RU2036928C1 - (1′r,5′r)-3′-aza-1′- (2-amino-1,6-dihydro-6- oxopurinyl-9)-3′-deoxy- 3′- (azidonaphthalene-1-sulfamido) -hexopyranosyl-6′-triphospha- te trilithium salts as specific photoactivated irreversible rna-polymerase inhibitors - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: product: (1′R,5′R)-3′-aza-1′- (2-amino-1,6-dihydro-6-oxopurinyl-9) -3′-deoxy- 3′- azidonaphthalene-1-sulfamido) -hexopyranosyl-6′-triphosphate trilithium salts, yield is 58-63%, empirical formula is C₂₀H₂₆Li₃N₁₀O₁₆P₃S·2H₂O. Reagent 1: 4- or 5-azidonaphthalene-1-sulfonylhydrazine. Reagent 2: oxoguanosine-5′-triphosphate sodium salt, in the presence of lithium-acetate buffer. Reaction condition: in dioxane medium, at pH 4.5-5. EFFECT: improved method of synthesis. 2 tbl

Description

O

где R₁ N₃ и R₂ Н (I) или R₁ Н и R₂ N₃ (II), как специфическим фотоактивируемым необратимым ингибиторам РНК-полимеразы.The invention relates to new chemical compounds, trilithium salts of (1 R, 5 R) -3-aza-1 - (2-amino-1,6-dihydro-6-oxo-purinyl-9) -az-3 deoxy-3 - ( azidonaphthalene-1-sufamido) hexopyranosyl-6-triphosphates of the general formula
H

O

where R ₁ N ₃ and R ₂ N (I) or R ₁ H and R ₂ N ₃ (II), as specific photoactivable irreversible RNA polymerase inhibitors.

 These compounds can be used in biochemistry, biophysics, and molecular biology to study the structure and mechanism of action of RNA polymerase.

 Closest to the described is the known compound (1 R, 5 R) -3-aza-1 - (2-amino-1,6-dihydro-6-oxopurinyl -9) -3-deoxy-3 - (2-hydroxybenzamido) hexopyranosyl-6 triphosphate, which has a specific inhibitory effect on RNA polymerase and has fluorescent properties (1).

However, this inhibitor has the following disadvantages:
insufficiently high inhibitory effect;
the absence of a reactive group in the molecule, which excludes the possibility of selective introduction into the active center of the enzyme of the fluorescent label forming a covalent bond with the protein, followed by separation from the excess of an unbound modifier, which significantly limits the scope of its application.

 The purpose of the invention is the creation of new compounds in a series of derivatives of 5-guanyl nucleotides, which have a more pronounced inhibitory effect on RNA polymerase and allow more efficient study of this enzyme using spectrofluorimetry.

 The goal is achieved by the trilithium salts of (1 R, 5 R) 3-aza-1 - (2-amino-1,6-dihydro-6-oxo-sopurinyl-9) -3 deoxy-3 - (azidonaphthalene-1-sulfamido) hexopyranosyl 6 -triphosphates of the above general formula as specific photoactivable irreversible RNA polymerase inhibitors.

Compounds I and II are obtained by a method based on the known cyclization reaction of products of the periodic oxidation of ribonucleotides with hydrazides of arenesulfonic acids in an aqueous organic medium at pH 4.5-5 [1]
The starting 4- and 5-azidonaphthalene-1-sulfonylhydrazines are prepared by a process based on the following known reactions:
substitution of the azide diazo group in the diazonium derivatives of the corresponding aminonaphthalene-1-sulfonic acids upon interaction with sodium azide [2]
chlorodehydroxylation of intermediate azidonaphthalene-1-sulfonic acids with phosphorus pentachloride [2]
the hydrazinolysis of arenesulfonyl chloride excess hydrazine [3]
Example 1. Trilithium salt of (1 R, 5 R) -3-aza-1 - (2-amino-1,6-dihydro-6-oxo-purinyl-9) -3 deoxy-3 - (4-azidonaphthalene-1-sulfamido) hexopyranosyl-6-triphosphate (I).

 To a solution of 64 mg (0.1 mmol) of sodium salt of oxo-guanosine-5-triphosphate, purified from impurity of sodium iodate, in 5 ml of 1 M lithium acetate buffer (pH 5.0), a solution of 34 mg is added with stirring and red light (0.13 mmol) of 4-azidonaphthalene-1-sulfonylhydrazine in 7 ml of dioxane. The resulting solution was stirred for 2 hours at room temperature, protected from light, then evaporated under reduced pressure to a volume of ≈0.5 ml and treated with 5 ml of a cooled mixture anhydrous ethanol, anhydrous diethyl ether (3: 1, vol.). The precipitate was separated by centrifugation, washed with anhydrous dioxane, then anhydrous diethyl ether and dried in vacuum in the presence of phosphoric anhydride. 50 mg of expected product is obtained (yield 63.5%).

 Physico-chemical properties of the obtained compounds are presented in table 1.

