JPH04103597A - 8-hydroxyadenosine-5'-phosphate-containing adenosine monophosphate-based trimer, its production and protein synthesis inhibitor consisting of the same compound - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a novel adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate, a method for producing the same, and a method for inhibiting protein synthesis using the compound. Regarding drugs.

[Prior art and problems to be solved by the invention] It is widely known that interferon has a virus growth inhibitory effect, but in early research using living cell systems, it was found that this effect occurs at the mRNA translation stage. was suggested. Then, when double-stranded RNA (dsRNA) is added to the interferon-treated cell extract, ATP-dependent mRNA degradation (inhibition of mRNA translation) occurs, and a low-molecular-weight protein synthesis inhibitor is produced depending on the presence of ATP. It was found that Furthermore, subsequent research revealed that this protein synthesis inhibitor was biosynthesized by a 2-5A-dependent enzyme induced within cells by interferon treatment, and was a mixture of oligoadenylic acids with 2'5' bonds. was discovered and named 2-5A.

This interferon treatment produces 2-5A,
In the process of inhibiting protein synthesis, first, inactive 2-5A synthetase is induced by interferon treatment, and this enzyme is activated in the presence of dsRNA, resulting in AT
P-dependent 2-5A is synthesized. This 2-5A is an inactive endoribonuclease (RNase a
L) and degrades mRNA, resulting in inhibition of protein synthesis.

This 2-5A is an extremely unstable substance within cells.

This is 2'PDEase, in which 2-5A is a degrading enzyme.
For example, in the case of trimeric triphosphate (ppp5'A2'p5'A2'p5'A), the half-life in a mouse L cell-free system is only about 15 minutes; There was a problem in that it was degraded before sufficient activity against RNaseL was exerted. For this reason, many studies have been conducted to increase resistance to 2'-PDEase. On the other hand, 2
Adenosine monophosphate trimer, which is a -5A indigestion compound, has high resistance to 2' PDEase, but
There was a problem that the binding ability to aseL was insufficient, and as a result, the ability to inhibit protein synthesis was inferior to that of trisic acid trimer.

As a result of intensive research by the present inventors to solve the above problems,
Since the adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate is a monophosphate trimer, it has an ability to inhibit protein stiffness equivalent to that of the trisulfate trimer. The present invention was completed based on the discovery that it has high resistance to 2'-PDEase.

[Means for Solving the Problems] One of the adenosine monophosphate trimers containing 8-hydroxyadenosine-5'-phosphate of the present invention is 8-hydroxyadenosine represented by -Funenin (a) -(2'5')-
8-hydroxyadenyl-(2'-5')8-hydroxyadenosine-57-monophosphate. Another adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate of the present invention is -
Adenyl-(2'-5'), shown in Shipman (b)
-7te=ru(2'-51-8-hydroxyadenosine 5'-monophosphate).

The present invention also provides the following formula (d) by reacting 8-hydroxyadenosine-5'-monophosphate represented by the following formula (C) with imidazole in the presence of triphenylphosphine and dipyridyl disulfide. Adenyl-(2'-5')-adenosine-5
'-monophosphate is reacted with phorpholine in the presence of triphenylphosphine and dipyridyl disulfide to obtain 8-hydroxyadenosine-5'-phosphoroimidazolitate represented by the following formula (f); -Hydroxyadenosine-5'-phosphoroimidazolitate is trimerized in the presence of a uranyl acetate catalyst in a buffer solution of pH = 6.8 to 7.0, and hydrolyzed in the above (a). The gist of this invention is a method for producing the adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate. Adenyl-(2'-5')-adenosine-phosphoromorpholitate shown up to is obtained, and then this adenyl-(2'-5')-adenosine-phosphoromorpholitate and -Funenin (d) A phosphoroimidazolitate derivative represented by
= 6.6 to 6.8 in a buffer solution in the presence of a uranyl acetate catalyst, and then hydrolyzed to produce 8-hydroxyadenosyl 7:-5'- represented by the general formula (b).
The summary is a method for producing an adenosine monophosphate dimer containing phosphoric acid.

