KR20160032914A - Benzenesulfonamide compounds - Google Patents

Abstract

The present invention provides a benzenesulfonamide compound represented by chemical formula 1 or a biotinylated compound thereof. The compounds of the present invention selectively react with a sulfhydryl group (-SH) having unoxidized cysteine, and do not react with an oxidative modified product. Thus, the compounds of the present invention can detect the sulfhydryl group having unoxidized cysteine. In addition, the compounds of the present invention can measure reactivity with respect to an oxidation reaction of each cysteine, and thus can screen target proteins of reactive oxygen species.

Description

Benzene sulfonamide compounds < RTI ID = 0.0 >

The present invention relates to benzenesulfonamide compounds, processes for their preparation and compositions comprising them.

More particularly, the present invention relates to a compound capable of selectively detecting a protein that reacts selectively with active cysteine and sensitively reacts with oxidation-reduction, a method for producing the same, a composition for detecting active cysteine comprising the same, To a composition for screening active oxygen target proteins and a screening method.

The Reactive Oxygen Species (ROS) are constantly produced during normal human metabolism and play an important role in various biological processes in the cell.

However, when normal cells are exposed to the environment such as ultraviolet rays, virus infection, and hyperglycemia and the concentration of active oxygen is excessively increased, a specific gene or protein in the cell reacts with active oxygen to lose its original function, Causing oxidative stress, which is known to act as a cause of inflammatory diseases such as cancer and diabetes. In addition, intracellular reactive oxygen is also implicated in Alzheimer's disease, Parkinson's disease, and aging.

Specifically, the active oxygen species (ROS) are derived from sulfhydryl groups (-SH) of cysteine, sulfenic acid (Cys-SOH), sulfinic acid (Cys-SO ₂ H) , Sulfonic acid (Cys-SO ₃ H) or disulfide bond (Cys-SS-Cys), etc., to participate in cell proliferation, death, cell migration and inflammation.

However, the cysteine oxidation sensitivity to oxidation stress is determined by the surrounding environment such as the acid dissociation constant (pKa), and the cysteine having a low acid dissociation constant (pKa) is called a 'redox sensitive cyst' A protein containing a sensitive cysteine is called a 'ROS target protein' since it is a major target of active oxygen.

Therefore, the detection of redox-sensitive cysteine and screening of an active oxygen target protein can play a very important role in studying the ROS-mediated cell signaling pathway of ROS-related diseases, It can be helpful.

Conventionally, an alkylating agent such as iodoacetamide (IAA) or N-ethylmaleimide (NEM) was used to detect cysteine. However, since the alkylating agents can react not only with unoxidized sulfhydryl groups (-SH) of cysteine but also with sulfoxylic acid (-SOH) and sulfinic acid (-SO ₂ H), which are oxidized forms of sulfhydryl groups, It is possible to simply detect cysteine, and there is a limit in detecting redox-sensitive cysteine.

Thus, in order to detect redox sensitive cysteine and screen for active oxygen target proteins, the development of new compounds that selectively react with sulfhydryl groups of cysteine and do not react with oxidative modifications has been very urgent.

Korean Patent Publication No. 2012-0074843

It is an object of the present invention to provide a benzenesulfonamide compound which selectively reacts with a sulfhydryl group of cysteine and a process for producing the same.

Another object of the present invention is to provide a composition for detecting active cysteine and a method for detecting active cysteine using the same.

Another object of the present invention is to provide an active oxygen target protein screening composition comprising the same and a method for screening an active oxygen target protein using the same.

In order to solve the above problems, the present invention provides a benzenesulfonamide compound, a method for preparing the same, a composition for detecting active cysteine comprising the same, and a method for detecting active cysteine using the same, a composition for screening active oxygen target proteins containing the same, A method for screening an oxygen target protein is provided. Hereinafter, each invention will be described in detail.

Benzenesulfonamide  compound

The present invention provides a benzenesulfonamide compound represented by the following formula (1) or a biotinylated compound thereof:

[Chemical Formula 1]

In Formula 1,

R ¹ Or R ² are each hydrogen, C _{1 -6} straight or branched alkyl, C _{3 -7-cycloalkyl,} or C _{6 -10} aryl, (wherein the C _{3 -7-cycloalkyl} or hydrogen of C _{6 -10} aryl are halogen, may be substituted with -OH or C _{1 -4} alkyl), or R ¹ and R ² are connected to each other H of the 5-membered to 6 (wherein the heterocyclic ring together with the nitrogen atom of the heterocyclic ring include halogen, -OH or it forms a C _{1 -4} may be substituted with alkyl),

R ³ or R ⁴ are each -COR ⁵ , -CN, -SO ₃ H, -NO ₂ , -CF ₃ or -CCl ₃ ,

R ⁵ is hydrogen, C _{1 -4} alkyl, -OH, halogen, C _{1 -4} alkoxy, -NR ⁶ R ^7,

R ⁶ Or R ⁷ are each hydrogen or C _{1 -6} straight or branched alkyl.

