JPH0477495A - Oxetanosine-3'-carboxylic acids and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (B is formula II to formula IV). USE:An antiviral agent. PREPARATION:Oxetanosines expressed by formula V are treated with a microorganism [e.g. Pseudomanas sp. NKO76161 strain (FERM P-11528), etc.] belonging to genus Pseudomonas or with a treated material thereof, preferably at 20-50 deg.C for 12-48hr.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to oxetanosine-3'-carboxylic acids that exhibit physiological activities such as antiviral activity.

(Prior Art) Nucleic acid derivatives are conventionally known as antiviral agents. Oxetanosine itself has also been published in the Journal of Antibiotics.
tics) Vol. 39, No. 11, 1623-25 (1986), describes Oxetanosin A in JP-A No. 61-293992.
Oxetanosine-H and G, 2-aminooxetanosine-G are disclosed in JP-A-1-100192.

(Problems to be Solved by the Invention) Conventional antiviral agents are accompanied by various side effects such as cytotoxicity. Antiviral agents with as few side effects as possible are desired.

(Means for Solving the Problems) As a result of various studies, the present inventors have developed oxetanosine-3°-carboxylic acids represented by the general formula (I) (wherein B means a group represented by !) and their The present invention was completed by discovering that a pharmacologically acceptable salt has an antiviral effect. General formula (I) of the present invention
Examples of the compounds are as follows.

Compound abbreviation B (name of base) in general formula (1) Thorium, potassium hydroxide, etc. are preferred.

Next, the compounds 0XT-AC, 0XT-HC, 0XT-GC
The manufacturing method will be briefly explained.

General formula (■) (where B is oxetanosine-3''-carboxylic acids of the present invention can be obtained by reacting oxetanosine with a microorganism belonging to the genus Pseudomonas.

The compound of general formula (1) of the present invention forms a salt with an acid or a base. The acid or base for forming a salt may be any pharmacologically acceptable acid or base.9 For example, the acid is hydrochloric acid, sulfuric acid, phosphoric acid, etc., and the base is hydroxylated. . ) Pseudomonas to the oxetanosines shown in
A microorganism that belongs to the genus and has the ability to oxidize the hydroxymethyl group at the 3°-position of the oxetane ring to a carboxyl group is used to react with the general formula (■) (in the formula, B means a group represented by (I)). Oxetanosine-3'-carboxylic acids represented by the formula are obtained.

In the present invention, a culture (bacterial body) obtained by culturing a microorganism belonging to the genus Pseudomonas and having the ability to convert the hydroxymethyl group at the 3'-position of the oxetane ring into a carboxyl group in a nutrient medium can be used as is, Processed products such as acetone-dried microbial cells, crushed microbial cells, products treated with surfactants, zozyme, etc., and microbial cells immobilized on natural or synthetic polymers can be similarly used.

Specifically, Pseudomonas sp.N, which was isolated as a microorganism from soil in Shiojiri City, Nagano Prefecture in July 1989,
KO76161 strain [FER
MP-11528)] is used.

Hereinafter, Pseudomonas sp, NKO
The mycological properties of strain 76161 are shown.

1. Morphological characteristics■Cell shape: Rod-shaped cell size = 0.5-0.7χ1.4-1.9μm■
μm property: None ■Motility: Yes, polar flagella ■Spores: None ■Durham staining: Negative ■Acid-fastness: None 2. Growth status on each medium (276C, cultured for 1 to 7 days and observed by standard method. ) ■ Broth agar plate culture: Growth is good, with circular or irregular periphery as the colonies multiply, and the colonies have a dull luster and a milky white color, and no soluble pigment is observed. It also emits an aromatic odor.

■ Broth agar slant culture: Growth is good. ■ Broth liquid culture: Growth is uniform throughout, but the upper part is slightly better, and as the culture time progresses, bacterial cells accumulate at the bottom of the tube.

■Meat juice gelatin puncture culture: The upper part liquefies in a layer.