Furthermore, the present invention provides the following formula (a) It combines with the hydroxyl group at the 2' position to form a dimer.

This dimer is then hydrolyzed, and the free phosphoroimidazolitate group portion becomes a phosphate, yielding the compound (a) of the present invention. Furthermore, in the production method of the present invention for obtaining the compound represented by formula (b) above, adenyl-(2'-5')-adenosine-5'-monophosphate represented by formula (e) is adenosine represented by the following formula. The gist of the present invention is a protein synthesis inhibitor comprising an adenosine monophosphate dimer containing 8-hydroxyadenosine-5'-phosphate represented by monophosphate.

In the production method of the present invention for obtaining a compound represented by formula (a) above, a compound represented by formula (d) is obtained from a compound represented by formula (C), and this compound (d) is mixed with a uranyl acetate catalyst in a buffer solution. When reacted in the presence of , the phosphoroimidazolitate group of compound (d) reacts with imidazole in the presence of triphenylphosphine and dipyridyl disulfide of another molecule to form adenosine-5 as shown below.
'-phosphoroimidazolitate was obtained, and this adenosine-
5'-phosphoroimidazolitate at pH=6.8-7.
It is obtained by reacting in the presence of a uranyl acetate catalyst in a 0.0 buffer solution. At this time, the molar ratio of uranyl acetate catalyst/adenosine-5'-phosphoroimidazolitate is 1 to 1.
When it is set to 100, the by-product of dimers and tetramers is suppressed, and the compound shown by (e), which is a dimer, is obtained.

The compound (b) of the present invention is obtained by reacting the compound represented by formula (formula) obtained from the above compound (e) with the compound represented by the above formula (d) in a buffer solution, and then hydrolyzing it. I can do it.

To hydrolyze the compound obtained by reacting compound (d) and compound (f), adjust the pH to 4.0 by adding 10% aqueous acetic acid, 10% aqueous hydrochloric acid, etc., and heat at 37°C. 8
A method of incubating for ~14 hours can be adopted.

As the buffer used in the above method of the present invention, use N-ethylmorpholine-acetic acid buffer.

I can do it. Also, in the method for producing compound (a), there is a step of dimerizing compound (d), and in the method for producing compound et al., a step of reacting the compound represented by compound (f) with the compound represented by (d). When the uranyl acetate used in the above is added in a molar ratio of uranyl acetate/Compound (d) (or Compound (d) 10 compounds) = 1150 to 1/100, it forms tetramers, dimers, etc. This is preferable because it produces less by-products. In addition, the reaction temperature of the dimerization reaction is 20 to 3
The temperature is preferably 7°C, and the reaction time is preferably 10 to 24 hours. After the reaction is completed, the catalyst, free imidazole, etc. are removed, and the compound of the present invention can be further separated and recovered by HPLC. The structure of the obtained compound of the present invention is '
This can be confirmed by H-NMR.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 8-hydroxyadenosine-5' phosphoroimidazolitate (compound (d)) was produced by a conventional method, and 0.28 g of this compound (d) was dissolved in 0.5 mM uranyl acetate. Add to M's N-ethylmorpholine-acetate buffer (pH = 7.0) to a concentration of 50mM,
It was left at 22°C for 24 hours. Anion exchange - HPL
After confirming that the monomer has disappeared at step C, add cation exchange resin (Dowex 50W-X8.Na” type) 2.5
1! i! was added to adsorb and remove uranyl ions, followed by filtration. After drying the filtrate under reduced pressure, an aqueous solution of the residue was added dropwise to ethanol to cause reprecipitation. After separating the precipitate using a centrifuge, it was washed with ethanol and diethyl ether,
Dry under reduced pressure in a desiccator, then 0.02
M was dissolved in ammonium acetate (pH=5.75).

Add 2.5 ta of Nuclease P 1 to this solution.
150 μρ of g/d solution was added and incubated at 37°C. After 24 hours, a solution 201 mixed with isoamyl alcohol:chloroform=3=7 was added and shaken vigorously to deactivate Nuclease P 1, and then the aqueous layer was washed with diethyl ether and concentrated under reduced pressure. Dissolve the residue in 200 d of water and apply it to an anion exchange resin column (S
ephadex A-25, tICOs type, 1.6
Compound flyJ (a) was separated by linear gradient method using 0-0.8 M triethylamine-carbonate buffer (p)I = 7.5), and further purified by anion exchange-HPLC. did.

