JP3059479B2 - Novel antiviral agent and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] An object of the present invention is a novel antiviral agent represented by the general formula [I] and a method for producing the same.

Wherein X is a hydrogen atom or an amino group, and R is a hydrogen atom or a pharmaceutically acceptable ester-forming group, preferably an acetyl group, a propionyl group, a benzoyl group, or another acyl group up to C20. [Prior art] 2 ', 3'-dideoxypurine nucleosides having a hydroxyamino group (-NHOH) in the purine base as shown in the general formula [I], and their synthesis No report is known.

Also, no report on the antiviral effect is known.

[Problems to be solved by the invention]

Viruses such as HBV (hepatitis B virus), CMV (cytomegalovirus), and HSV (herpes simplex virus)
It can infect humans and sometimes cause serious illness. Influenza, which is a pandemic every year, is also a disease caused by viruses, and viral diseases are now a major social problem for modern people.

The most problematic of the viral diseases known to date is AIDS (acquired immunodeficiency syndrome) caused by HIV (AIDS virus).

AIDS says that the treatment method has not yet been established,
Due to its high mortality, it is now one of the most feared diseases of mankind.

Some HIV-infected patients include HBV, CMV and HHV-6.
There are known cases of co-infection with AIDS (a type of herpes virus that has been linked to the onset of AIDS). In this sense, AIDS is a serious disease.

Although a complete cure for AIDS is not yet known, it prevents AIDS in carrier people or slows disease progression.
Drugs that have a prolonged effect on the life of the patient are known.

AZT (Proc. Natl. Acad. Sci. USA 82 , 7096 (1985))
Is one of the anti-HIV agents showing such effects.

AZT is currently the only approved drug, has been used many around the world, bone marrow toxicity (N.Eng.J.Med. 317, 192 (198
7)) and other side effects are known. Furthermore, it has been shown that AZT-resistant strains of HIV emerge when AZT is used for a long time (Science 243 , 1731 (1989)).
The next anti-HIV agent to replace AZT is awaited.

An object of the present invention is to provide a new antiviral agent which overcomes the above-mentioned various drawbacks found in existing drugs, and a method for synthesizing the same.

[Means for solving the problem]

The present invention provides a compound represented by the general formula [I]: (Where X is a hydrogen atom or an amino group, R is a hydrogen atom,
Or an acetyl group, a propionyl group, a benzoyl group, or another acyl group up to C20. ) Are based on the finding that the novel nucleic acid derivative has an antiviral effect.

The present invention relates to 2 ′, 3 ′ represented by the above general formula [I].
The present invention relates to a novel antiviral agent comprising at least one of dideoxypurine nucleosides as an active ingredient, and a method for producing the same.

In the present invention, the purine derivative represented by the general formula [II] used as a starting material can be easily obtained by using a base exchange reaction with a microorganism [Japanese Patent Application No. 01-46].
No. 183 (JP-A-2-308797)].

If the immobilized microorganism is used, it can be obtained industrially at low cost [Japanese Patent Application No. 01-181885 (Japanese Unexamined Patent Application Publication No.
086)].

[Operation] Hereinafter, the novel antiviral agent of the present invention, namely, 6-
The antiviral action of hydroxyaminopurine-2 ', 3'-dideoxyribofuranosides will be described.

The antiviral effect of the novel antiviral agent of the present invention is:
It is a synergistic effect between the antagonistic effect and the DNA chain termination effect.

These effects indicate that the novel antiviral agent of the present invention
It is derived from having a special structure called 2 ', 3'-dideoxypurine ribofuranoside skeleton.

That is, the novel antiviral agent of the present invention has a structure
It is very similar to the nucleosides that are components of DNA (especially 2'-deoxyadenosine, 2'-deoxyguanosine, etc.).

For this reason, the affinity for the enzyme derived from virus is good, and the enzyme of virus is different from the nucleic acid (2'-deoxyadenosine, 2'-deoxyguanosine, etc.) which is the original substrate, and the novel antiviral agent of the present invention is used. Incorporation and, consequently, hinders the synthesis of virus DNA (antagonistic inhibition), indicating a first antiviral effect.