According to one embodiment of the present invention, the heterocyclic ring is preferably a pyrrolidine ring, a piperidine ring or a morpholine ring. Further, the heterocyclic ring is more preferably a piperidine ring.

According to another embodiment of the present invention, R ³ is preferably -NO ₂ .

Further, according to another embodiment of the invention, R ⁴ is -COR ^5, or -NO _2, R ⁵ is preferably a C _{1 -4} alkyl, C _{1 -4} alkoxy, or -NH _2. Further, R ⁴ is more preferably -COMe, -COOMe, -CONH ₂ or -NO ₂ .

According to another embodiment of the present invention, the biotinylated compound may be biotinylated at the R ⁴ position.

According to one embodiment of the present invention, the compound of formula (I) may be selected from the group consisting of:

1) methyl 3-nitro-4- (piperidin-1-ylsulfonyl) benzoate;

2) 1- (3-Nitro-4- (piperidin-1-ylsulfonyl) phenyl) ethanone;

3) 1- (2,4-dinitrophenylsulfonyl) piperidine; And

4) 3-Nitro-4- (piperidin-1-ylsulfonyl) benzamide.

Further, according to another embodiment of the present invention, the biotinylated compound may be selected from the group consisting of the following compounds:

1) 3-Nitro-N- (2- (4- (2-oxohexahydro-1H-thieno [ 1-ylsulfonyl) < / RTI >benzamide; And

2) Preparation of 3-Nitro-N- (16-oxo-20 - ((3R, 6S) -2-oxohexahydro- 6,9,12-tetraoxa-15-azaicol) -4- (piperidin-1-ylsulfonyl) benzamide.

The benzenesulfonamide compounds of the present invention selectively react with unoxidized sulfhydryl groups (-SH) of cysteine and do not react with oxidative modifications. Accordingly, the benzenesulfonamide compounds of the present invention can be used for the detection of cysteine, particularly for the detection of redox sensitive cysteine, and further can be used to screen active oxygen target proteins.

Benzenesulfonamide  Method for producing a compound

The benzenesulfonamide compound of the present invention can be prepared by reacting a sulfonyl halide compound of the following formula (2) with an amine compound of the following formula (3).

(2)

(3)

(R ¹ to R ⁷ are as defined above and X is halogen)

The reaction can be carried out under the usual reaction conditions of a sulfonyl halide compound and an amine compound.

According to one embodiment of the present invention, the biotinylation may be performed by reacting a biotinylation reagent with a carboxylic acid compound of the following formula (I-1).

(I-1)

The biotinylating reagent used in the reaction may be biotin, biotin ethylenediamine, biotin-PEG4-amine, biotin-PEG4-alkyne, biotin hydrazide, biotin poly (ethylene oxide) amine, biotin poly (ethylene oxide) Acetamide, and the like, and it is preferable to use biotin ethylenediamine or biotin-PEG 4 -amine having an amine group at the terminal group.

The biotinylation reaction can be carried out using conventional coupling reagents such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDCI), 1-hydroxybenzotriazole hydrate (HOBt), biotin ethylenediamine, diethyl In the presence of isopropylamine.

Composition and method for detecting cysteine

According to one embodiment of the present invention, the benzenesulfonamide compound of the present invention or a biotinylated compound thereof can react with an unoxidized sulfhydryl group (-SH) of cysteine, and thus can be included in a composition for detecting cysteine.

In particular, the composition of the present invention can confirm whether or not the sulfhydryl group of cysteine is oxidized according to pH change or oxidative stress condition.

According to another embodiment of the present invention, there is provided a method for detecting a sulfhydryl group (-SH) of cysteine using the benzenesulfonamide compound of the present invention or a biotinylated compound thereof.

In addition, the method of the present invention can confirm the oxidation of the sulfhydryl group of cysteine according to pH change or oxidative stress condition.

The detection method of the present invention comprises the steps of (S1) contacting a protein with a compound of formula (I) or a biotinylated compound thereof while changing pH or oxidative stress conditions and (S2) confirming labeling of the protein in each reaction .

The oxidative stress condition may be, but is not limited to, varying the conditions by varying the concentration of H ₂ O ₂ .

Active oxygen target  COMPOSITION FOR SCREENING PROTEIN AND SCREENING METHOD

According to one embodiment of the present invention, the benzenesulfonamide compound of the present invention or a biotinylated compound thereof can be included in a composition for screening a target protein of an active oxygen-mediated disease.

The active oxygen-mediated disease refers to any disease caused by active oxygen. For example, reactive oxygen-mediated diseases can be selected from the group consisting of inflammatory diseases, degenerative brain diseases and aging. The inflammatory disease may be cancer or diabetes, and the degenerative brain disease may be Alzheimer's disease (AD) or Parkinson's disease (PD).

