RU2380417C2 - Agent for inhibiting human 8-oxoguanine-dna-glycosylase enzyme - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention covers compound 6-amino-4-chloropyrazolo[3,4-d]pyrimidine. Offered compound is made of 2-amino-4,6-dichloropyrimidine by the two-staged method based on a common reaction of selective replacement of hydroxyl group with chlorine atom (chlorodehydroxylation) and replacement of hydrogen atom with carbonyl group followed with ring closure of the product with hydrazine. The declared compound provides a specific inhibiting action on human 8-oxoguanine-DNA-glycosylase enzyme (hOggl).

EFFECT: low-price and available compound, extended range of specific inhibitors of said enzyme and possibility for effective application in medicine.

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Description

The invention relates to molecular biology, biochemistry and biotechnology, and specifically to a tool for inhibiting the enzyme 8-oxoguanine-DNA-glycosylase human (hOggl), which is a 6-amino-4-chloropyrazolo [3,4-d] pyrimidine of the General formula (I) :

In the process of functioning, cellular DNA is exposed to various factors leading to its damage. One of the most aggressive environmental factors is reactive oxygen species that induce oxidative damage to DNA. The main product of the oxidative modification of purine DNA bases is 7,8-dihydro-8-oxoguanine (8-oxoguanine). Such damage to the genetic apparatus has a cytotoxic and mutagenic effect and can lead to the development of cardiovascular, neurodegenerative and oncological diseases. It has been shown that the level of 8-oxoguanine in DNA increases with diseases such as leukemia, Parkinson's disease, Alzheimer's disease, multiple sclerosis, hepatitis, breast cancer, lung cancer and many others (Cooke MS et al. FASEB J. 2003, 17, 1195 -1214; Evans MD et al. Mutat. Res. 2004, 567, 1-61).

Removal of 8-oxoguanine residues from human DNA is carried out by the enzyme 8-oxoguanine-DNA glycosylase (hOgg1) (Radicella J.P. et al. Proc. Natl. Acad. Sci. USA 1997, 94, 8010-8015). The hOggl enzyme is a bifunctional enzyme that cleaves the N-glycosidic bond of the damaged base to form the free 8-oxoguanine residue and apurin / apyrimidine site in DNA. Then it destroys the phosphodiester bond from the 3'-carbon atom of the 2'-deoxyribose residue by β-elimination (AP-lyase activity), forming a single-strand break in DNA (B. Karahalil et al. Nucleic Acids Res. 1998, 26, 1228 -1232). An alternative pathway for the breakdown of a potentially mutagenic apuric / apyrimidine site is through the interaction of DNA with Apel human AP endonuclease.

Inhibitors of DNA repair enzymes, in particular human 8-oxoguanine-DNA glycosylase (hOgg1), can be used in chemotherapy of oncological diseases, since a decrease in the body's repair resistance is necessary for more effective action of antitumor drugs.

Known inhibitors of DNA glycosylases (US Patent No. 006369237 B1, class C07D 207/00, op. 09.04.2002), which are a number of compounds with the General formula (II)

where B is a purine or pyrimidine nitrogenous base or its derivative;

X is O, N, S or CH ₂ ;

R ₁ is hydrogen or a protective amino group;

R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ - this, regardless of the environment, as far as valency and stability allows, hydrogen, nucleotide or oligonucleotide, phosphoryl, phosphonate, phosphoamidate, carbamate, phosphothioate, halogen, alkyl, alkenyl, alkynyl, carbonyl, thiocarbonyl, ketyl, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxide, sulfate, sulfonate;

p - may be 0-6.

These inhibitors are analogues of nucleosides modified at the ribose residue.

A disadvantage of the known DNA glycosylase inhibitors is that these compounds are difficult to access due to the complexity of the synthesis of derivatives of nucleosides bearing substituents on the ribose residue and their subsequent isolation from the reaction mixture.

An object of the invention is the creation of a highly specific agent for inhibiting human 8-oxoguanine-DNA glycosylase that does not contain a ribose residue in its composition.

The technical problem is solved by the proposed tool for inhibiting the enzyme 8-oxoguanine-DNA glycosylase of the person, which is 6-amino-4-chloropyrazolo [3,4-d] pyrimidine of the above general formula (I).

The proposed compound is an analog of heterocyclic bases of nucleic acids, namely purines, which are bicyclic nitrogen-containing molecules.

The proposed compound is obtained from 2-amino-4,6-dichloropyrimidin-5-aldehyde in two stages by a method based on the known reaction of selective replacement of a hydroxyl group with a chlorine atom (chlorodehydroxylation) and replacement of a hydrogen atom with a carbonyl group, followed by cyclization of the product with hydrazine.

The invention is illustrated by the following examples of specific performance.

Example 1

The proposed compound 6-amino-4-chloropyrazolo [3,4-d] pyrimidine is obtained in two stages. In step 1, the intermediate 2-amino-4,6-dichloropyrimidin-5-aldehyde is obtained.

To this end, 21 ml (0.138 mol) of dry dimethylformamide are added dropwise to VOCl ₃ (90 ml) cooled to 0 ° C with vigorous stirring over 20 minutes. The cooling bath was removed and 15 g (0.117 mol) were added in 1 g portions of dry 2-amino-4,6-dihydroxypyrimidine. The reaction mixture was heated to 100 ° C and stirred at this temperature for 4 hours. The solution was cooled to room temperature and poured into 0.6 L of H ₂ O and 400 g of ice. The aqueous solution was then heated to 50 ° C and stirred for 2 hours. The suspension was cooled (+ 6 ° C) overnight, the precipitate was filtered off and dried. Yield 16.0 g (71%).