■ Lidomus milk: unchanged (top blue) 3, physiological properties ■ Nitrate reduction: negative ■ Denitrification reaction: negative ■ MR test: negative ■ VP test: negative ■ Indole production: negative ■ Hydrogen sulfide production: negative ■ Starch Hydrolysis of: Negative ■ Use of citric acid Koser's medium: Positive Christensen's medium: Positive Simmons' medium: Positive ■ Use of inorganic nitrogen sources Ammonium bromide, ammonium sulfate, ammonium chloride, potassium nitrate and sodium nitrate are used.

[Phase] Pigment production King A: Negative King B: Negative (water-soluble fluorescent yellow) ■ Amino acid decomposition Arginine: Negative Lysine: Negative Ornithine: Negative ■ Urease: Negative ■ Oxidase: Positive ■ Catalase: Positive [Phase] Growth range Temperature = 4-37°C Optimum temperature: 24-27°C pH: 5-IO Optimal pH: 6-8 [Phase] Attitude towards oxygen: Aerobic @ 0-F test Dioxide [Phase] Sodium chloride Resistance of 5%: +7%: [Phase] Formation of acids and gases in response to various sugars The presence or absence of the production of acids and gases in response to various sugars is as shown in Table 1.

Based on the findings in Table M1 and above, I referred to Purge AIDS Manual of Determinative Bacteriology 8th edition and Purge AIDS Manual of Systematic Bacteriology Volume 1, and found that Pseudomonas.

This strain was identified as Pseudomonas sp., NK0761.
It was named 61.

Next, the method for producing the compound of general formula (I) of the present invention will be explained in more detail.

Belongs to the genus Pseudomonas and has a 3” oxetane ring.
Microorganisms that have the ability to oxidize hydroxymethyl groups to carboxyl groups at 25-35°C in a nutrient medium
After culturing for 248 to 72 hours, live bacterial cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7), and the compound of general formula (U) was added to this suspension.
20-50℃.

By reacting for 12 to 48 hours, the desired compound of general formula (I) is produced in the reaction solution. The product can be collected from the reaction solution using known methods, such as removing bacterial cells by centrifugation and utilizing the difference in solubility in water or organic solvents, or using activated carbon, adsorption resin, or ion exchange resin to absorb the product. Appropriate combinations of desorption methods and the like can be used.

(Test Example) Anti-HIV (Human I
Approximately 100,000 MT-4 cells/ml were placed in a 24-well tray, and 100 μl of a solution containing a certain amount of the compound of the present invention was added at 37°C in a 5% (V/V) carbon dioxide gas chamber. After culturing for 2 hours, HIV 103-104 infectious units were added, and after culturing for 4 days, a portion of the culture solution was smeared on a slide glass and fixed with acetone, and the expression of viral antigen was observed using a fluorescent antibody method. Note that serum from an AIDS patient was used as the primary antibody for the fluorescent antibody method, and human IgG labeled with FITC was used as the secondary antibody. Further, the cytopathic effect of the compound of the present invention on MT-4 cells was carried out without adding virus and observed under a microscope.

Table 2 Activity of compounds of the present invention against HIV *Cytotoxicity: indicates no toxicity.

As is clear from Table 2, the compounds of the present invention exhibit a growth inhibiting effect on HIV and cause very little cell degeneration, and are therefore expected to be used as anti-AIDS therapeutic agents.