According to another embodiment of the present invention, there is provided a method for screening a target protein of an active oxygen-mediated disease using the benzenesulfonamide compound of the present invention or a biotinylated compound thereof.

The screening method comprises the steps of (S1) contacting a protein with a compound of formula (I) or a biotinylated compound thereof while changing oxidative stress conditions, (S2) confirming labeling of the protein in each reaction, and (S3) Lt; RTI ID = 0.0 > a < / RTI > pattern of the protein.

The compounds of the present invention selectively react with unoxidized sulfhydryl groups (-SH) of cysteine, and exhibit the action and effect of not reacting with oxidative modifications. Therefore, the compound of the present invention can detect the sulfhydryl group of cysteine.

In addition, the compounds of the present invention can measure the reactivity of each cysteine to an oxidation reaction, thereby screening an active oxygen target protein

1 is a Streptavidin-HRP result confirming whether the compound of the present invention reacts with a sulfhydryl group of cysteine in a protein (Prx6, DJ-1, UCH-L1).
Figures 2a to 2d show nano UPLC-ESI-q-TOF tandem mass spectrometry, which confirms whether the compounds of the present invention react with the sulfhydryl groups of cysteine in the protein (Prx6, DJ-1, UCH-L1) The results of the mass spectrometry used.
Figures 3a and 3b are Streptavidin-HRP results confirming changes in labeling of NPSB-B for UCH-L1 wild type (WT) and UCH-L1 C90S mutations with pH change or H ₂ O ₂ concentration increase.
FIG. 4A is a result of Streptavidin-HRP confirming the change of NPSB-B labeling to Prx1 and Prx6 with increasing H ₂ O ₂ concentration. FIG. 4B is a graph showing a quantitative result of FIG. 4A. FIG.
FIG. 5A shows the labeling of Nm23-H1 wild type (WT), Nm23-H1 C4S mutation and Nm23-H2 according to H ₂ O ₂ concentration increase. FIG. 5B is a graph quantitatively showing the result of FIG. 5A. FIG. Figure 5c shows Streptavidin-HRP results confirming the change in labeling for Nm23-H1 wild type (WT), Nm23-Hl mutations (C4S, C109A, C145S) and Nm23-H2.
FIG. 6A is a heat map of the relative redox reactivity of a protein with changes in H ₂ O ₂ concentration. FIG. FIG. 6B shows the results of Streptavidin-HRP and the relative redox reactivity to confirm the change of labeling of NPSB-B against Hsp60, GAPDH and STAT1 with increasing H ₂ O ₂ concentration. FIG. 6C is a result of Streptavidin-HRP confirming the change of labeling of NPSB-B with respect to Nm23-H1 with increasing H ₂ O ₂ concentration. FIG. 6D shows the result of Streptavidin-HRP which confirms the change of labeling of NPSB-B with respect to GAPDH with increasing H ₂ O ₂ concentration.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples.

< Example  1> methyl  3-Nitro-4- (piperidin-l- IlSilfoil ) Benzoate ( NPSB -1)

[Reaction Scheme 1]

Methyl 4- (chlorosulfonyl) -3-nitrobenzoate (104 mg, 0.37 mmol) was dissolved in 3.6 mL of well-dried dichloromethane (DCM), and the solution was cooled to 0 ° C, and piperidine , 0.39 mmol) was added slowly. To this was added again pyridine (45 [mu] L, 0.56 mmol). Then the temperature was raised to room temperature and the mixture was stirred at room temperature for 5 hours. When the reaction was completed, 10 ml of a saturated ammonium chloride aqueous solution was added to terminate the reaction, and the reaction was terminated. The organic layer was extracted with DCM (4 mL each) for 3 times. The obtained organic layer was treated with anhydrous magnesium sulfate to remove moisture and sift out the precipitate. The resulting solution was distilled under reduced pressure, concentrated and purified by column chromatography to obtain 84 mg (0.27 mmol, 72%) of the desired compound.

^{1 H NMR (400 MHz, Chloroform} -d) δ 8.26 (dd, J = 8.2, 1.6Hz, 1H), 8.20 (s, J = 1.6Hz, 1H), 8.02 (d, J = 8.2Hz, 1H), 3.96 (s, 3H), 3.26-3.22 (m, 4H), 1.63 - 1.51 (m, 6H)

< Example  2> 3-Nitro-N- (2- (4- (2- Oxohexahydro -1H- Thieno [3,4-d] imidazole Yl) Butylamino ) Ethyl) -4- (piperidin-l- IlSilfoil ) Benzamide ( NPSB -B)

[Reaction Scheme 2]