In stage 2, the target compound 6-amino-4-chloropyrazolo [3,4-d] pyrimidine is obtained.

To a suspension of 2-amino-4,6-dichloropyrimidin-5-aldehyde (15.0 g, 78.5 mmol) in 270 ml of THF and 12.5 ml of triethylamine, 2.55 ml of hydrazine in 60 ml of H ₂ O are added dropwise with stirring over 25 minutes. The mixture was stirred for 1 hour. The solution was filtered and the filtrate was evaporated by 80% and 100 ml of H ₂ O were added. The precipitate formed was filtered off and dried in vacuo. After reprecipitation with water from a minimum amount of hot DMF (90 ° C), 11.2 g (85%) of 6-amino-6-chloropyrazolo [3,4-d] pyrimidine are obtained in the form of a yellow crystalline solid. Figure 1 presents the scheme for obtaining the claimed compounds.

NMR analysis data: (DMSO-D ₆ ) δ: H ¹ 8.48 (1H, s); C ¹³ 100.91 (C9), 131.14 (C8), 149.64 (C3), 153.92 (C4), 156.92 (C6).

Mass spectrometry data: calculated M = 169.0155, found M = 169.0335.

Example 2. Inhibition of the enzyme 8-oxoguanine DNA glycosylase hOggl (IC ₅₀ ) with various concentrations of the compound of the above general formula (I).

The determination of IC _{50 was} performed by quantifying the activity of the 8-oxoguanine DNA glycosylase hOggl enzyme in a sample that contains a buffer (50 mM Tris-HCl, (pH 7.5), 50 mM KCl, 1 mM dithiothreit, 1 mM EDTA, 9 % glycerol) enzyme (1 μM), AP substrate (2 μM) and the test compound of the present invention in different concentrations (0-500 μM) at 25 ° C.

Used ³² P-labeled substrate containing the AP site. For this, oligonucleotide d (CTCTCUCCTTCC) (0.1 μmol) was treated with uracil-DNA glycosylase (15 act units) (SibEnzyme, Russia) in 150 μl of buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitis, 0.1 mg / ml BSA, pH 8.0). The reaction mixture was kept for 14 hours at 37 ° C. Reverse phase chromatography on Nucleosil 100-10-C ₁₈ resin in 0.1 M TEA-Ac buffer solution, pH 7.0 and a linear gradient of 0-20% acetonitrile was used to isolate the reaction product containing the AP site. The introduction of the ³² P label at the 5'-end of the obtained oligonucleotide was carried out according to (Maniatis T. et al. Methods of genetic engineering. Molecular cloning. M .: Mir. 1984. S. 402). To do this, 30 pmol of the oligonucleotide in 30 μl of a buffer solution of 50 mM Tris-HCl, pH 7.6, 10 mM MgCl ₂ , 5 mM dithiothreit, 30 pmol [γ- ³² P] -ATP (specific act.3.3 μCi / pmol) was held for 60 min with 10-20 units. active polynucleotide kinase of phage T4 at 37 ° C. The reaction mixture was precipitated with a 10-fold volume of 2% LiClO ₄ in acetone. The labeled oligonucleotide was isolated by electrophoresis in denaturing 20% PAG, the radioactive band was detected by radio-autography and excised from the gel. Next, the oligonucleotide was transferred by electroelution to a DE-81 paper filter (Whatman, UK), from where it was washed off with a solution of 3 M LiClO ₄ heated to 56 ° C and precipitated with a 2% solution of LiClO ₄ in acetone. The precipitate was washed three times with 200 μl of acetone, dried in vacuo and dissolved in 100 μl of twice distilled water.

The reaction in the test sample (4 μl) was initiated by adding an enzyme solution to the sample mixture and after incubation for 10 min at 25 ° C, the reaction was stopped by adding 4 μl of a solution containing 7 M urea, 0.1% bromophenol blue and 0.1% xylencyanol .

The activity of the 8-oxoguanine DNA glycosylase hOggl enzyme was quantified by analysis of the reaction products by electrophoresis in a 20% polyacrylamide gel containing 7 M urea. Aliquots of 4 μl were applied to the pockets of the gel and separated at a voltage of 50 V / cm.

The gel was autographed on an Agfa CP-BU New X-ray film (Agfa-Geveart, Belgium) for 10-20 hours at a temperature of -10 ° С. The degree of substrate cleavage was determined by processing a digital autograph, previously digitized, in the Gel-Pro Analyzer 4.0 software package (Media Cybernetics, USA). The degree of cleavage was calculated as the ratio of the peak area corresponding to the reaction product to the sum of the peak areas of the product and the peak of the starting oligonucleotide.

The value of 1C ₅₀ represents the concentration of the test compound, which causes a 50% decrease in the activity of the enzyme 8-oxoguanine DNA glycosylase hOggl. The data obtained are shown in figure 2. The IC ₅₀ value is 12.5 μM.

Figure 2 shows that the described compound has a pronounced inhibitory effect on the enzyme 8-oxoguanine-DNA glycosylase hOggl at micromolar concentrations.

Thus, the proposed compound has a specific inhibitory effect on the enzyme 8-oxoguanine DNA glycosylase (hOggl) and, being a cheaper and more affordable compound, expands the arsenal of specific inhibitors of this enzyme and can be successfully used in medicine.

Claims (1)

The agent for inhibiting the enzyme 8-oxoguanine DNA-glycosylase of a person, which is 6-amino-4-chloropyrazolo [3,4-d] pyrimidine of the general formula:

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