The obtained compound was dissolved in heavy water and heated at 25°C.
-NMR measurements were performed. The obtained ')l-NMR spectrum is shown in FIG. This result 5.1 ppffi~5
．． Three signals of protons at the 2' position appear around 4 ppm, and two of them are broad, confirming that the hydroxyl group and phosphate group at the 2' position are bonded. 5, 1 appearing around 7 to 6.0 ppm+
Signal of proton at position 7.8-8. From the three signals caused by the 2-position proton appearing near Oppm, 8-hydroxyadenyl-(
2'-5')-8-hydroxyadenyl (2'-5')
-8-hydroxyadenosine-5'-monophosphate (hereinafter, this compound will be referred to as
It is referred to as the compound of Example 1. ).

Example 2 Adenosine-5'-monophosphoroimidazolitate was produced by a conventional method, and 1.46 g of this compound was added to 0.5 m
M uranyl acetate was dissolved at 4.0 mM in 0.2 M N ethylmorpholine-acetate buffer (pH = 7.0).
The mixture was added so that

After confirming that the monomer has disappeared by anion exchange-HPLC, cation exchange resin (Dosmex 50W-X
8. After adsorbing and removing uranyl ions by adding Na'' type) 2.5+d, the mixture was filtered to obtain adenyl-(2'-5')-adenosine-5'-monophosphate (compound (e)). This compound ( e) 250 g, 480 g of triphenylphosphine, 200 g of dipyridyl sulfide and 0.32 d of phorforin, DMSoo, 74 d and DMFo, 7
4alt in a mixed solvent, then add 0.18d of triethylamine and react at room temperature for 12 hours to obtain adenyl-(2'-5,000-adenosine-5'-phosphoromorpholitate (compound)). Obtained. Next, the above compound (d) and the compound (d) obtained in the same manner as in Example 1 were mixed into 4:
350 as a mixture of both in a molar ratio of 1
■ 0.2M in which 0.5mM uranyl acetate was dissolved
of N-ethylmorpholine-acetate buffer (pf (=7.0
) at 22°C to a concentration of 50mM.
It was left for 4 hours. After confirming that compound (d) disappeared by anion exchange-HPLC, cation exchange resin (Dowex 50W-X8.Na'' type)2.
5Mfl was added to adsorb and remove uranyl ions, followed by filtration.

Next, the obtained compound was treated with an enzyme in the same manner as in Example 1, and acetic acid was added to an aqueous solution of the obtained residue to adjust the pH=
and incubated at 37°C for 10 hours. After drying the remaining aqueous solution under reduced pressure, the residue was dissolved in 100 Id of water.
and anion exchange resin column (Sephadex).
A-251HCOj type, 1.6
Compound (a) was separated by the linear gradient method using H=7.5) 242, and further anion exchange HPL
Purified by C.

The obtained compound was dissolved in heavy water and heated at 25 °C')
l-N? IR measurement was performed. The obtained 'H-NMR spectrum is shown in FIG. The result was 4.8 ppm to 5.
Around 2 ppm, three signals of protons at the 2' position appear, and two of them are broad, confirming that the hydroxyl group and phosphate group at the 2' position are bonded. 5, signal of proton at position 1 appearing around 6-6.2 ppm, 7.9-8. Two signals resulting from the 2nd proton appearing near Opp■ and 8.
From the two signals caused by the proton at position 8 appearing around 1 to 8.2 pp-, adenyl-(2'-5')-adenyl-(2'-5'L8- It was confirmed to be hydroxyadenosine-5'-monophosphate (hereinafter, for convenience, this compound will be referred to as the compound of Example 2).

