JPS6281387A - Novel compound om-3223 and production thereof - Google Patents

Abstract

NEW MATERIAL:Compound OM-3223 shown by the formula or its salt having the following physical and chemical properties. White needle-like crystal. Elemental analysis(%): C 50.77, H 5.65, N 19.87. Molecular formula: C12H16N4O4. Molecular weight: 280 (mass spectrum, m/Z). Specific rotatory power: [alpha]D=-31.6 deg. (C=1.0, H2O). Color reaction: positive in sulfuric acid and KMnO4 and negative in ninhydrin, FeCl3, etc. USE:An adenosine deaminase inhibitor. PREPARATION:A microorganism such as Streptomyces sp.OM-3223 (FERM P-8447), etc., is cultivated in a medium and a compound shown by the formula is formed and accumulated in the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel compound 0M-3223 useful as an adenosine deaminase inhibitor and a pharmaceutically acceptable salt thereof, and a method for producing the above compound.

Conventional technology adenosine deaminase inhibitors include, for example, coformycin (Journal of Antibiotics,
Series Knee, 20 volumes, 227-231 true, 1967
), deoxycoformycin (covidarabine, Annals of the New York Academy of Sciences, Vol. 284, pp. 21-29, 1978), and the synthetic agent erythro-9-(2-hydroxyl-3- Nonyl) adenine (Journal on Medicinal Chemistry, Vol. 17, 6-8L 1974) is known, and these drugs have anti-acute lymphocytic leukemia, immunosuppressive effects, and adenosine-related drugs in combination with adenosine-related drugs. It is known to have the effect of prolonging and increasing the efficacy of drugs.

Problems to be Solved by Drug I However, known adenosine deaminase inhibitors have strong acute toxicity, and there are problems in using them as medicines.

Therefore, there is a demand for adenosine deaminase inhibitors with stronger activity and lower toxicity. The present inventors have learned that a certain type of microorganism belonging to the genus Streptomyces isolated from soil produces a new compound having strong adenosine deaminase inhibitory activity.

The present invention was completed based on the above findings, and
The purpose of the invention was to develop the OM-3223
An object of the present invention is to provide a novel compound useful as an adenosine deaminase inhibitor, a pharmaceutically acceptable salt thereof, and a method for producing the compound.

To solve the problems r According to the present invention, a novel compound 0M-322 represented by the following formula
3 and pharmaceutically acceptable salts thereof are provided.

H The novel compound OM-3223 provided by the present invention is
0M belonging to the genus Streptomyces and represented by formula (1)
It is produced by culturing a microorganism capable of producing -3223 in a medium, producing and accumulating OM-3223 in the culture, and collecting it.

The physicochemical properties of 0M-3223 are as follows.

(1) White needle crystals (2) Elemental analysis value C+ t HIb Na Oa Calculated value: C51,42; H5,75; N19.99% Experimental value: C50,77; H5,65; N19.87% ( 3) Molecular weight and molecular formula 0M-3223 substance mass spectrum A molecular ion peak was observed at earthwork/z280, and the molecular formula CIz H+ b N404 (actual value 280.11?, calculated value 280°117) was observed from the high molecular weight mass spectrum. Decided.

(4) Specific rotation [α] D = -31.6° (C = 1.01H20) (5) Ultraviolet absorption spectrum As shown in Figure 1, it exhibits a characteristic absorption maximum at 279 nm in water.

(6) Infrared absorption spectrum The infrared absorption spectrum measured by the KBr tablet method is as shown in Figure 2, and is 3400.1640.1380.1
The main absorption band is shown at 200.1120 cm-'.

(7) Nuclear Magnetic Resonance Spectrum The 'H NMR spectrum measured in a heavy water solution using a Parian XL-400, 400 MHz NMR spectrometer is shown in Figure 3.

(8) Color reaction sulfuric acid Positive potassium permanganate Positive ninhydrin Negative ferric chloride Negative The structure shown in formula (1) is the result of comparing the above physicochemical properties with those of known similar compounds. It has been decided.

As an example, the strain used to produce the antibiotic 0M-3223 substance of the present invention is Streptomyces sp. 0M-3223 strain, which was newly isolated by the present inventors from the soil of Inokashira Park, Tokyo. can be mentioned.

The mycological properties of this strain are as follows.

(1) Morphological properties Vegetative hyphae develop well on various agar media, and no division is observed. Aerial mycelium grows abundantly on yeast, malt agar, starch, inorganic salt agar, etc., and has a velvety appearance. When observed under a microscope, aerial hyphae appear linear, and chains of 20 or more spores are observed. Spore size is 0.5X0.8
It is cylindrical in μm. The surface of the spore is smooth. sclerotia,
No sporangia and zoospores are found.