(Example-1) Manufacturing method of compound 0XT-AC Peptone 2
%, powdered yeast extract 1%, meat extract 1%, NaC10
, 500 ml of 100 ml of pH 7.0 medium containing 5%
After dispensing the mixture into Erlenmeyer flasks, the mixture was sterilized by autoclaving at 120°C for 120 minutes. Pseudo in this flask
One loopful of Monas sp, NKO76161 was inoculated and cultured aerobically with shaking at 30°C for 224 hours. Separately, 100 ml of a medium having the same composition was dispensed into a 500 ml Erlenmeyer flask, and then sterilized by autoclaving at 120° C. for 20 minutes. This flask was inoculated with 1 ml of the above culture solution and cultured with shaking at 30°C for 160 to 70 hours. Next, 5L of this culture solution was centrifuged at 10,000 rpm for 10 minutes to collect viable bacterial cells, and 0.85% saline (pH 7.0) was added to the solution.
After washing twice with 0ml, add 50mM phosphate buffer (pH 7).
,0) suspended in IL. 50ml each of the two suspensions 5
Dispense into 20 Erlenmeyer flasks each with a volume of 0.
20 mg of XT-A was added and shaken at 35°C for 24 hours. Next, the pH of this reaction solution was adjusted to 5°0 with 3N hydrochloric acid, and then centrifuged at 100 rpm for 10 minutes to eliminate bacteria.
．． 0XT-AC and unreacted 0XT-A are adsorbed. In this reaction, a small amount of 0XT-H and 0X
T-HC is produced as a by-product, but since it is not adsorbed by the strongly acidic ion exchange resin, it can be easily separated and removed by washing the column with water. Adsorbed 0XT-AC and 0XT-
A was eluted using 0.5N ammonia water, the resulting eluate was concentrated under reduced pressure to remove ammonia, and then a weakly basic ion exchange resin (WA30.CH type, 1190-300μ
) Pass the liquid through a column packed with 100 ml to adsorb 0XT-AC. Since OXT-A is not adsorbed by the weakly basic ion exchange resin, it can be easily separated and removed by washing the column with water. The adsorbed 0XT-AC was eluted using an IN ammonium chloride solution, and the resulting eluate was passed through a column packed with 50 ml of activated carbon powder (for Wako Tsumugi chromatography) to adsorb 0XT-AC and washed with water. After 0. Desalination is carried out by passing IN hydrochloric acid through the solution, followed by washing with water. The adsorbed and desalted OXT-AC was eluted using 50% acetone-water, and the resulting eluate was concentrated to dryness to obtain 235 mg of OXT-AC crude powder. This crude powder was dissolved in distilled water, concentrated, cooled and recrystallized, and the resulting crystals were filtered and dried to obtain 220 mg of colorless crystalline OXT-AC (e ratio 55.0%).

FAB-MS; 266 (M+H)”Uv;λ□g
259 nm (H2O, pl(='7.0)'H
-NMR (200MHz, DMSO) δppm; 3.
70 (2H, d, 2”-cH2), 3.98 (18,
m, 2'-CI (), 4.90 (LH, d, 3'-C
) I), 5.30 (IH, 2”-0H), 6.48
(IH, d, 1'-CH), 7°40 (2H, brs
, 6-NH2), 8.17(IH,s,2-CH),
8.48 (IH, s, 8-CH), 13.2 (1
M, s, 3”-COOH) (Example-2) Compound 0
Pseu was produced in the same manner as in XT-HC manufacturing method Example-1.
domonas sp.

NKO76161 was cultured and a bacterial cell suspension IL was prepared from 5 L of the culture solution in the same manner. This suspension was dispensed into 20 Erlenmeyer flasks each having a volume of 500 ml, and 20 mg of 0XT-H was added to each flask and shaken at 35° C. for 24 hours. Next, this reaction solution was adjusted to pH 5.0 with 3N hydrochloric acid, centrifuged at 10,000 rpm for 10 minutes to eliminate bacteria, and the resulting supernatant was mixed with a weakly basic ion exchange resin (WA30°C1 0XT-HC is adsorbed through a column filled with 100 ml of 0XT-HC (1190-300.cz). No response 0
Since XT-H is not adsorbed by the weakly basic ion exchange resin, it can be easily separated and removed by washing the column with water. The adsorbed 0X THC was eluted with IN asiamonium chloride solution, and the resulting eluate was passed through a column packed with 50 ml of activated carbon powder (Wako Pure Chemical, for chromatography).
After adsorbing C and washing with water, 0. Desalination is carried out by passing IN hydrochloric acid through the solution, followed by washing with water. The adsorbed and desalted OXT-HC was eluted using 50% acetone-water, and the resulting eluate was concentrated to dryness to obtain 120 mg of OXT-HC crude powder.