 PRI me R 2. Trilithium salt of (1 R, 5 R) -3-aza-1 - (2-amino-1,6-dihydro-6-oxo-purinyl-9) -3-deoxy- 3 - (5-azidonaphthalene-1-sulfonamido) hexopyranosyl-6-triphosphate (II).

 Under the conditions described in example 1, using 34 mg (0.13 mmol) of 5-azidonaphthalene-1-sulfonylhydrazine, 46 mg of the expected product are obtained (yield 58%).

 Physico-chemical properties of the obtained compounds are presented in table 1.

 The study of the inhibition of RNA polymerase by the described compounds was carried out as follows.

DNA-dependent RNA polymerase of phage T7 was isolated from the producer strain E. coli. The enzyme activity was determined by the formation of / ³² P / -RNA from / ³² P / -nucleoside triphosphates.

The incubation mixture for determining the activity contains 50 mm Tris-Hcl, pH 7.8; 10 mM MgCl ₂ ; 5 mM LiCl; 10 mM β-mercaptoethanol, solution of plasmid pGEM-2 ("Promega Biochem.", USA) 20-40 μg / ml; adenosine-5-triphosphate (ATP), uridine-5-triphosphate (UTP), cytidine-5-triphosphate (CTF) and guanosine-5-triphosphate (GTP) at 0.4 μmol / ml; / α - ³² P / -ATP or / α - ³² P / -UTP (200,000 cpm / sample); the enzyme is 0.2-0.5 μg in the sample. When determining the inhibitory effect, the test compound is introduced into the incubation medium under red light to obtain a solution of the desired concentration. The total sample volume is 25 μl. The reaction is carried out at 37 ^about C for 20 minutes The determination of the amount of formed RNA (acid-insoluble material) is carried out in a known manner.

The value of I _{50 is} determined from a graph of the dependence of enzyme activity on the concentration of inhibitor.

The used preparation of DNA-dependent RNA polymerase of phage T7 has an activity of 130,000-160000 units. and is characterized by the following values of K _m for nucleoside-5-triphosphates: K _{m GTP} 0.16 _mm ; K _{m ATP} 0.04 _mm ; K _{m CTF} 0.08 mM; K _{m utf} 0.06 _mm . The nucleoside triphosphate initiating the reaction is GTP.

Under these conditions (red light), the I ₅₀ value for compounds I and II was 0.35 and 0.18 mm, respectively. Inhibition was reversible.

 Under the action of light (λ320 ± 10 nm), the inhibitors are irreversibly bound to the enzyme as a result of the generation of a highly reactive nitrene radical. The free radical reaction mechanism makes possible the covalent interaction of inhibitors with virtually any amino acid residue in the protein binding site.

The effect of inhibition of RNA polymerase by the described compounds under photolysis conditions increases over time and does not decrease after they are removed from the preincubation medium (gel filtration), which indicates the irreversible binding of inhibitors to the enzyme. After gel filtration, the preparation of the enzyme modified by compounds I and II had pronounced fluorescence properties (λ _{max exc.} = 350 and 360 nm, λ _{max fluor.} 425 and 450 nm, respectively), which also indicates the irreversible binding of inhibitors I and II with RNA polymerase.

 With the joint preincubation of RNA polymerase with compounds I and II and 5 mM (substrate of RNA polymerase), a protective effect from the action of inhibitors is observed, which indicates their specific interaction with the active center of the enzyme.

 Inhibition of RNA polymerase by compounds I and II (under photolysis conditions), as well as the closest structural analogue of (1 R, 5 R) -3-aza-1 - (2-amino-1,6-dihydro-6-oxopurinyl-9 ) -3-deoxy-3 - (2-hydroxybenzamido) hexopyranosyl-6-triphosphate (III), characterized by the data given in table. 2. The inhibitory effect of compounds I, II and III on RNA polymerase.

When studying the change in fluorescence intensity of the covalent enzyme-inhibitor complexes as a result of alkaline hydrolysis (pH 12, 0 ° ^C, 48 h) morpholino residues fragment inhibitors I and II have been found to increase the fluorescence intensity ≈v 1.5.

 As follows from a comparison of the indicated data, the described compounds I and II have a significantly more pronounced irreversible inhibitory effect on RNA polymerase compared to the known compound III and expand the arsenal of specific irreversible enzyme inhibitors, allowing its study using spectrofluorimetry. The described compounds I and II can be used to localize GTP-binding centers in proteins.

Claims (1)

1. Trilithium salts of (1 'R, 5' R) -3'-aza-1 '- (2-amino-1,6-dihydro-6-oxopurinyl-6) -3'deoxy-3'- (azidonaphthalene - 1-sulfamido) - hexopyranosyl-6'-triphosphates of the general formula

where R ₁ N ₃ ; R ₂ H;
or R ₁ H;
R ₂ N ₃ ,
as specific photoactivated irreversible RNA polymerase inhibitors.

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