Step 1: 3-Nitro-4- (piperidin-l- IlSilfoil ) Preparation of benzoic acid

(60 mg, 0.18 mmol) prepared in Example 1 was dissolved in dry THF (0.9 mL) under argon atmosphere, and then 5.0 N sodium hydroxide solution 0.1 mL. The temperature was then raised to room temperature and stirred for 30 minutes at room temperature. When the reaction was completed, the solution was neutralized with 1.0 N aqueous hydrogen chloride solution to adjust the pH to 4 to 5. The organic layer was extracted with ethyl acetate (3 times each with a total of 5 times), and the obtained magnesium layer was treated with anhydrous magnesium sulfate to remove moisture. The precipitate was filtered out, and the obtained solution was concentrated by distillation under reduced pressure and purified by column chromatography to obtain 56 mg (0.18 mmol, 99%) of the desired compound.

^{1 H NMR (300 MHz, Methanol} -d4) δ 8.30-8.15 (m, 1H), 8.15-8.04 (m, 1H), 8.01-7.87 (m, 1H), 3.28 (s, 4H), 1.70-1.45 ( m, 6H)

Step 2: 2,5- Dioxopyrrolidine -1-yl-3-nitro-4- (piperidin-1- IlSilfoil ) Benzo Manufacture of Eight

Nitro-4- (piperidin-1-ylsulfonyl) benzoic acid (25 mg, 0.08 mmol) prepared in Step 1 was dissolved in 0.8 mL of dry THF under argon atmosphere and then 1-hydroxypyrrolidin- , 5-dione (19 mg, 0.16 mmol) and DCC (19 μL, 0.12 mmol) were added and the mixture was stirred at room temperature for 14 hours. When the reaction was completed, the reaction mixture was filtered and washed with ethyl acetate. The filtrate was dried over anhydrous magnesium sulfate, and the precipitate was filtered. The resulting solution was concentrated by reduced pressure distillation and purified by column chromatography to obtain the desired compound (4 mg, 0.010 mmol, 10%).

^{1 H NMR (400 MHz, Chloroform} -d) δ 8.40 (dd, J = 8.3, 1.7Hz, 1H), 8.34 (dd, J = 1.7, 0.4Hz, 1H), 8.17 (dd, J = 8.3, 0.4Hz (M, 4H), 2.97 (s, 4H), 1.73 - 1.55 (m, 6H)

Step 3: 3-Nitro-N- (2- (4- (2- Oxohexahydro -1H- Cyano [3,4-d] imidazole 4-yl) Butylamino ) Ethyl) -4- (piperidin-l- IlSilfoil ) Benzamide  Produce

To a solution of the 2,5-dioxopyrrolidin-1-yl-3-nitro-4- (piperidin-1-ylsulfonyl) benzoate (1.6 mg, 0.004 mmol), and triethylamine (Et ₃ N; 4 μL, 0.028 mmol) was added thereto. Biotin ethylamine hydrobromide (2 mg, 0.005 mmol) was added thereto, followed by stirring at room temperature for one day. When the reaction was completed, the solvent was distilled off under reduced pressure. The residue was purified by column chromatography to obtain the desired compound (3 mg, 0.005 mmol, 97%).

^{1 H NMR (400 MHz, Chloroform} -d) δ 8.38 (t, J = 5.1Hz, 1H), 8.24-8.16 (m, 1H), 7.96 (d, J = 8.7Hz, 1H), 6.81 (t, J = 5.5Hz, 1H), 6.24 ( s, 1H), 5.35 (s, 1H), 5.27 (s, 2H), 4.47 (dd, J = 7.8, 4.8Hz, 1H), 4.27 (dt, J = 7.8, 3.0Hz, 1H), 3.69 (s , 4H), 3.50 (t, J = 5.0Hz, 2H), 3.36 (q, J = 5.2Hz, 2H), 3.26-3.18 (m, 5H), 3.09 (td, J = 7.4, 4.5Hz, 1H) , 2.87 (dd, J = 12.8, 4.9Hz, 1H), 2.69 (d, J = 12.8Hz, 1H), 2.17 (t, J = 7.4Hz, 2H), 1.73- 1.54 (m, 8H), 1.52 (tt, J = 6.8, 4.0 Hz, 2H), 1.38 (p, J = 7.5 Hz, 2H)

< Example  3> 3-Nitro-N- (16-oxo-20 - ((3R, 6S) -2- Oxohexahydro -1H- Thieno [3,4-d] imidazole Yl) -3,6,9,12- Tetraoxa -15- Azaikosil ) -4- (Piperidin-l- One &Lt; / RTI & Benzamide  Produce

[Reaction Scheme 3]

(28 mg, 0.09 mmol) was dissolved in dry DMF (0.9 mL) under argon atmosphere, diisopropylethylamine (62 L, 0.36 mmol) was added, and 5 Lt; / RTI &gt; PyBop (93 mg, 0.18 mmol) and HOBt (24 mg, 0.18 mmol) were added thereto, followed by stirring at room temperature for 5 minutes. Then, Biotin-PEG ₄ -NH ₂ (41 mg, 0.09 mmol) was added thereto, followed by stirring at room temperature for 30 hours. When the reaction was completed, the solvent was removed by distillation under reduced pressure, and the residue was purified by column chromatography to obtain 27 mg (0.036 mmol, 40%) of the desired compound.

