JP3042540B2 - Adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate, method for producing the same, and protein synthesis inhibitor comprising the compound - Google Patents

Description

The present invention relates to a novel adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate, a method for producing the trimer, and a protein synthesis inhibitor comprising the compound. Agent.

[Problems to be solved by conventional technology and invention]

It is widely known that interferon has a virus growth inhibitory effect, but studies in early living cell lines have suggested that this effect occurs at the mRNA translation stage. Subsequently, when double-stranded RNA (dsRNA) is added to the interferon-treated cell extract, ATP-dependent mRNA degradation (inhibition of mRNA translation) occurs, and low-molecular-weight protein synthesis inhibitors are produced depending on the presence of ATP. Was found to work. Further studies have shown that this protein synthesis inhibitor was biosynthesized by a 2-5A-dependent enzyme induced in cells by interferon treatment.
It was found to be a mixture of oligoadenylic acids having 2 ', 5' linkages and was named 2-5A.

This interferon treatment produces 2-5A,
In the process of inhibiting protein synthesis, an inactive 2-5A synthetase is first induced by interferon treatment, and this enzyme is activated in the presence of dsRNA to synthesize ATP-dependent 2-5A. The 2-5A activates an inactive endoribonuclease (RNaseaL) and degrades mRNA, resulting in inhibition of protein synthesis.

This 2-5A is an extremely unstable substance in cells.
This is because 2-5A is rapidly degraded by 2'-PDEase, which is a degrading enzyme. For example, in the case of trimer triphosphate (ppp5'A2'p5'A2'p5'A), mouse L cells The half-life in a cell line is only about 15 minutes, and there is a problem that RNaseL is degraded before a sufficient activity is exhibited. Therefore, many studies have been conducted to increase the resistance to 2'-PDEase. On the other hand, adenosine monophosphate trimer, which is a 2-5A analog, has high resistance to 2'-PDEase,
The ability to bind to aseL was not sufficient, and as a result, there was a problem that the ability to inhibit protein synthesis was inferior to that of the triphosphate trimer.

The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, the adenosine monophosphate-based trimer containing 8-hydroxyadenosine-5'-phosphate is a monophosphate-based trimer, while being a monophosphate-based trimer. The present inventors have found that they have the same protein rigidity inhibiting ability as the dimer, and that they have high resistance to 2'-PDEase, and have completed the present invention.

[Means for Solving the Problems] One of the adenosine monophosphate trimers containing 8-hydroxyadenosine-5'-phosphate of the present invention is represented by the general formula (a): 8-hydroxyadenyl- (2'-5 ')-represented by
8-hydroxyadenyl- (2'-5 ')-8-hydroxyadenosine-5'-monophosphate. Another of the adenosine monophosphate trimers containing 8-hydroxyadenosine-5'-phosphate of the present invention is:
General formula (b) Adenyl- (2'-5 ')-adenyl-
(2'-5 ')-8-hydroxyadenosine-5'-monophosphate.

Further, the present invention provides the following formula (c): Is reacted with imidazole in the presence of triphenylphosphine and dipyridyl disulfide to give the following formula (d): Is obtained, and then the 8-hydroxyadenosine-5'-phosphoroimidazolyte is converted to a pH = 6.8
Trimerization in the presence of a uranyl acetate catalyst in a buffer of ~ 7.0 and hydrolysis to give the 8
The gist of the present invention is a method for producing adenosine monophosphate trimer containing -hydroxyadenosine-5'-phosphate.
Also, the following formula (e), Adenyl- (2'-5 ')-adenosine represented by
5'-monophosphate is reacted with forphorin in the presence of triphenylphosphine and dipyridyl disulfide to give the following formula (f): To obtain adenyl- (2'-5 ')-adenosine-phosphoromorpholate represented by the formula:
(2'-5 ')-adenosine-phosphoromorpholinate and a compound represented by the general formula (d) And a phosphoroimidazolate derivative represented by the formula:
An adenosine monophosphate system containing 8-hydroxyadenosine-5'-phosphate represented by the general formula (b) by trimerizing in a buffer solution of 6.6 to 6.8 in the presence of a uranyl acetate catalyst and then hydrolyzing it. The gist is a method for producing a trimer.