This crude powder was dissolved in distilled water, concentrated, cooled and recrystallized, and the resulting crystals were filtered and dried to obtain 105 mg of colorless crystalline 0XT-HC (yield 26.3%).

FAB-MS; 267 (M1H)+UV;λwn
ax 248 nm (H2O, pH=7.0) 1H
-NMR(200MB2.DMSO)δppm;
3.71 (2H2d, 2”-CH2), 3.89
(IH+ m, 2'-CH), 4.91 (IH9d
, 3'CH), 5.33 (IH, 2''-0H), 6
．． 46 (IH, d, 1'-CH), 8゜09 (IH,
d, 2-CH), 8.48 (IH, s, 8-CH
), 12.45 (IH, s, 1-NH), 13.2
(IH,s,3”-COOH) (Example-3) Compound 0
Pseu was produced in the same manner as in XT-GC manufacturing method Example-1.
domonas sp.

NKO76161 was cultured and a bacterial cell suspension IL was prepared from 5 L of the culture solution in the same manner. This suspension was dispensed into 20 Erlenmeyer flasks each having a volume of 500 ml, and 20 mg of 0XT-G was added to each flask, followed by shaking at 35° C. for 24 hours. Next, this reaction solution was adjusted to pH 5.0 with 3N hydrochloric acid, centrifuged at 10,000 rpm for 10 minutes to eliminate bacteria, and the resulting supernatant was collected using a weakly basic ion exchange resin (WA3).
The solution is passed through a column filled with 80 ml of 0C1 type, 1190-300 μ) to adsorb 0XT-GC. Unreacted 0XT
Since -G is not adsorbed by the weakly basic ion exchange resin, it can be easily separated and removed by washing the column with water. The adsorbed 0XT-GC was eluted with IN ammonium chloride solution, and the resulting eluate was passed through a column packed with 40'ml of activated carbon powder (Wako Pure Chemical, for chromatography) to obtain 0XT-GC.
After adsorbing GC and washing with water, 0°IN hydrochloric acid was passed through it to desalt it, followed by washing with water. Adsorbed and desalted 0XT-GC
was eluted using 50% acetone-water, and the resulting eluate was concentrated to dryness to obtain 139 mg of 0XT-GC crude powder.
(yield: 69.5%).

FAB-MS; 2 8 2 (M+H)”U
V; λt=ax 254 nm (H2O,p
H4,0)'H-NMR (200MB2.DMSO)
δppm; 3.40 (182m, 2'-CH),
3.65 (2H9d, 2”-CH2), 4.65
(I H1d+ 3'-CH), 5.30 (IH, 2
”-0H), 6.21 (IH, d, 1°-CH),
6°72 (2H, brs, 2-NH2), 8.29
(IH, s, 8-CH), 10.60 (IH, s,
1-NH), 13.2(IH,s, 3”-CO
OH) Patent applicant Nippon Kayaku Co., Ltd.

Claims (2)

[Claims] (1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, B is ▲There are mathematical formulas, chemical formulas, tables, etc.▼▲There are mathematical formulas, chemical formulas, tables, etc.▼ ▲Mathematical formulas, chemical formulas Oxetanosine-3'-carboxylic acids and pharmacologically acceptable salts represented by ▼ means the group represented by . (2) General formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (In the formula, B is ▲There are mathematical formulas, chemical formulas, tables, etc.▼▲There are mathematical formulas, chemical formulas, tables, etc.▼ ▲Mathematical formulas, chemical formulas , tables, etc. ▼ means the group shown) Pseudomonas
A method for producing oxetanosine-3'-carboxylic acids, which comprises converting them into oxetanosine-3'-carboxylic acids represented by the general formula (I) by reacting with a microorganism belonging to the genus or a treated product thereof.