^{1 H NMR (400 MHz, Chloroform} -d) δ 8.38 (t, J = 5.2Hz, 1H), 8.22-8.17 (m, 1H), 7.99-7.94 (m, 1H), 6.81 (t, J = 5.6Hz , 1H), 6.25 (s, 1H), 5.35 (s, 1H), 5.27 (s, 2H), 4.47 (dd, J = 7.8, 4.8Hz, 1H), 4.27 (dt, J = 7.8, 3.0Hz, 1H), 3.72-3.53 (m, 20H ), 3.50 (t, J = 5.0Hz, 2H), 3.36 (q, J = 5.2Hz, 2H), 3.25-3.18 (m, 5H), 3.09 (td, J = 7.4, 4.5Hz, 1H), 2.87 (dd, J = 12.8, 4.9Hz, 1H), 2.69 (d, J = 12.9Hz, 1H), 2.17 (t, J = 7.4Hz, 2H), 1.73-1.55 (m, 8H), 1.52 (tt, J = 6.8, 4.0 Hz, 2H), 1.43-1.33

< Example  4 > 1- (2,4- Dinitrophenylsulfonyl ) Preparation of piperidine

[Reaction Scheme 4]

After dissolving 2,4-dinitrobenzene-1-sulfonyl chloride (506 mg, 1.90 mmol) in 9.5 mL of well-dried dichloromethane (DCM) under argon, the solution was cooled to 0 ° C and piperidine (197 μL, 1.99 mmol) was added slowly. To this was added pyridine (230 [mu] L, 2.85 mmol). Then the temperature was raised to room temperature and the mixture was stirred at room temperature for 4 hours. When the reaction was completed, 10 ml of a saturated ammonium chloride aqueous solution was added to terminate the reaction, and the reaction was terminated. The organic layer was extracted with DCM (4 mL each) for 3 times. The obtained organic layer was treated with anhydrous magnesium sulfate to remove moisture and sift out the precipitate. The resulting solution was distilled under reduced pressure, concentrated, and purified by column chromatography to obtain 162 mg (0.51 mmol, 27%) of the desired compound.

^{1 H NMR (400 MHz, Chloroform} -d) δ 8.47 (dd, J = 8.6, 2.2Hz, 1H), 8.42 (d, J = 2.2Hz, 1H), 8.18 (d, J = 8.6Hz, 1H), (M, 4H), 1.70-1.61 (m, 4H), 1.53 (s, 2H)

< Example  5> 1- (3-Nitro-4- ( Ferritin -One- IlSilfoil ) Phenyl ) Ethanone  Produce

[Reaction Scheme 5]

Acetyl-2-nitrobenzene-1-sulfonyl chloride (213 mg, 0.81 mmol) was dissolved in 3.6 mL of well-dried DCM under argon, and then cooled to 0 ° C, piperidine (84 μL, 0.85 mmol) Slowly. To this was added pyridine (98 L, 1.21 mmol). Then the temperature was raised to room temperature and the mixture was stirred at room temperature for 8 hours. When the reaction was completed, 10 ml of a saturated ammonium chloride aqueous solution was added to terminate the reaction, and the reaction was terminated. The organic layer was extracted with DCM (4 mL each) for 3 times. The obtained organic layer was treated with anhydrous magnesium sulfate to remove moisture and sift out the precipitate. The resulting solution was distilled under reduced pressure, concentrated, and purified by column chromatography to obtain the title compound (67 mg, 0.21 mmol, 26%).

^{1 H NMR (400 MHz, Chloroform} -d) δ 8.15 (dd, J = 8.2, 1.7Hz, 1H), 8.08 (d, J = 1.6Hz, 1H), 8.03 (dd, J = 8.2, 0.3Hz, 1H ), 3.28-3.20 (m, 4H), 2.64 (s, 3H), 1.66-1.57 (m, 4H), 1.56-1.

< Example  6> 3-Nitro-4- (piperidin-1- IlSilfoil ) Benzamide  Produce

[Reaction Scheme 6]

Methyl 3-nitro-4- (piperidin-1-ylsulfonyl) benzoate (13.76 mg, 0.042 mol) prepared in Example 1 was dissolved in dry THF under argon atmosphere and 0.4 ml of ammonium hydroxide was added. The temperature was raised to 50 ° C and 0.2 ml of ammonium hydroxide solution was further added every 12 hours while stirring for 4 days. When the reaction was completed, 1 M aqueous solution of hydrogen chloride was added to adjust pH to 5. The reaction was terminated and extracted with ethyl acetate (EA) (total of 3 times in 4 mL portions). The obtained organic layer was treated with anhydrous magnesium sulfate to remove water and sieve the precipitate. The resulting solution was distilled under reduced pressure, concentrated, and purified by column chromatography to obtain the desired compound (4.3 mg, 0.014 mmol, 33%).