Further, the novel antiviral agent of the present invention has a structure in which no hydroxyl group is present at the 3'-position. Therefore, once incorporated into the viral DNA, the next nucleic acid cannot be ligated, and the viral DNA strand ends here.
Therefore, the virus cannot grow any further and will die.

In this manner, the novel antiviral agent of the present invention
Acts as a chain terminator and exhibits a second antiviral effect.

Fortunately, human enzymes are much more advanced than viral enzymes, so they do not suffer from competitive inhibition or chain termination in the synthesis of human DNA or RNA. That is, the antiviral agent of the present invention has very weak substrate specificity and does not interact with human enzymes.

Therefore, the novel antiviral agent of the present invention is useful as a medicine and a reagent for a dideoxy method experiment (lab manual genetic engineering) used in genetic engineering.

Currently, AZT has already been approved as an AIDS treatment,
The antiviral effect of the clinical drug ddI and the like is also attributed to the synergistic effect of antagonistic inhibition and DNA chain termination, but the novel antiviral agent of the present invention (ie, 6-hydroxyaminopurine-2 ', 3 As described below, '-dideoxyribofuranosides) have properties and advantages not possessed by conventionally known drugs (such as AZT and ddI).

Its properties are due to the fact that the novel antiviral agent of the present invention can be changed into four tautomers.

For simplicity, the general formula [I] (In the formula, X is a hydrogen atom or an amino group, and R is a hydrogen atom, or an acetyl group, a propionyl group, a benzoyl group, or another acyl group up to C20.) Of these, the case of 6-hydroxyaminopurine-2 ', 3'-dideoxyribofuranoside will be described as an example.

As shown below, 6-hydroxyaminopurine-2 ', 3'-dideoxyribofuranoside represented by the formula (1) has four types of isomers represented by the formulas (2) to (5). It can take the form of a body as a stable structure (tautomerism).

That is, the novel antiviral agent of the present invention can have the following three effects that are advantageous as an antiviral agent because of this property.

Increases affinity for virus-derived enzymes and enhances competitive inhibitory effects.

In other words, the same effect as the administration of four types of drugs can be expected with the administration of one type of drug.

Enzyme metabolic pathways are generally
e passway is basic.

For example, phosphorylation and metabolism of adenosine, inosine, and guanosine are performed by different enzymes. However, the novel antiviral agents of the present invention can be substrates for several enzymes due to their tautomeric effects.

That is, taking phosphorylation at the 5 'position as an example, it is phosphorylated by many enzymes and is more efficiently incorporated into viral DNA. As a result, the DNA chain termination effect can be enhanced.

Since the four tautomers change instantaneously, it is difficult to generate virus-resistant strains against this drug.

As described above, the novel antiviral agent of the present invention has a special structure of 6-hydroxyaminopurine in the molecule, so that it can quickly tautomerize, and has a low enzyme affinity for viruses and resistance to virus-resistant strains. It is an excellent antiviral agent with new properties.

The novel antiviral agent of the present invention has a partial structure
Because it has a 2 ', 3'-dideoxyribofuranoside skeleton, it is particularly effective against retroviruses such as HIV (AIDS virus) and RSV (avian sarcoma virus). However, other viruses such as hepatitis B virus and It also works effectively against herpes virus.

The 2 ', 3'-dideoxypurine nucleoside having a halogen at the 6-position, which is used as a starting material in the present invention, can be easily obtained by using a base exchange reaction with a microorganism as described above [Japanese Patent Application No. Hei 11 (1994)]. -46183 (JP-A-2-308797)]. For example, when 6-chloroguanine and dideoxyuridine are used as raw materials, when a base exchange reaction is performed using a microorganism (for example, Escherichia coli),
The reaction product 2 ', 3'-dideoxy-6-chloroguanosine is obtained in good yield. Further, by using an immobilized microorganism, it can be more easily obtained [Japanese Patent Application No. 01-181885 (Japanese Patent Application Laid-Open No. 3-47086)].