(I I) Properties on various media E, B, Shi-rlin
g) and De Gottlieb (D.Gottlfe)
b) method (International Journal of Systemic Bacteriolone-.Volume 16, 3)
The following table shows the culture properties of this producing bacterium, which were investigated by 134 (1966). The color tone was determined as a standard color using the Color Harmony Manual, 4th edition (Container Corporation of America, Chicago, 1958), and the code is also written in parentheses along with the color chart base. The following are the results of observations on each culture medium after 12 weeks at 27°C, unless otherwise specified.

Culture properties (E) Physiological properties (1) Production of melanin pigment (A) Tyrosine agar negative (B) Peptone/yeast iron agar negative (C) Glucose/peptone/gelatin medium (21-23'C) Negative ( d) Tryptone yeast solution Negative (2) Tyrosinase reaction Negative (3) Production of hydrogen sulfide Negative (4) Reduction of nitrate Negative (5) Liquefaction of gelatin (21 -23@C) (Glucose peptone gelatin medium) Positive (6) Hydrolysis of starch Positive (7) Growth temperature range 13-34'C (8) Utilization of carbon sources Use: D-glu (Pleadum Goss, D-Pheasant Streeb Agar, i-inositol) Slightly used: L-arabinose, raffinose, D-fructose, shootalose Not used: melibiose, D-mannitol, L-rhamnos (9) Degradation of cellulose Mediastinal (IV')
Cell Wall Composition Diaminopimelic acid in the cell wall is of the LL type.

The mycological properties of this bacterium are summarized as follows.

Diaminopimelic acid in the cell wall is of the LL type. Aerial hyphae have a linear morphology and form long spore chains. The surface of the spore is smooth. The vegetative hyphae are gold or ipoly
The aerial mycelium is gray or pinkish in color. Does not produce soluble pigments.

From these results, this bacterial strain belongs to the genus Streptomyces, and is classified as gray or gray according to Pridham and Tresner's classification (Versis Manual on Determinative Bacteriology, 8th edition, 748-8291, 1974). This strain, which is considered to belong to the red series, is Streptomyces sp.
p, 0M-3223), and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (Huikou Research Institute Bacteria Repository No. 84).
No. 47). This strain also simultaneously produces coformycin, a known adenosine deaminase inhibitor.

Mutant strains of the above strains can also be used in the method of the present invention as long as they have the ability to produce OM-3223.

Other strains belonging to the genus Streptomyces that are capable of producing OM-3223 can also be used.

As the medium, a synthetic medium or a natural medium containing appropriate amounts of carbon sources, nitrogen sources, and inorganic substances suitable for ordinary actinomycetes culture, and other nutrients as necessary can be used.

The carbon source and nitrogen source used in the culture medium may be any type that is available to the strain used. In other words, carbon sources include, for example, glucose, glycerol,
fructose, maltose, mannitol, xylose,
Various carbohydrates can be used, such as galactose, ribose, starch or their hydrolysates. Its concentration is usually preferably 0.1-5% relative to the medium. In addition, various organic acids such as gluconic acid, pyruvic acid, lactose, and acetic acid, various amino acids such as glycine, glutamic acid, and alanine, as well as various non-90% alcohols such as methanol and ethanol, and normal paraffin. Aromatic hydrocarbons or various vegetable or animal fats and oils can also be used.

Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, and ammonium nitrate, peptone, NZ-7mine, meat extract, yeast extract, dried yeast, corn steep liquor, Nitrogen-containing organic substances such as casein hydrolyzate, fishmeal or its extinguishing agent, soybean powder or its extinguishing agent, defatted soybean or its extinguishing agent, hydrolyzate, and various amino acids such as glycine, glutamic acid, and alanine are used. It is possible. Culture conditions such as stirring speed and aeration are appropriately adjusted and selected depending on the type of bacterial strain used and external conditions. If foaming occurs even in liquid culture, for example, silicone fat,
Antifoaming agents such as vegetable fats and surfactants are used as appropriate.

Zero M− accumulated in the culture thus obtained.
3223 is normally produced in the culture filtrate. However, about 10% of the production amount may be produced in the bacterial body.

To collect 0M-3223 from the culture filtrate, the means normally used for collecting metabolites from microbial cultures may be used singly, in combination in any order, or repeatedly. For example, filtration, centrifugation, dialysis, concentration, drying, freeze-drying, adsorption, desorption, methods that utilize differences in solubility in various solvents (e.g., precipitation, crystallization,
Methods such as recrystallization, dissolution, countercurrent distribution), chromatography, etc. are used.

Since OM-3223 is mainly produced and accumulated in the culture filtrate, in order to separate and collect this compound, it is preferable to collect it from the culture filtrate after removing the bacterial cells from the culture solution. For example, for chromatography, cation exchange resins, Sephadex ion exchangers, ion exchange cellulose, etc.), gel filtration media, activated carbon, high porous polymers (e.g. Amberlite XAD-2, manufactured by Rohm & Haas), and Diaion HP -10 (manufactured by Mitsubishi Chemical Industries, Ltd.), alumina, florisil, silica gel, cellulose, etc. chromatography is effectively used.