¹ H NMR (400 MHz, Chloroform-d) 8 8.07 (dd, J = 1.1, 0.7 Hz, 2H), 8.05 (t, J = 1.1 Hz, 1H), 3.33-3.27 , J = 5.7 Hz, 4H), 1.62 - 1.54 (m, 2H)

< Example  7> Identification of Selective Reactivity of Compounds of the Present Invention

NPSB-1 prepared in Example 1 was reacted with Cys-SH, Cys-SOH, and Cys-SO ₂ H as shown in the following Reaction Scheme 7.

[Reaction Scheme 7]

As a result, NPSB-1 selectively reacted with Cys-SH, while the sulfhydryl group did not react with the oxidized strain.

To further confirm this selective reactivity, NPSB-1 was reacted with thiophenol, benzenesulfinic acid, benzenesulfinic acid as shown in Scheme 8 below.

[Reaction Scheme 8]

As a result, NPSB-1 similarly reacted selectively with thiophenol, but not with benzene sulfexane or benzenesulfinic acid.

Meanwhile, in order to confirm the selectivity of IAA, which is a conventional cysteine probe, NPSB-1 was reacted with thiophenol, benzenesulfuric acid, and benzenesulfinic acid as shown in Reaction Scheme 9 below.

[Reaction Scheme 9]

As a result, unlike NPSB-1, IAA was found to react with both thiophenol, benzenesulfinic acid and benzenesulfinic acid. Therefore, it can be seen that IAA does not selectively react with the sulfhydryl group of cysteine.

< Example  8> Using the compounds of the present invention, detection of redox sensitive cysteine

In order to confirm the detection of the redox-sensitive cysteine of the compound of the present invention, Prx6, DJ-1 and UCH-L1 known as active oxygen target proteins containing redox-sensitive cysteine, NPSB-1 prepared in Example 1 and And reacted with NPSB-B prepared in Example 2.

Prx6 is one antioxidant protein as a sensor of the H ₂ O _2, it is known to be a Cys47 active site. As shown in FIG. 1A, it was confirmed that NPSB-B binds to Prx6, and a mass increase of 179.02 in Cys47 was confirmed as shown in FIG. 2A, indicating that NPSB-1 binds to Cys47.

DJ-1 is a neurodegenerative disease-related protein, and Cys106 is known to be an active site. As shown in FIG. 1B, NPSB-B was confirmed to bind with DJ-1, and mass increase of 179.02 was confirmed in Cys106 as shown in FIG. 2B, indicating that NPSB-1 binds to Cys106 have.

UCH-L1 plays an important role in the invasion and metastasis of cancer cells mediated by UCH-L1 hydrolytic enzyme activity, and Cys90 among Cys90 and Cys152 is known to be active cysteine. As shown in Fig. 1C, NPSB-B was found to bind with UCH-L1 and a mass increase of 179.02 was observed in both Cys90 and Cys152, as shown in Figures 2c and 2d, so that NPSB-1 binds .

Accordingly, the compound of the present invention can confirm the presence of active cysteine and can be found in its position.

< Example  9> Determine the difference in reactivity of each cysteine

Experiment 1. Comparison of the reactivity of several cysteines in one protein

The pKa of cysteine is 8.4, but the pKa of cysteine in the protein varies from 4 to 9 depending on the environment of the protein. Basic residues near the -SH of cysteine, the lower the pKa of the cysteine thiol -SH rate (-S ^-) and is ionized to have a high reactivity with the result ROS.

To determine the difference in the reactivity of each cysteine in the protein, wild type (WT) and C90S mutants of UCH-L1 were treated with various H ₂ O ₂ concentrations (pH 6.5 and 7.4).

As shown in Figure 3A, the UCH-L1 WT bound to NPSB-B at pH 6.5 and 7.4 and showed a higher intensity at pH 7.4 than pH 6.5, while the UCH-L1 C90S mutation was associated with NPSB-B at pH 7.4 And at pH 6.5 it showed lower strength than the wild type, indicating that it binds NPSB-B in Cys152 in the case of UCH-L1 C90S mutation. These results are consistent with previous studies in which Cys90 of UCH-L1 has a lower pKa and Cys152 has a higher pKa.