Using the 2 ′, 3′-dideoxypurine nucleoside having a halogen at the 6-position synthesized as described above, a suitable organic solvent (eg, ethanol) and monohydroxylamine, the novel antiviral agent of the present invention It can be easily synthesized. For example, when 6-chloro-2 ', 3'-dideoxypurine ribofuranoside is added to ethanol in which monohydroxylamine is dissolved, and stirred under reflux conditions for several hours, chlorine at the 6-position reacts with monohydroxylamine. The desired product, 6-hydroxyaminopurine-
2 ', 3'-Dideoxyribofuranoside is produced.
After completion of the reaction, the reaction solution is returned to room temperature and blown into ethyl ether in the same amount or more as the reaction solution to obtain a white precipitate of the desired product.

As described above, according to the synthesis method of the present invention,
The antiviral agent of the present invention can be synthesized from easily available raw materials by a simple technique.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are for illustrative purposes only, and are not intended to limit the scope of the present invention in any case.

(1) Production Example Production Example 1 6-hydroxyaminopurine-9-β-D-2 ′, 3′-
Synthesis of dideoxyribofuranoside In a 300 ml eggplant-shaped flask, 12.0 g (173 mmol) of monohydroxyamine hydrochloride and 11.2 g (20 mmo) of potassium hydroxide were added.
l) Add 200 ml of absolute ethanol and reflux for 2 hours. After cooling to room temperature, the precipitated KCl was removed by filtration, and the solution was transferred to another 300 ml eggplant-shaped flask. This was followed by 6-chloropurine-2 ', 3'-dideoxyribofuranoside 1.45.
g (5.7 mmol) is added and refluxed for 10 hours. Then return to room temperature and continue stirring for 2 hours.

After completion of the reaction, the ethanol was distilled off, and the residue was stirred in 300 ml of ethyl ether to obtain a white solid. This was collected by filtration under reduced pressure and subjected to column treatment (mobile solvent).
CHCl ₃ : MeOH = 9: 1), fractions with UV absorption are collected and concentrated to give colorless flaky crystals.
The crystals were dried under reduced pressure (50 ° C., 0.1 mmHg, 10 h) to obtain colorless flaky crystals of 6-hydroxyaminopurine-2 ′, 3′-dideoxyribofuranoside. 86% yield.

Colorless flaky crystal Melting point 112 ° C NMR spectrum (DMSO-d6); δ 1.66 to 2.60 (4H, m, C
2'and C3'-H) 3.15 to 3.80 (2H, m, C5'-H) 3.80 to 4.37 (1H, m, C4'-H) 6.11 (1H, t, C1'-H) 7.80 (1H, s , C8-H) 8.13 (1H , s, C2-H) 6.25~8.95 (3H, broad s, C5'-OH and C6-NHOH) MS spectrum (FAB-MS; m / z ) M + 1 252 (C 10 H ₁₃ O ₃ as N ₅ +1) Rf value (Silica Gel HF 254) 0.16 [ CHCl 3 / MeOH = 9/1] 0.44 [CHCl 3 MeOH = 4/1] production example 2 2-amino-6-hydroxy-aminopurine - 9-β-D
Synthesis of -2 ', 3'0 dideoxyribofuranoside In a 300 ml eggplant-shaped flask, 12.0 g (173 mmol) of monohydroxyamine hydrochloride and 11.2 g (20 mmo) of potassium hydroxide were added.
l) Add 200 ml of absolute ethanol and reflux for 2 hours. After cooling to room temperature, the precipitated KCl was removed by filtration, and the solution was transferred to another 300 ml eggplant-shaped flask. 6-chloropurine-2'-3'-dideoxyribofuranoside 2.
69 g (10 mmol) are added and the mixture is refluxed for 10 hours. Then return to room temperature and continue stirring for 2 hours. After the completion of the reaction, the reaction solution is blown into 300 ml of ethyl ether to obtain a white precipitate. This was collected by filtration under reduced pressure, washed well with ether, and dried under reduced pressure (50 ° C., 0.1 mmHg, 10 h) to give 2-amino-6-hydroxyaminopurine-white powder.
2 ', 3'-Dideoxyribofuranoside was obtained. Yield 91
%. White microcrystal Melting point 156 ° C NMR spectrum (DMSO-d6); δ 1.72 to 2.63 (4H, m, C
2'and C3'-H) 3.32 to 3.80 (2H, m, C5'-H) 3.80 to 4.30 (1H, m, C4'-H) 5.97 (1H, t, C1'-H) 6.63 (2H, s _{, C6-NH 2) 7.87 (} 1H, s, C8-H) 5.0~8.5 (3H, broad s, C5'-OH and C6-NHOH) MS spectrum (FAB-MS; m / z ) M + 1 267 (C 10 H ₁₄ O ₃ as N ₆ +1) Rf value (typically by Silica Gel HF 254) 0.09 [CHCl 3 / MeOH = 9/1] 0.32 [CHCl 3 MeOH = 4/1] (2) antiviral test the invention The antiviral data for the compounds are shown below. The compound numbers are indicated by the numbers of the above and the examples, which exemplify the production method in the examples of the present invention.