Further, the present invention provides the following formula (a) Or the following formula (b) The gist is a protein synthesis inhibitor comprising an adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate represented by

In the production method of the present invention for obtaining a compound represented by the above formula (a), a compound represented by the formula (d) is obtained from a compound represented by the formula (c), and the compound (d) is converted into a uranyl acetate catalyst in a buffer solution. When the compound (d) is reacted in the presence of a compound (d), the phosphoroimidazolyte group of the compound (d) is bonded to the hydroxyl group at the 2'-position of another molecule to form a trimer. Next, this trimer is hydrolyzed, and the free phosphoroimidazolyte group moiety becomes a phosphate to obtain the compound (a) of the present invention. Further, in the production method of the present invention for obtaining a compound represented by the above formula (b), the adenyl-
(2'-5 ')-adenosine-5'-monophosphate is adenosine monophosphate represented by the following formula: Is reacted with imidazole in the presence of triphenylphosphine and dipyridyl disulfide to give the following formula Is obtained by reacting this adenosine 5'-phosphoroimidazolyte in a buffer having a pH of 6.8 to 7.0 in the presence of a uranyl acetate catalyst. Can be At this time, when the molar ratio of uranyl acetate catalyst / adenosine-5'-phosphoroimidazolyte is adjusted to 1 to 100, trimer and tetramer by-products are suppressed and the dimer is obtained (e). Is obtained.

The compound of the present invention (b) is obtained by reacting the compound of the formula (f) obtained from the compound (e) with the compound of the formula (d) in a buffer solution, followed by hydrolysis. be able to.

In order to hydrolyze the compound obtained by reacting the compound (d) with the compound (f), the pH is adjusted to 4.0 by adding a 10% aqueous acetic acid solution, a 10% aqueous hydrochloric acid solution, and the like. A time incubation method can be adopted.

As the buffer used in the method of the present invention, N-
Ethyl morpholine-acetate buffer can be used.
Also, a step of trimerizing compound (d) in the method for producing compound (a), or reacting a compound represented by compound (f) with a compound represented by (d) in a method for producing compound (b). When uranyl acetate used in the step is added in a molar ratio of uranyl acetate / compound (d) (or compound (d) + compound (f)) = 1/50 to 1/100, a tetramer, It is preferable because by-products such as pentamer are small. The reaction temperature of the trimerization reaction is preferably from 20 to 37 ° C, and the reaction time is preferably from 10 to 24 hours. After completion of the reaction, the compound of the present invention can be separated and recovered by HPLC after removing the catalyst, free imidazole and the like. The structure of the obtained compound of the present invention can be confirmed by ¹ H-NMR.

〔Example〕

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

Example 1 8-Hydroxyadenosine-5'-phosphoroimidazolyte (compound (d)) was produced by a conventional method, and 0.28 g of this compound (d) was dissolved in 0.5 mM uranyl acetate.
It was added to a 2M N-ethylmorpholine-acetate buffer (pH = 7.0) to a concentration of 50 mM and left at 22 ° C. for 24 hours.
After confirming the disappearance of the monomer by anion exchange-HPLC, 2.5 ml of cation exchange resin (Dowex 50W-X8, Na ⁺ type)
Was added to remove adsorbed uranyl ions, followed by filtration. After the filtrate was evaporated to dryness under reduced pressure, an aqueous solution of the residue was dropped into ethanol to cause reprecipitation. The precipitate was separated by centrifugation, washed with ethanol and diethyl ether, dried in a desiccator under reduced pressure, and then dissolved in 15 ml of 0.02M ammonium acetate (pH = 5.75). Add Nuclease to this solution
150 μl of a 2.5 mg / ml solution of P1 was added and incubated at 37 ° C. Twenty-four hours later, 20 ml of a solution of isoamyl alcohol: chloroform = 3: 7 was added, and the mixture was shaken vigorously.
After deactivating lease P1, the aqueous layer was washed with diethyl ether and concentrated under reduced pressure. Dissolve the residue in 200 ml of water,
Anion exchange resin column (Sephadex A-25, HCO ₃ type, 1.6
× 30 cm), and the compound (a) was separated by a linear gradient method using 0 to 0.8 M triethylamine-carbonate buffer (pH = 7.5) 2, and further purified by anion exchange-HPLC.