In addition, as shown in FIG. 3B, UCH-L1 WT showed a gradual decrease in labeling by NPSB-B, while UCH-L1 C90S mutation showed almost no decrease in labeling by NPSB-B. These results indicate that Cys90 of UCH-L1 is reactive and readily oxidized in response to oxidative stress, while Cys152 is reactive but not readily oxidized.

Experiment 2. Comparison of the reactivity of two or more proteins with cysteine

In order to compare the reactivity of cysteine in two or more proteins, the reactivity between 2-Cys Prx, Prx6, and 1-Cys Prx, Prx1, on the change of H ₂ O ₂ concentration was examined.

As shown in FIGS. 4A and 4B, it can be seen that Prx1-labeled NPSB-B is more readily reduced than Prx6-labeled NPSB-B for H ₂ O ₂ concentration changes. These results show that Prx1 and Prx6 have different reactivities to H ₂ O ₂ and Prx 1 is more reactive than Prx 6 and easily oxidized. These results are in agreement with the previous report that Prx6 is more slowly oxidized than Prx1 and that the compounds of the present invention are able to detect the difference in reactivity of each cysteine.

< Example  9> Identification of the control cysteine in the redox signal transduction pathway

It is necessary to detect regulatory Cys in the redox signal transduction pathway because regulatory cysteine plays an important role in influencing protein activity and structure depending on the redox state.

In order to confirm the specific reactivity of NPSB-1 with the regulatory cysteine residues in the redox signaling pathway, Nm23-H1 was used.

Cys4 of Nm23-H1 reacts with Cys145 to form a disulfide bond and changes its structure to easily expose Cys109 to the oxidizing environment. NDPK activity and tumor metastasis inhibitory activity of Nm23-H1 are inhibited by oxidation and glutathioneation of Cys109 residues, while NDPK activity of Nm23-H2 is not affected by oxidative stress (see FIGS. 5a and 5b). A notable difference between Nm23-H1 and Nm23-H2 is the presence or absence of Cys4.

To test the reactivity of the regulatory cysteine residues at Nm23-H1, Nm23-H1 cysteine mutations (C4S, C109A, C145S) and Nm23-H2 (without Cys4) were labeled at pH 7.4.

As shown in FIG. 5C, NPSB-B only labeled wild type (WT), C109A mutation, C145S mutation of Nm23-H1, Nm23-H1 C4S mutation and Nm23-H2 were not labeled. These results show that NPSB-B only captures Cys4 of Nm23-H1 and Cys4 is the most reactive Cys residue affecting stepwise oxidation. These results are consistent with previous studies in which Nm23-H1 is regulated by the formation of Cys4-Cys145 disulfide bonds and consequent oxidation at Cys109.

< Example  10> Protein screening using the compounds of the present invention

APSB-B represented by the following formula 4 wherein the nitro group of NPSB-B is substituted with an amino group can not bind cysteine because the electron-withdrawing group is substituted for the electron withdrawing group and is used as a control group of the compound of the present invention.

[Chemical Formula 4]

Except for the protein detected with APSB-B, a protein with an emPAI value of 1.5 times or more as compared to APSB-B was considered as a heat. Based on the largest emPAI value, the relative redox reactivity of the protein to the concentration change of H ₂ O ₂ was converted from 0 to 1 according to the emPAI value.

FIG. 6A is a heat map showing the relative redox reactivity of proteins according to the concentration of H ₂ O ₂ , and based on this, proteins can be classified into four types as shown in Table 1 below.

[Table 1]

Type 1 is strongly labeled with NPSB-1 and labeling decreases with increasing concentration of H ₂ O ₂ . Thus, Type 1 proteins have intrinsic redox-sensitive cysteine residues that are characterized by their ability to be readily oxidized by active oxygen. This type includes a number of proteins known to have redox regulation, such as NDKA / Nm23-H1, Prx1, PDIAI, Hsp60, CLIC1, and the like. Hsp60 was identified by immunoblotting in an independent pulldown experiment (see top of FIG. 6b), and Prx1 was found to be abruptly reduced in labeling with increasing H ₂ O ₂ concentration (see FIGS. 4a and 4b) ) And Nm23-H1 also have the same pattern (see Fig. 6C).

Type 2 has a reactive cysteine residue only when the concentration of active oxygen is low, and this type is fine-tuned by oxidative stress.

Type 3 increases labeling by NPSB-B as the concentration of H ₂ O ₂ increases. The cysteine residue of type 3 does not have reactivity in the reduced form, but has reactivity due to structural modification by oxidative stress. Proteasome subunits b1 and b3, GAPDH, PDIA3, PDIA6, and PARP1 were detected in this type. GAPDH was identified by immunoblotting in an independent pulldown experiment (see Figure 6b), and GAPDH was found to increase labeling with increasing H ₂ O ₂ concentration (see Figure 6d). These results indicate the possibility that these types of proteins, including GAPDH, may play a modulating role in oxidized form. Thus, type 3 proteins show that the responsive cysteine is dependent on oxidative stress.

