JPH0725682B2 - Antiprotozoal agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antiprotozoal agents. More specifically, the present invention relates to an antiprotozoal agent useful for prevention and treatment of diseases caused by parasitic pathogenic protozoa belonging to the genus Leishmania, the genus Trypanosoma, and the like.

Leishmania (Leishmania) and Trypanosoma (Tripanosoma) are diseases caused by parasitic pathogenic protozoa belonging to the genus Leishmania and the genus Trypanosoma, respectively, and are chronic infectious diseases that prevail in specific regions from the temperate zone to the tropical zone.

Parasitic protozoa belonging to the genus Leishmania include, for example, Leishmania donovani, a pathogenic protozoa of black fever (Kala-azar), and Oriental sor.
e) Pathogenic protozoa Leishmania tropica (Leishmania)
tropica) and the pathogenic protozoan of Espundia in South America Leishmania brasiliensis (Leishmania)
braziliensis) and Reishminia mexicana (L
eishmania mexicana) is known. Parasitic protozoa belonging to the genus Trypanosoma include, for example, Trypanosoma gambiensis (Trypanpsoma gambiensis), a pathogenic protozoan of African Trypanosomiasis.
mbiense), Trypano Soma Rhodesiens (Trypano)
somarhodesiense), and trypanosoma cruzi, the pathogenic protozoa of American trypanosomiasis.

Currently, drugs used as therapeutic agents for diseases caused by these parasitic pathogens include pentamidine (Pentamid).
ine), pentostane (Pentostane) or amphotericine B (amphotericine B), suramin (suramin), stilbamine isethonate (Stilbamin isethonate), triparsamide (Tryparsamide), etc. Are required to be hospitalized to receive and be treated with these drugs under the strict control of a doctor.

However, there are relatively many developing countries in the areas where Leishmaniasis and trypanosomiasis are endemic, and the lack of skilled medical personnel in these countries makes them more effective, has fewer side effects, and is easy to use. There is a strong demand for the development of a possible antiparasitic protozoal agent.

Nucleoside compounds are known as antiprotozoal agents. For example, the inventors have previously found that 3'-deoxyinosine is effective as an antiprotozoal agent (see Japanese Patent Application No. 60-184018). Also, 4'-fluoronucleosides have been proposed as antiprotozoal agents (Japanese Patent Application No. 48).
-22479). However, these do not always have sufficient drug efficacy, and more effective antiprotozoal agents are required. The present inventor has conducted extensive studies to develop an effective drug for treating a disease caused by a pathogenic protozoa, and as a result, specific 3′-deoxy-3′-fluoropurine nucleosides have been identified as leishmaniasis and trypanosomes. It was found that the protozoa of Alzheimer's disease show remarkable drug efficacy, and the present invention has been completed based on this finding.

That is, the present invention provides an antiprotozoal agent containing 3'-deoxy-3'-fluoroadenosine or 3'-deoxy-3'-fluoroinosine.

3'-deoxy-3'-fluoro purine nucleosides are compounds unknown in the literature. Details of this compound and a method for producing this compound are described in the prior application (Japanese Patent Application No. 60-220186) filed by the present applicant. The specific 3'-deoxy-3'-fluoropurine nucleosides in the present invention are compounds in which the purine residue is an adenine residue or a hypoxanthine residue. The outline of the 3'-deoxy-3'-fluoro purine nucleosides and the production method thereof described in the above-mentioned prior application will be described below, but the production method of the compound in the present invention is not necessarily limited thereto. .

3'-deoxy-3'-fluoro purine nucleosides are compounds represented by the following (I) (wherein R represents a purine residue).

In the compound of the present invention, R in the formula (I) is an adenine residue or a hypoxanthine residue. In this compound, the 1-position is a halogen atom, an alkoxy group, a hydroxyl group,
Alternatively, the above-mentioned 1 of 3-deoxy-3-fluoro-β-D-ribofuranoside derivative which is a group such as a protected hydroxyl group.
It is produced by introducing a purine residue at the position. In addition, the fluoro-β-D-ribofuranoside derivative is 1
By fluorinating the 3-position hydroxyl group of the β-D-xylofuranoside derivative in which the 2-position is a halogen atom, an alkoxy group, or a protected hydroxyl group and the 2- and 5-position hydroxyl groups are protected, with a fluorinating agent can get. If a leaving group is introduced into the 3-position hydroxyl group, fluorination is easy.
By fluorination, a fluorine atom is stereospecifically introduced at the position opposite to the hydroxyl group at the 3-position, and the hydroxyl group is eliminated. Β-D above
In the xylofuranoside derivative, only the hydroxyl group at the 3-position is not selectively protected, and all other hydroxyl groups must be protected. Since the reactivity of the hydroxyl group with respect to the protective agent is slightly different, the β-D-xylofuranoside derivative can be produced by utilizing this. 3-deoxy-3-
The flow of the above-mentioned production method of the fluoro-β-D-ribofuranoside derivative is shown below.