The obtained compound was dissolved in heavy water, and ¹ H-NM was added at 25 ° C.
An R measurement was performed. The obtained ¹ H-NMR spectrum is shown in FIG. As a result, three signals of the 2'-position proton appeared around 5.1 ppm to 5.4 ppm, and two of them were broad, indicating that the 2'-position hydroxyl group and the phosphate group were bonded. Is confirmed, and from the signal of the 1'-position proton appearing around 5.7 to 6.0 ppm and the three signals derived from the second-position proton appearing around 7.8 to 8.0 ppm, etc.
8-hydroxyadenyl- (2′-) represented by the formula (a)
5 ')-8-Hydroxyadenyl- (2'-5')-8
-Hydroxyadenosine-5'-monophosphate was confirmed (hereinafter, for convenience, this compound is referred to as the compound of Example 1). Example 2 Adenosine-5'-monophosphorimidazolyte was prepared by a conventional method. Then, 1.46 g of this compound was added to a 0.2 M N-ethylmorpholine-acetate buffer (pH = 7.0) in which 0.5 mM uranyl acetate was dissolved to be 4.0 mM, and the mixture was added at 22 ° C.
Left for 24 hours. After confirming the disappearance of the monomer by anion exchange-HPLC, the cation exchange resin (Dowex50W-
X8, Na ⁺ type) was added to remove 2.5 ml of adsorbed uranyl ion, followed by filtration, followed by adenyl- (2'-5 ')-adenosine-
5'-monophosphate (compound (e)) was obtained. 250 mg of this compound (e), 480 mg of triphenylphosphine, 200 mm of dipyridyl sulfide and 0.32 ml of forphorin were added to DMSO
Dissolved in a mixed solvent of 0.74 ml and 0.74 ml of DMF, then added 0.18 ml of triethylamine and reacted at room temperature for 12 hours,
Adenyl- (2'-5 ')-adenosine-5'-phosphoromorpholinate (compound (f)) was obtained. Next, 350 mg of a mixture of the compound (f) and the compound (d) obtained in the same manner as in Example 1 was dissolved in 0.5 mM uranyl acetate at a molar ratio of 4: 1. N-
It was added to an ethyl morpholine-acetate buffer (pH = 7.0) to a concentration of 50 mM and left at 22 ° C. for 24 hours. After confirming that the compound (f) and the compound (d) have disappeared by anion exchange-HPLC, the cation exchange resin (Dowex50W-X8, N
(a ⁺ type) 2.5 ml was added to remove adsorbed uranyl ion, followed by filtration. Next, acetic acid was added to an aqueous solution of the residue obtained after enzymatically treating the obtained compound in the same manner as in Example 1.
The pH was adjusted to 4.0 and incubated at 37 ° C for 10 hours.
After the residue aqueous solution was evaporated to dryness under reduced pressure, the residue was dissolved in 100 ml of water, and an anion exchange resin column (Sephadex A-25, HCO ₃ type,
1.6 × 30 cm), the compound (a) was separated by a linear gradient method using 0 to 0.8 M triethylamine-carbonate buffer (pH = 7.5) 2, and further anion exchange-HP
Purified by LC.

The obtained compound was dissolved in heavy water, and ¹ H-NM was added at 25 ° C.
An R measurement was performed. The obtained ¹ H-NMR spectrum is shown in FIG. As a result, three signals of the 2'-position proton appeared around 4.8 ppm to 5.2 ppm, and two of them were broad, indicating that the hydroxyl group and the phosphate group at the 2'-position were bonded. Was confirmed, and a signal of the 1'-position proton appearing around 5.6 to 6.2 ppm, two signals derived from the second-position proton appearing around 7.9 to 8.0 ppm, and 8.1
From the two signals derived from the proton at the 8-position appearing at about 8.2 ppm, the adenyl- (2 '
-5 ')-Adenyl- (2'-5')-8-hydroxyadenosine-5'-monophosphate was confirmed (hereinafter, this compound is referred to as the compound of Example 2 for convenience).