Type 4 does not depend on the concentration of H ₂ O ₂ in reactivity. These proteins have reactive cysteine residues but are not oxidized by oxidative stress. This type includes STAT1, annexin A2, the GTP-binding nuclear protein Ran, and G6PD. GAPDH was confirmed by immunoblotting in an independent pulldown experiment (see bottom of FIG. 6B).

As described above, the compounds of the present invention can screen for oxidation-reduction reactivity of proteins, and thus it is possible to confirm whether or not the protein is a target protein of ROS-mediated diseases.

Claims (16)

A benzenesulfonamide compound represented by the following formula (1) or a biotinylated compound thereof:
[Chemical Formula 1]

In Formula 1,
R ¹ Or R ² are each hydrogen, C _{1 -6} straight or branched alkyl, C _{3 -7-cycloalkyl,} or C _{6 -10} aryl, (wherein the C _{3 -7-cycloalkyl} or hydrogen of C _{6 -10} aryl are halogen, may be substituted with -OH or C _{1 -4} alkyl), or R ¹ And R ² are connected to each other to form a (which may be substituted with a hydrogen of the heterocyclic group is halogen, -OH, or C _{1 -4} alkyl), 5-membered to 6-membered heterocyclic together with the nitrogen atom,
R ³ or R ⁴ are each -COR ⁵ , -CN, -SO ₃ H, -NO ₂ , -CF ₃ or -CCl ₃ ,
R ⁵ is hydrogen, C _{1 -4} alkyl, -OH, halogen, C _{1 -4} alkoxy, -NR ⁶ R ^7,
R ⁶ Or R ⁷ are each hydrogen or C _{1 -6} straight or branched alkyl. 2. The compound according to claim 1, wherein the heterocycle is a piperidine ring. The compound according to claim 1, wherein R ³ is -NO ₂ . The method of claim 1, wherein, R ⁴ is -COR ^5, or -NO _2, R ⁵ is C _{1 -4} alkyl, C _{1 -4} alkoxy or -NH ₂ compounds. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of:
1) methyl 3-nitro-4- (piperidin-1-ylsulfonyl) benzoate;
2) 1- (3-Nitro-4- (piperidin-1-ylsulfonyl) phenyl) ethanone;
3) 1- (2,4-dinitrophenylsulfonyl) piperidine; And
4) 3-Nitro-4- (piperidin-1-ylsulfonyl) benzamide.
5) Preparation of 3-nitro-N- (2- (4- (2-oxohexahydro-1H-thieno [3,4- d] imidazol- 1-ylsulfonyl) &lt; / RTI &gt;benzamide; And
6) 3-Nitro-N- (16-oxo-20 - ((3R, 6S) 6,9,12-tetraoxa-15-azaicol) -4- (piperidin-1-ylsulfonyl) benzamide. A process for preparing a compound of formula (I) according to any one of claims 1 to 5 or a biotinylated compound thereof, comprising the step of reacting a sulfonyl halide compound of formula (2) with an amine compound of formula Way:
(2)

(3)

Wherein R ¹ to R ⁷ are the same as defined in claim 1, and X is halogen. The method according to claim 6, wherein the biotinylation is performed by reacting a biotinylation reagent with a carboxylic acid compound represented by the following formula (I-1).
(I-1)

A composition for detecting sulfhydryl groups (-SH) of cysteine comprising a compound of formula (I) according to any one of claims 1 to 5 or a biotinylated compound thereof. A method for detecting a sulfhydryl group (-SH) of cysteine using the compound of formula (I) according to any one of claims 1 to 5 or a biotinylated compound thereof. 10. The method of claim 9,
(S1) contacting a protein with a compound of formula (I) or a biotinylated compound thereof, with varying pH or oxidative stress conditions; And
(S2) confirming labeling of the protein in each reaction;
&Lt; / RTI &gt; A composition for screening a target protein of an active oxygen-mediated disease, comprising a compound of formula (I) according to any one of claims 1 to 5 or a biotinylated compound thereof. 11. The composition of claim 10, wherein the ROS-mediated disease is selected from the group consisting of inflammatory diseases, degenerative brain diseases, and aging. 13. The composition of claim 12, wherein the inflammatory disease is cancer or diabetes. 13. The composition of claim 12, wherein said degenerative brain disease is Alzheimer's disease, Parkinson's disease or dementia. Use of a compound of formula (I) according to any one of claims 1 to 5 or a biotinylated compound thereof for screening a target protein of an active oxygen-mediated disease. 16. The method of claim 15,
(S1) contacting a protein with a compound of formula (I) or a biotinylated compound thereof, while varying oxidative stress conditions;
(S2) confirming labeling of the protein in each reaction; And
(S3) sorting the proteins according to a pattern of reactivity;
&Lt; / RTI &gt;

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