Pharmaceutically acceptable salts of the present compounds, such as hydrochlorides, sulfates, nitrates, phosphates, carbonates, tartrates, citrates, and succinates, can be prepared by conventional methods. Since this type of manufacturing method is well known, its explanation will be omitted.

Effects of the invention Adenosine deaminase inhibitory activity Using adenosine deaminase derived from bovine preintestinal mucosa (Sigma Chemical Co., Ltd., A-1155), the method of Mayer Conscience (Analytical Biochemistry Vol. 105,
334, 1980), the adenosine deaminase inhibitory effect was measured as an IC5° value.
The IC3° value of 223 was 1.6×10 −′M.

When 0M-3223 was administered intravenously in a pre-acute test using bumper mice as test animals, even administration of 100 mg/kg did not lead to death. No particular abnormality was observed in observation for 10 days after administration and in autopsy.

Similar administration experiments were conducted with known adenosine deaminase inhibitors coformycin and deoxycoformycin, and as a result, coformycin caused death in mice at a dose of 12.5 mg/kg. In addition, a decrease in body weight was observed when administering 25 mg/kg of deoxycoformycin.

Therefore, it can be said that this substance has lower toxicity than known adenosine deaminase inhibitors.

As described above, since the present invention 'ff0M-3223 has inhibitory activity against adenosine deaminase, it is possible to induce remission of acute lymphocytic leukemia by administering this to humans. It can also be used as a combination agent to significantly prolong the efficacy of known immunosuppressants and adenosine related drugs.

EXAMPLES The present invention will be explained in detail with reference to Examples below, but the present invention is not limited thereby.

Example.

In a 500m flask, glucose 2.0%, peptone 0.5%, yeast extract 0.1%, meat extract 0.5%,
Dispense 100 ml of liquid medium (pH 7.0) containing 0.5% sodium chloride and 0.3% calcium carbonate, and
Steam sterilized at °C for 15 minutes. These were inoculated with 0M-3223 cells grown on agar plates using a platinum loop, and cultured with shaking at 30''C for 3 days to obtain seeds.

Glucose 2.0 per unit of 3ON Jia Noah Mentor
%, peptone 0.5%, yeast extract 0.1%, meat extract 0.5%, sodium chloride 0. A liquid medium (pH 7.0) containing 20A containing 5% calcium carbonate and 0.3% calcium carbonate was prepared and steam sterilized at 121°C for 15 minutes. The above-mentioned 6 seedlings were transplanted to this, and cultured with aeration at 30° C. for 28 hours under conditions of a stirring speed of 200 r, p, m, and an aeration rate of 15 β/min. Centrifuge the culture solution using a Sharpless centrifuge (10
,00Or,p,m,) and separated into bacterial cells and culture supernatant.

Approximately 20j2 of the obtained culture supernatant was filtered using Celite as a filter aid, and then adsorbed on an activated carbon column (1, O4). After washing with water (10/2), 7% acetone water (71 ) and 50% acetone water (10 liters).

The obtained eluate was dried under reduced pressure for 500 m 7! After concentrating to 100%, the mixture was stirred and dropped into ethanol (21), filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 8.0 g of powder. After dissolving this in a small amount of water, use the weak cation exchange resin Amberlite IRC.
(H type; manufactured by Rohm and Haas Co., Ltd.: LOOML) was adsorbed through the column, and after washing with water (300 mJ),
0. It was eluted with IN aqueous ammonia (500 mj). The obtained active portion was freeze-dried to obtain 4.1 g of powder.

Next, this was divided into several times and subjected to reverse phase silica gel column chromatography (manufactured by Merck & Co., Ltd., Art, 9302.20g
) was developed with water and the active portion was concentrated to obtain 19 mg of 0M-3223 substance in the form of colorless needles.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the novel compound OM-3223 according to the present invention measured in water, Figure 2 shows the infrared absorption spectrum of the compound measured by the KBrBr method, and Figure 3 shows the proton nuclear magnetic resonance spectrum of the compound measured in heavy water. The spectrum is shown.

Claims (2)

[Claims] (1) Compound OM-3223 or a pharmaceutically acceptable salt thereof represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I). (2) A microorganism that belongs to the genus Streptomyces and has the ability to produce the compound OM-3223 represented by the formula ▼mathematical formula, chemical formula, table, etc. -3
223 was produced and accumulated, and the compound OM-32 was produced and accumulated from the culture.
Compound OM-3223 characterized by collecting 23
or a method for producing a pharmaceutically acceptable salt thereof.