Example 3 Compound of Example 1, compound of Example 2 and adenyl-(2'-5')-adenyl-(2'-5') as a comparative example
- Adenosine-5'-triphosphate (hereinafter, for convenience, this compound will be referred to as a compound of a comparative example). A cleavage test for RNase eL liposomal RNA was conducted on three compounds. FIG. 1 shows the concentration dependence of the ability of each compound to bind to RNaseL. In FIG. 1, M indicates the compound of Example 1, M indicates the compound of Example 2, and * indicates the compound of Comparative Example. As a result, the compound of Example 1, the compound of Example 2
Although the compound was a monophosphate trimer, it had a binding ability equivalent to that of the comparative example compound, which was a triphosphate trimer. Moreover, the IC5° of each compound is the compound of Example 1,
Both the compound of Example 2 has a value of 3.0X10-'M, the compound of Comparative Example has a value of 1.5X10-'M, and the compound of Example 1 and the compound of Example 2 have twice the value of the compound of Comparative Example. had.

Then, 5VPD
(Snake Venom Phosphodiesterase) 0.2un
An it/d solution was prepared, 100 μl of this solution was added to each compound (1,0 OD) and incubated in a constant temperature bath at 37°C, and then the enzyme was inactivated at 90°C for 30 minutes.
Qualitative and quantitative determinations were performed 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.

C Effects of the Invention] The adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate of the present invention is a new compound, and according to the method of the present invention, the above-mentioned 8-hydroxyadenosine-5'- It has the effect of reliably capturing a novel adenosine monophosphate trimer containing phosphoric acid.

Furthermore, the protein synthesis inhibitor of the present invention has strong resistance to decomposition by degrading enzymes and has an excellent protein synthesis inhibitory effect, and is useful as an antiviral agent, an anticancer agent, and the like.

[Brief explanation of drawings]

Figure 1 is a chart showing the 'H-NMR spectrum of the compound of Example 1, Figure 2 is a chart showing the 'I-NMR spectrum of the compound of Example 2, and Figure 3 is a chart showing the RNaseL spectrum of the compound of Example and Comparative Example. FIG. 2 is a graph showing the concentration dependence of the binding ability for Figure 1 8.0 7.0 5.0 4.0 3.0 2.0

Claims (1)

[Claims] 1) An adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate represented by the following formula (a): ▲Mathical formula, chemical formula, table, etc. ▼(a). 2) Adenosine monophosphate trimer containing 8-hydroxyadenosine-5'-phosphate represented by the following formula (b), ▲Mathematical formula, chemical formula, table, etc.▼(b). 3) 8-hydroxyadenosine-5'-monophosphate represented by the following formula (c), ▲Mathematical formula, chemical formula, table, etc.▼(c) is reacted with imidazole in the presence of triphenylphosphine and dipyridyl disulfide. Then, 8-hydroxyadenosine-5'-phosphoroimidazolitate represented by the following formula (d), ▲Mathematical formula, chemical formula, table, etc.▼(d) is obtained, and then this 8-hydroxyadenosine-5'- phosphoroimidazolitate, pH=6
．． The adenosine monophosphate containing 8-hydroxyadenosine-5'-phosphate according to claim 1, which is trimerized and hydrolyzed in the presence of a uranyl acetate catalyst in a 8-7.0 buffer solution. A method for producing a trimer. 4) Adenyl-(2'-5')-adenosine-5 shown by the following formula (e), ▲Mathematical formulas, chemical formulas, tables, etc.▼(e)
By reacting ′-monophosphate with phorpholine in the presence of triphenylphosphine and dipyridyl disulfide, adenyl-(2′- 5′)-adenosine-phosphoromorpholitate, and then this adenyl-(2
'-5')-Adenosine phosphoromorphoritate and the phosphoroimidazolitate derivative represented by the following formula (d) ▲Mathematical formula, chemical formula, table, etc.▼(d)
8-Hydroxyadenosine-5'-phosphate according to claim 1, characterized in that the 8-hydroxyadenosine-5'-phosphate according to claim 1 is trimerized in the presence of a uranyl acetate catalyst in a buffer solution of 6.6 to 6.8 and then hydrolyzed. A method for producing an adenosine monophosphate trimer comprising: (5) 8-hydroxyadenosine-5′ shown by the following formula (a) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (a) or the following formula (b) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (b) - A protein synthesis inhibitor consisting of an adenosine monophosphate trimer containing phosphoric acid.