Example 3 The compound of Example 1, the compound of Example 2 and, as a comparative example, adenyl- (2'-5 ')-adenyl- (2'-
RNaseL ribosomal RNA cleavage test was performed on three compounds of 5 ')-adenosine-5'-triphosphate (hereinafter, this compound is referred to as a compound of a comparative example for convenience). First, the concentration dependence of the binding ability of each compound to RNaseL
Shown in the figure. In FIG. 1, ▲ indicates the compound of Example 1, ▼ indicates the compound of Example 2, and ● indicates the compound of Comparative Example. As a result, although the compound of Example 1 and the compound of Example 2 were monophosphate trimers, they had the same binding ability as the compound of the comparative example which was a triphosphate trimer. The IC _{50 of} each compound was 3.0 × 10 ⁻⁹ M for both the compound of Example 1 and the compound of Example 2, and 1.5 × 10 ⁻⁹ M for the compound of Comparative Example. Compound 2 had twice the value of the compound of Comparative Example.

4 A 0.2 unit / ml solution of SVPD (Snake Venom Phosphodiesterase) was adjusted with a 0.01 M Tris acetate buffer (pH = 8.8) containing 0.01 M magnesium chloride.
After adding μ to each compound (1.0 OD) and incubating in a thermostat at 37 ° C., the enzyme was inactivated at 90 ° C. for 30 minutes, and qualitative and quantified by anion exchange-HPLC.

As a result, it was found that the compound of Comparative Example was almost completely decomposed in about 10 minutes, whereas the compound of Example 1 and the compound of Example 2 were hardly decomposed in about 10 minutes.

〔The invention's effect〕

The adenosine monophosphate-based trimer containing 8-hydroxyadenosine-5'-phosphate according to the present invention is a novel compound, and according to the method of the present invention, a novel compound containing 8-hydroxyadenosine-5'-phosphate is used. It has the effect of reliably providing a novel adenosine monophosphate trimer. In addition, the protein synthesis inhibitor of the present invention has strong resistance to the decomposing action by a degrading enzyme and has an excellent protein synthesis inhibiting action, and is useful as an antiviral agent, an anticancer agent and the like.

[Brief description of the drawings]

The first figure is the ¹ H-NMR spectrum of the compound of Example 1, the chart Fig. 2 shows a ¹ H-NMR spectrum of the compound of Example 2, FIG. 3 embodiment, with respect RNaseL of the compound of Comparative Example It is a graph which shows the concentration dependency of a binding ability.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07H 21/02 A61K 31/7115 A61P 31/12, 35/00, 43/00 CA (STN) REGISTRY (STN )

Claims (5)

(57) [Claims] (1) The following formula (a): Adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate represented by the formula: 2. The following formula (b): Adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate represented by the formula: 3. The following formula (c): Is reacted with imidazole in the presence of triphenylphosphine and dipyridyl disulfide to give the following formula (d): Is obtained, and then the 8-hydroxyadenosine-5'-phosphoroimidazolyte is converted to a pH = 6.8
2. The method according to claim 1, wherein the compound is trimerized and hydrolyzed in the presence of a uranyl acetate catalyst in a buffer solution of -7.0.
A method for producing adenosine monophosphate trimers containing hydroxyadenosine-5'-phosphate. 4. The following formula (e): Adenyl- (2'-5 ')-adenosine represented by
5'-monophosphate is reacted with forphorin in the presence of triphenylphosphine and dipyridyl disulfide to give the following formula (f): To obtain adenyl- (2'-5 ')-adenosine-phosphoromorpholate represented by the formula:
(2'-5 ')-adenosine phosphoromorpholate and the following formula (d) And a phosphoroimidazolate derivative represented by the formula:
2. The process according to claim 1, wherein the trimerization is carried out in the presence of a uranyl acetate catalyst in a buffer solution of 6.6 to 6.8, followed by hydrolysis.
A method for producing an adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate according to the above. 5. The following formula (a): Or the following formula (b) A protein synthesis inhibitor comprising an adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate represented by the formula: