KR20170082372A - Composition for regulating activity of Sirt6 - Google Patents

Abstract

The present invention provides a pharmaceutical composition for inhibiting the synthesis of anticancer and cholesterol, which comprises the compound represented by the above formula (1) as an active ingredient, which is excellent in the activity of increasing the activity of the Sirt6 enzyme. It can be used for treatment.

Description

Sirt6 < / RTI > (Composition for regulating activity of Sirt6)

The present invention relates to a compound that modulates Sirt6, a pharmaceutically acceptable salt thereof, and a composition for preventing or treating cancer and metabolic diseases containing the same as an active ingredient.

SIRT (sirtuin) is known to regulate lifespan from yeast to niacinamide amide (NAD) -dependent deacetylation activity in a variety of species.

The first SIR2 (Silent Information Regulator 2) gene was discovered in yeast in 1992, and it was found to play a role in increasing yeast life. Because of similarities with mammals such as humans who repeatedly divide somatic cells in the adult body, they have been studied continuously since they can be good models for human aging research. SIR2 was subsequently NAD-dependent protein deacetylase and one of the important substrates was found to be a histone protein.

SIRT, which is involved in mammals, is now identified from Sirt1 to Sirt7, and is involved in energy metabolism, cell survival, aging, inflammation, and gene stability.

sirtuins have been reported to play an important role in preventing diabetes, inflammation and degenerative diseases through regulation of metabolism, increased stress resistance, regulation of energy levels in the body, and prevention of DNA damage.

 When the caloric restriction, that is, the lack of food, changes in the intracellular metabolic pathway, the longevity gene is activated. The protein that is made by the expression of SIR2 gene is a histone deacetylase that breaks DNA double helix and removes the acetyl group of histone so that the double helix of DNA tightens and tightens gene expression.

 Sirt1 is the most studied of the mammalian sirtuin family. Several studies have reported that chromosome anomalies die in model mice that inhibit the expression of sirt1. Thus, sirt1 is involved in resistance to stress and in gene stability and metabolism. Like sirt1, Sirt2, first discovered in the cytoplasm, also performs multi-functions such as DNA modification, cell metabolism, and aging. sirt 3,4,5 exists in mitochondria and functions in energy metabolism. It plays a role in the TCA cycle that produces energy, plays a role in fatty acid oxidation, oxidative oxidation, and inhibits the production of toxic substances that are generated as the metabolism of tumor cells increases.

sirt7 is reported to cause cardiac hypertrophy and cardiomyopathic inflammation in insufficient rats, stabilizing cells and functioning in their lifespan.

sirt6 is also a sirtuin family of NAD + dependent deacetylases and is involved in metabolic regulation, inflammation, DNA stability, and senescence.

Sirt6 overexpressed rat models have been reported to have an increased lifespan, and inadequate genetic instability and metabolic defects shorten their lifespan. In addition, by binding to telomeric chromatin, deacetylation of histone H3 and lysine residue 9 is controlled by deacetylation. When sirt6 is deficient, the symptoms of werner sydrome due to telomeres result in premature aging.

 Over-expression of Sirt6 in 2012 has been shown to reduce obesity by reducing triglyceride, low-density lipoprotein cholesterol (LDL cholesterol) and inducing obesity in transgenic rats.

sirt6 activates PARP1, a mono-ADP-ribosylate, to protect and repair DNA against oxidative stress.

Therefore, it is very important to find new drug candidates exhibiting deacetylation activity of SIRT6 in the study of aging - related life phenomena, and these binding or activity analyzes are indispensable in the development of therapeutic agents for diseases.

 Brett Langley & Anthony Sauve., &Quot; Sirtuin Deacetylases as Therapeutic Targets in the Nervous System " Neurotherapeutics Vol. 10, pp. 605620, 2013.  Anna, C. et al., &Quot; SIRT6 protects human endothelial cells from DNA damage, telomere dysfunction, and senescence " Cardiovascular Research Vol. 97, pp. 571579, 2013.  Yariv, K. et al., &Quot; The sirtuin SIRT6 regulates lifespan in male mice " NATURE Vol. 483, pp. 218-221, 2012.

The present inventors aim to screen for new compounds having an excellent effect of increasing Sirt6 activity, and further to clarify EC50 values which can be referred to when conducting experiments using cells. The present invention provides a pharmaceutical composition for the prevention and treatment of cancer or metabolic diseases.

In order to accomplish the object of the present invention, there is provided a pharmaceutical composition for prevention and treatment of cancer or metabolic diseases comprising as an active ingredient a derivative represented by Chemical Formulas 1 and 2, a pharmaceutically acceptable salt thereof, .

From the various candidate substances, it was found that a new substance having an excellent effect of increasing the activity of Sirt6 was screened, and that Formulas 1 and 2 increased sirt6 enzyme activity and the EC50 value was measured to complete the invention.

 ≪ Formula 1 >

(2)

The present inventors have made extensive efforts to develop an effective therapeutic composition for various cancer or metabolic diseases caused by Sirt 6 by controlling Sirt 6 activity. As a result, it was confirmed that derivatives represented by the structures of Formulas 1 and 2 efficiently regulate the activity of Sirt 6 activity, thereby completing the present invention.

According to the present invention, the compounds of formulas (1) and (2) of the present invention significantly modulate the activity of Sirt 6. Accordingly, the compounds of the present invention can be usefully used for the treatment or prevention of cancer or metabolic diseases.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for the prevention or treatment of Sirt 6-related cancer and metabolic diseases comprising the compounds represented by formulas (1) and (2) as an active ingredient.

(b) The compounds of formulas (1) and (2) of the present invention can be effectively used for the prevention or treatment of cancer and metabolic diseases by effectively controlling the activity of Sirt 6.

1 and 2 show the results obtained by the following method.
The sirt6 activity compounds were selected using the cyclex Sirt6 deacetylase Fluotometic assay kit (cat. CY-1156) from Cyclex. Sirt6 deacetylates substrate acetylated lysine by treating sirt6 enzyme with fluorescence attached acetylated lysine substrate and lysylendopeptidase fluoresces by cutting C - terminal side peptide bond connected to lysine. At this time, activity is calculated by measuring the intensity of fluorescence emitted when the compounds are treated together. To increase the selectivity for sirt6, add Trichostatin A, a histone deacetylase (HDAC) enzyme that is not sirtuin. When the compounds are treated together, the compound exhibits a higher fluorescence value than the sirt6 enzyme. Therefore, the activity is calculated based on the untreated fluorescence value.
Figure 3 is a schematic representation of a method for measuring the activity.

Example 1: Preparation of 5-hydrazinyl-3- (3- (trifluoromethyl) phenyl) - [1,2,3] triazolo [1,5- a] quinazoline

Step 1: 2-azidobenzoic acid

Anthranilic acid (5 g, 36.5 mmol) was dissolved in 6N HCl (125 mL), cooled to -10 ^° C, and sodium nitrite (2.7 g, 39.0 mmol) was dissolved in 40 mL of H ₂ O . After stirring for 2 hours, an aqueous solution of sodium acetate trihydrate (147.0 g, 1.08 mol) and sodium azide (2.69 g, 39.0 mmol) dissolved in H ₂ O (220 mL) was slowly added at 0 ° C., stirred for 3 hours, And the mixture was stirred for 12 hours. After the completion of the reaction was confirmed, the reaction mixture was cooled to 0 ° C, 6N-HCl was added thereto to adjust the pH to 2 to 3, and the resulting white crystals were washed with water. The obtained crystals were dried and dried to give 2-azidobenzoic acid (4.8 g, 80%).

Step 2: 3- (3- (trifluoromethyl) phenyl) - [1,2,3] triazolo [1,5-a] quinazolin-5 (4H) -one

(1.2 g, 53.6 mmol) was dissolved in EtOH (35 mL), cooled to 0 ^° C, and 2- (3- (trifluoromethyl) phenyl) acetonitrile (3.1 mL, 24.5 mmol) was slowly added thereto and stirred for 30 minutes. A solution of 2-azidobenzoic acid (2.5 g, 15.3 mmol) in EtOH (100 mL) was slowly added to the reaction mixture and the mixture was refluxed for 12 hours. After the reaction was completed, the excess EtOH was concentrated under reduced pressure, extracted with CHCl ₃ and H ₂ O, the H ₂ O layer was adjusted to pH 2 with 2N HCl, filtered, and dried to obtain 3- (trifluoromethyl) - [1,2,3] triazolo [1,5-a] quinazolin-5 (4H) -one (3.5 g, 69%).

Step 3: 5-chloro-3- (3- (trifluoromethyl) phenyl) - [1,2,3] triazolo [1,5- a] quinazoline

5 (4H) -one (2 g, 6.06 mmol) was dissolved in TEA (42 mL, 303 mmol) at 0 [deg.] C, After cooling to 0 ° C, POCl ₃ (28.2 mL, 303 mmol) was slowly added and the mixture was stirred at 120 ° C for 14 hours. When the reaction was completed, the reaction solution was cooled, ice was added to terminate the reaction, diluted with CHCl ₃ , extracted with H ₂ O and saturated NaHCO ₃ , and dried over Na ₂ SO ₄ . The concentrate was crystallized to give 5-chloro-3- (3- (trifluoromethyl) phenyl) - [1,2,3] triazolo [1,5- a] quinazoline as a solid (1.8 g, 85%).

Step 4: 5-hydrazinyl-3- (3- (trifluoromethyl) phenyl) - [1,2,3] triazolo [1,5- a] quinazoline

1,5-a] quinazoline (500 mg, 1.43 mmol) was dissolved in DMF (14 mL), and hydrazine (0.32 g, mL, 10.04 mmol) and TEA (7.95 mL, 57.35 mmol) were added thereto, followed by reflux stirring for 14 hours. After confirming that the reaction was complete, it was diluted with MC, extracted with H ₂ O and dried with Na ₂ SO ₄ . The concentrate was crystallized to give 5-hydrazinyl-3- (3- (trifluoromethyl) phenyl) - [1,2,3] triazolo [1,5- a] quinazoline as a solid (150 mg, 38%).

Example 2: Preparation of 3- (4-methoxyphenyl) -N- (1-phenylethyl) - [1,2,3] triazolo [1,5- a] quinazolin-5-amine

Step 1: 3- (4-methoxyphenyl) - [1,2,3] triazolo [1,5-a] quinazolin-5 (4H) -one

Na (1.76 g, 76.4 mmol) was dissolved in EtOH (45 mL), cooled to 0 ^° C, and 2- (4-methoxyphenyl) acetonitrile (4.6 mL, 33.7 mmol) was slowly added thereto and stirred for 30 minutes. A solution of 2-azidobenzoic acid (3.5 g, 21.5 mmol) in EtOH (150 mL) was slowly added to the reaction mixture and the mixture was refluxed for 12 hours. After the reaction was completed, the excess EtOH was concentrated under reduced pressure, and extracted with CHCl ₃ and H ₂ O. The H ₂ O layer was adjusted to pH 2 with 2N HCl and filtered to obtain 3- (4-methoxyphenyl) - [ , 3] triazolo [1,5-a] quinazolin-5 (4H) -one (4.86 g, 78%).

Step 2: 5-chloro-3- (4-methoxyphenyl) - [1,2,3] triazolo [1,5- a] quinazoline

After dissolving 100 mg (0.34 mmol) of 3- (4-methoxyphenyl) - [1,2,3] triazolo [1,5-a] quinazolin-5 after cooling to 0 ℃, we were added to POCl ₃ (1.6mL, 16.8mmol) was slowly stirred under reflux for 12 hours. When the reaction was completed, the reaction solution was cooled, ice was added to terminate the reaction, diluted with CHCl ₃ , extracted with H ₂ O and saturated NaHCO ₃ , and dried over Na ₂ SO ₄ . The concentrate was crystallized to give 5-chloro-3- (4-methoxyphenyl) - [1,2,3] triazolo [1,5-a] quinazoline (75 mg) as a solid.

Step 3: 3- (4-methoxyphenyl) -N- (1-phenylethyl) - [1,2,3] triazolo [1,5- a] quinazolin-5-amine

(195 mg, 0.63 mmol) was dissolved in DMF (2 mL), and 1-phenylethan-1-ylmethyl- amine (0.53 mL, 4.18 mmol) and TEA (4 mL) were added thereto, followed by stirring at 120 ° C for 6 hours. After confirming that the reaction was complete, it was diluted with MC, extracted with H ₂ O and dried with Na ₂ SO ₄ . The concentrate was crystallized to give 3- (4-methoxyphenyl) -N- (1-phenylethyl) - [1,2,3] triazolo [1,5- a] quinazolin-5-amine (77 mg, 31% .

[Table 1]

[Example 3] Screening of agonists that increase Sirt6 activity

  1. In vitro assay for agonist screening

Sirt6 activity was measured using a cyclex Sirt6 deacetylase Fluotometic assay kit (Cat. CY-1156) from Cyclex.

Sirt6 deacetylates substrate acetylated lysine by treating sirt6 enzyme with fluorescence attached acetylated lysine substrate and lysylendopeptidase fluoresces by cutting C - terminal side peptide bond connected to lysine. When the compound is treated in the same manner as the fluorescence intensity measurement, the compound exhibits a higher fluorescence value than the sirt6 enzyme. Therefore, the activity is calculated based on the fluorescence value of the compound not treated.

Add 1ul of 10xsirt6 assay buffer, 0.2ul of 50x Fluoro-substrate peptide, 1xLysylendopeptidase, 1x TSA 1ul, and 3.3ul of H ₂ O. Add 6ul to 384 well by mixing the sample as many as the number of samples to be tested. No enzyme control group into more 1x NAD 1ul, H ₂ O 3ul as to the No test sample control group _{1x NAD 1ul, H 2 O 1ul} , recombinant Sirt6 enzyme 2ul insert No NAD control group H ₂ O 2ul to, recombinant Sirt6 enzyme Add 2ul.

 The 2,004 compounds of the KCCI dissolved in 5 mM of DMSO to be screened are diluted 1/1000 in PBS and prepared at 5 uM. Add 1ul of 1x NAD, 1ul of compound and 2ul of recombinant Sirt6 enzyme to 384 wells containing 6ul of mixture, and react at room temperature for 1 hour.

The fluorescence spectrometer excitation is measured at 490 ± 10 nm and the emission is measured at 530 ± 10 nm.

  2. Candidate Candidates

6,000 representative compounds were sold in Korea Chemical Bank.

  3. Screening of agents that increase the activity of Sirt6

The EC50 values of the compounds 1 and 2 among the compounds showing an activity of 200% or more out of 6000 species treated at a concentration of 5 uM were determined.

Cyclex Sirt6 deacetylase Fluotometic assay kit (Cat. CY-1156) was used. Sirt6 deacetylates substrate acetylated lysine by treating sirt6 enzyme with fluorescence attached acetylated lysine substrate and lysylendopeptidase fluoresces by cutting C - terminal side peptide bond connected to lysine. When the compound is treated in the same manner as the fluorescence intensity measurement, the compound exhibits a higher fluorescence value than the sirt6 enzyme. Therefore, the activity is calculated based on the fluorescence value of the compound not treated.

Add 1ul of 10xsirt6 assay buffer, 0.2ul of 50x Fluoro-substrate peptide, 1xLysylendopeptidase, 1x TSA 1ul, and 3.3ul of H ₂ O. Add 6ul to 384 well by mixing the sample as many as the number of samples to be tested. No enzyme control group into more 1x NAD 1ul, H ₂ O 3ul as to the No test sample control group _{1x NAD 1ul, H 2 O 1ul} , recombinant Sirt6 enzyme 2ul insert No NAD control group H ₂ O 2ul to, recombinant Sirt6 enzyme Add 2ul.

100uM, 10uM, 1uM, 100nM, 10nM, and 1nM concentrations of 10x for the concentration of 100uM, 10uM, 1uM, 100nM, 10nM, 1nM and 0.1nM of Fluvastatin (Sigma, cat, SML0038) Prepare to dissolve in PBS.

Add 1ul of 1x NAD, 1ul of compound and 2ul of recombinant Sirt6 enzyme to 384 wells containing 6ul of mixture, and react at room temperature for 1 hour.

The fluorescence spectrometer excitation is measured at 490 ± 10 nm and the emission is measured at 530 ± 10 nm.

 No test To calculate the value of the compound with the sample control group as 100

= (Value of compound / value of test sample in control group) * 100 to obtain% activity.

Calculate the% activity value using the Prism program.

[Example 4] Effect of compound

  1. Agents:

The EC50 value of the formula (1) is 5 uM.

The EC50 value of the formula (2) is 2.8 uM.

Claims (2)

1. A pharmaceutical composition for the prevention or treatment of metabolic diseases comprising a compound represented by the following formula 1 or 2, a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient.
≪ Formula 1 >

(2)

The method according to claim 1,
A pharmaceutical composition characterized by metabolic disease associated with Sirt6 activity.

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