilization Verification.

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