In the above, R ¹ represents a halogen atom, an alkoxy group, or a protected hydroxyl group. Particularly, a lower alkoxy group such as methoxy group is preferable. r ² represents an alkylidene group, and an isopropylidene group is particularly preferable. R ³ and
R ⁴ represents a hydroxyl-protecting group. For example, a tri (alkyl, aryl, or aralkyl) silyl group, an acyl group, an aralkyl group and the like are suitable. Particularly, R ³ is preferably an aralkyl group such as a benzyl group, and R ⁴ is preferably a trialkylsilyl group such as a t-butyldimethylsilyl group. Y represents a leaving group and the like, and examples thereof include a methanesulfonyl group, a trifluoromethanesulfonyl group, a p-toluenesulfonyl group, an acetyl group and the like, and a trifluoromethanesulfonyl group is particularly preferable. As the fluorinating agent, various fluorinating agents can be used, and among them, fluorinated tetra (alkyl or aralkyl) ammonium is preferable. Particularly, fluorinated tetraalkylammonium having a lower alkyl group is suitable, and fluorinated tetrabutylammonium is most preferable. A purine residue is introduced into the compound of the above (6) by a known method and then deprotected or a purine residue is introduced after deprotection to obtain the desired compound represented by the above formula (I). For example, the introduction of purine residues is described in the literature (Wright, Carbahydrate Reseach, 1
8 , 345 (1971)) can be used.

When the fluoronucleosides are administered to humans or other warm-blooded animals for the prophylaxis or treatment of diseases caused by the pathogens, by themselves, or by a commonly used method,
For example, a pharmaceutically acceptable carrier, excipient, diluent,
It can be orally or parenterally administered using a solubilizing agent and the like, for example, in the form of powder, granules, tablets, capsules, injections, suppositories and the like. The dose is symptom,
Depending on the nutritional status of the affected animal, the age, the route of administration of the drug, etc., for example, when administered orally to an adult suffering from Leishmaniasis or trypanosomiasis for treatment, about 1 to 5 times a day , About 5 to 100 mg / kg body weight per day as fluoronucleosides, and about 1 to 5 times a day for parenteral administration, about 1 to 40 mg / k per day
g body weight is preferred.

Next, details of the present invention will be described with reference to Synthesis Examples and Examples, but the present invention is not necessarily limited to these. In addition, in the synthesis example, 3'-deoxy-3'-fluoroadenosine and 3'-deoxy-3'-fluoroadenosine were prepared by producing compound (6) from compound (1) and then introducing a purine residue according to the above-mentioned production method.
This is an example of producing -deoxy-3'-fluoroinosine.

Synthesis Example Methyl 2-O-benzyl-3,5-O-isopropylidene-β-D-xylofuranoside [in the formula (2),
R ¹ is a methoxy group, R ³ is a benzyl group, and R ² is an isopropylidene group].

Methyl 3,5-O-isopropylidene-β-D-xylofuranoside 12.6 g (61.6 mmol) and silver oxide (15.0 g) N, N
-Add benzyl bromide (2
1.1 g) was added and the mixture was stirred at room temperature for 36 hours. The reaction solution was filtered, water was added, and the mixture was extracted with chloroform. The organic layer was washed with water, dried over magnesium sulfate, and concentrated. 12.6 g (yield 69%) of benzyl ether purified by column chromatography was obtained. ¹ H-NMR (CDCl ₃ ): δ1.38 (s, 6H), 3.41 (s, 3H), 3.8
−4.5 (m, 5H), 4.59 (s, 2H), 4.98 (s, 1H), 7.32 (s, 5
H).

Synthesis of methyl 2-O-benzyl-β-D-xylofuranoside [a compound of the formula (3), wherein R ¹ is a methoxy group and R ³ is a benzyl group].

Methyl 2-O-benzyl-3,5-O-isopropylidene-β-D-xylofuranoside 30.1 g (0.10 mol) was dissolved in acetic acid (60 ml) -water (24 ml) and reacted in a water bath at 50 ° C for 1 hour. Let Then, the low boiling point substances were distilled off while maintaining the water bath at 50 ° C. Purified by column chromatography, diol
20.9 g (yield 80%) was obtained.

Rf 0.40 (benzene-ethyl acetate = 1: 1).

Methyl 2-O-benzyl-5-Ot-butyldimethyl-β-D-xylofuranoside [in the formula (4),
R ¹ is a methoxy group, R ² is a t-butyldimethylsilyl group, R ³
Is a benzyl group].

20.9 g (82 mmol) of the diol synthesized in Synthesis Example was added to N, N-
It was dissolved in dimethylformamide (80 ml) and imidazole (16.8 g) was added. To this solution, 12.4 g of t-butyldimethylsilane chloride was added to N, N-dimethylformamide (60 ml).
Was added dropwise at 0 ° C. over 30 minutes. After stirring for 3 hours, post-treatment was carried out according to a conventional method. Purification by column chromatography gave 30.2 g of silyl ether (yield 100%). ¹ H-NMR (CDCl ₃ ): δ0.10 (s, 6H), 0.91 (s, 9H), 3.37
(S, 3H), 3.9-4.1 (m, 3H), 4.2-4.4 (m, 3H), 4.61
(S, 2H), 4.93 (s, 1H), 7.32 (s, 5H).

Methyl 2-O-benzyl-3-deoxy-3-fluoro-β-D-ribofuranoside [in the formula (6), R ¹
Is a methoxy group and R ³ is a benzyl group].

Methyl 2-O-benzyl-5-synthesized in the above synthesis example
11.4 g of 2.6-lutidine was added to a solution of 13.0 g (35.0 mmol) of Dt-butyldimethylsilyl-β-D-xylofuranoside in dichloromethane (80 ml), and the mixture was cooled to 0 ° C. Trifluoromethanesulfonic anhydride (20.0 g) was added dropwise over 15 minutes, and the reaction was continued for 30 minutes. After adding ice, post-treatment,
16.8 g of a crude product was taken out by short column chromatography.

This product was dissolved in tetrahydrofuran (60 ml), and 92 ml of a tetrahydrofuran solution (f = 1.0) of tetrabutylammonium fluoride was added dropwise at 0 ° C over 20 minutes. 0 ° C
After stirring for 24 hours at room temperature for 3 hours, tetrahydrofuran was distilled off, and the mixture was treated with saturated aqueous ammonium sulfate solution. Column chromatography purification was performed to obtain 5.4 g of the title fluorinated compound. ¹⁹ F-NMR (CDCl ₃ ): (CCl ₃ F standard) -207.1 (ddd, j = 5
3.7,22.0,13.4Hz), ¹ H-NMR (CDCl ₃ ): δ3.47 (s, 3H), 4.0-4.2 (m, 2H),
4.55 (s, 2H), 4.6-5.2 (m, 5H), 7.33 (s, 5H).

IR (CHCl ₃ ) 3300 cm ^-1 .

Synthesis of 3'-deoxy-3'-fluoroadenosine.

5.4g (21.1mm) of benzyl ether synthesized in the above synthesis example
ol) in 70 ml of ethanol and 5% -palladium black 5.
Hydrogenation was carried out at room temperature and normal pressure in the presence of 5 g. After 10 hours, the mixture was filtered through Celite 545 and concentrated.

The crude product was dissolved in 35 ml of pyridine and benzoyl chloride was added.
6.1 g was added and reacted at room temperature for 36 hours. After distilling off pyridine, the residue was purified by column chromatography to obtain 2.2 g of methyl 2.5-di-O-benzoyl-3-fluoro-β-D-ribofuranoside.
Obtained. ¹⁹ F-NMR (CDCl ₃ ): (CCl ₃ F standard) −211.6 (ddd, j = 5)
3.2, 18.1, 4.9Hz), 2.2g (5.9mmol) of this dibenzoyl compound is dissolved in acetic acid (0.4ml). A 30% -hydrogen bromide-acetic acid solution is added thereto, and the mixture is stirred at room temperature for 3 hours. After completely distilling off acetic acid and acetic anhydride,
It was dissolved in nitromethane (10 ml), added to a nitromethane solution (80 ml) containing 1.3 g of adenine monobenzoate, further added with 2 g of mercuric cyanide and heated under reflux for 1 hour. After the nitromethane was distilled off, the residue was washed with 30% aqueous potassium iodide solution and water and concentrated. It was roughly separated by a short column and used for the next reaction.

1.29 g of tribenzoyl compound was dissolved in methanol (38 ml), 1M-sodium methoxide-methanol solution was added thereto, and the mixture was heated under reflux for 1 hour. After distilling off methanol, water (40 ml) was added and the mixture was neutralized with a 2N-acetic acid aqueous solution. The aqueous layer was extracted with chloroform to remove organic substances and then concentrated.
Recrystallized from 99.5% -ethanol to give the final product, 3'-deoxy-3'-fluoroadenosine, 0.60 g.
Got ¹⁹ F-NMR (DMSO-d ₆ ): (CCl ₃ F standard) -197.8 (dt, 54.
4,22.1Hz), ¹ H-NMR (DMSO-d ₆ ): δ3.6-3.7 (m, 2H), 4.29 (dt, J
= 27.6, 3.7Kz, 1H), 4.8-5.0 (m, 1H), 5.09 (dd, J = 54.
4,4.2Hz, 1H), 5.69 (dd, J = 7.3,4.9Hz, 1H), 5.89 (d, J
= 6.3Hz, 1H), 5.93 (d, J = 8.1Hz, 1H), 7.39 (s, 2H), 8.
13 (s, 1H), 8.36 (s, 1H). ¹³ C-NMR (DMSO-d ₆ ): δ61.1 (d, J = 12.2 Hz, C-
5 '), 72.0 (d, J = 15.9 Hz, C-2'), 83.9 (D, J = 22.0
Hz, C-4 '), 86.9 (C-1'), 93.1 (d, J = 181.8Hz, C
-3 '), 119.4 (C-5), 140.1 (C-8), 149.11
(C-4), 152.4 (C-2), 156.2 (C-6).

IR (KBr tablet) 3300,1650 cm ^-1 .

Melting point 205.6 ° C.

Synthesis of 3'-deoxy-3'-fluoroinosine 3'-deoxy-3'-fluoroadenosine (900 mg) and adenosine deaminase (4.2
mg, manufactured by Sigma) was added to a 100 mM bicarbonate-triethylamine solution (90 ml, pH 7.1), and the reaction was carried out at 37 ° C for 1 hour. The reaction solution was evaporated to dryness under reduced pressure at 30 ° C., and then hot ethanol (300 ml) was added to dissolve it, and the insoluble matter was separated. The liquid was evaporated to dryness to obtain 750 mg of crystals of 3'-deoxy-3'-fluoroinosine.

λmax λmin UV absorption pH0 (in 1N-HCl) 251nm 221nm pH7 (65mm phosphate buffer) 248nm 223nm Example 1 Growth inhibition activity of 3'-deoxy-3'-fluoroadenosine against Leishmania tropica The assay was performed as follows. L.tropica
Dulbecco's Modified Eagle Medium (20% fetal bovine serum,
The cells were cultured at 25 ° C using 25 mM hepes buffer, containing hemin and hypoxanthine. L.tr at a cell density of 1 × 10 ⁵ cells / ml
3'-deoxy-3'-fluoroadenosine was added to 1 ml of a medium containing opica so that the final concentration was 114 x 10 ^-4 M to 3.6 x 10 ^-9 M (addition group), and 3'-deoxy-3 'was added. -The cells were cultured at 25 ° C for 72 hours together with a control group containing no fluoroadenosine, and the cell density was measured. Proliferation% was calculated by the following formula.

3'-deoxy- which inhibits growth by 50% compared to the control group
The molar concentration of 3'-fluoroadenosine EC ₅₀ is 3.2 × 10 ^-8 M
(Average of two experiments).

Example 2 The same test as in Example 1 was carried out by substituting 3'-deoxy-3'-fluoroinosine for 3'-deoxy-3'-fluoroadenosine in Example 1. Consequently, EC ₅₀ of 3'-deoxy-3'-fluoro inosine
It was 2.3 × 10 ^-7 M.

Example 3 Assay of the growth inhibitory activity of 3'-deoxy-3'-fluoroadenosine against Leishmania (L. donovani) was carried out. L. donovan instead of L. tropica in Example 1
i was cultured, and the EC ₅₀ of 3′-deoxy-3′-fluoroadenosine against L. donovani was measured in the same manner as in Example 1. As a result, the EC ₅₀ was 2.5 × 10 ^-7 M.

Example 4 In the same manner as in Example 3, the EC ₅₀ of 3′-deoxy-3′-fluoroinosine against L. donovani was measured. As a result, the EC ₅₀ was 1.0 × 10 ^-6 M.

Claims (1)

[Claims] 1. An antiprotozoal agent comprising 3'-deoxy-3'-fluoroadenosine or 3'-deoxy-3'-fluoroinosine.