Test Example 1 The antiviral effect of the compound represented by the general formula [I] of the present invention was tested using Rous sarcoma virus (RSV).

Using primary cultured cells (Chich Embyo Fibrofast)
RSV infection was carried out for about 30 minutes. Then, a serially diluted sample was added, and after 4 to 7 days, the stage at which the transformation of the cells due to the RSV infection was controlled was determined by microscopy. The results are shown in Table 1.

As shown in Table 1, the compound of the present invention has an excellent antiviral effect, and has very good solubility in water, so that the compound of the present invention is effective as a drug for treating viral diseases such as AIDS and hepatitis B. Clearly there is.

[Effects of the Invention] As described above, according to the present invention, a novel compound having an excellent antiviral action is provided. For example, it is extremely effective as a drug for treating viral diseases such as AIDS, hepatitis B and herpes.

────────────────────────────────────────────────── ─── Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07D 473/16 C07D 473/34 361 A61K 31/52 C12Q 1/68 CAPLUS (STN) REGISTRY (STN)

Claims (12)

(57) [Claims] 1. A compound of the general formula [I] (In the formula, X is either a hydrogen atom or an amino group, and R is a hydrogen atom or any one of an acetyl group, a propionyl group, a benzoyl group and other acyl groups up to C20.) , 3'-dideoxypurine ribonucleosides. 2. The compound according to claim 1, which is 2-amino-6-hydroxyaminopurine-9-.beta.-D-2 ', 3'-dideoxyribofuranoside. 3. A 6-hydroxyaminopurine-9-β-D
The compound according to claim 1, which is -2 ', 3'-dideoxyribofuranoside. 4. A stable structure having four tautomeric forms represented by the following formulas (2) to (5):
A compound as described. 5. A compound of the general formula [II] (Where X is either a hydrogen atom or an amino group, Y is a halogen atom, R is a hydrogen atom or an acetyl, propionyl, benzoyl, other C2
Any of up to 0 acyl groups. A) producing a 2 ′, 3′-dideoxypurine ribonucleoside represented by the general formula [I] by reacting a purine compound represented by the formula (1) with monohydroxylamine in an organic solvent; Law. (In the formula, X is a hydrogen atom or an amino group, and R is a hydrogen atom or any one of an acetyl group, a propionyl group, a benzoyl group, and other acyl groups up to C20.) 6. An organic solvent used in the reaction according to claim 5, wherein the purine compound represented by the general formula [II] can be completely dissolved and an alcoholic solvent of methanol or ethanol which does not hinder the reaction. A method for producing 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I], which is used. 7. The reaction product according to claim 5, wherein the reaction product, 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I], is dissolved in liquid benzene or ethyl ether which is difficult to dissolve. A method for producing a nucleic acid derivative, comprising directly blowing a reaction solution after completion of the reaction to precipitate 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I]. 8. An antiviral agent comprising at least one of the 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I] according to claim 1 as an active ingredient. 9. An antiretroviral agent comprising at least one of the 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I] according to claim 1 as an active ingredient. 10. A therapeutic agent for acquired immunodeficiency syndrome (AIDS) comprising at least one of the 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I] according to claim 1 as an active ingredient. And prophylactic drugs. 11. An experimental method for the dideoxy method used for genetic engineering, comprising at least one of the 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I] according to claim 1 as an active ingredient. Medicine. 12. An experiment for the dideoxy method used for genetic engineering, comprising at least one of the 2 ', 3'-dideoxypurine nucleosides represented by the general formula [I] according to claim 1 as an active ingredient. reagent.