JPH01309688A - Ws-7587 substance, its production and drug preparation containing the same substance - Google Patents

Abstract

NEW MATERIAL:WS-7587 substance having the following physical and chemical properties. Morphology and color, white powder; molecular weight, 403 (FAB- MS); elemental analysis (%), C 43.20, H 7.57, N 18.97; color reactions, positive to ninhydrin reaction, iodine reaction and potassium permanganate reaction and negative to ferric chloride reaction, Ehrlich reaction and Molisch reaction; solubility, soluble in water and methanol, slightly soluble in ethanol and acetone and insoluble in ethyl acetate and chloroform; specific rotation, [delta] =-5.7 deg. (c=1, methanol); nature, ampholytic substance; etc. USE:Antibacterial agent. PREPARATION:A bacterial strain belonging to genus Striptomyces [e.g., Streptomyces viridochromogenes No.7587 (FERM BP-1639)] is cultured preferably at 25-35 deg.C for 50-150hr.

Description

DETAILED DESCRIPTION OF THE INVENTION 'Industrial Application Field' The present invention relates to a novel compound having pharmacological activity, hereinafter referred to as MS-7587, a method for producing the same, and a formulation containing the same.

More specifically, the present invention relates to a novel compound MS-7587 substance having pharmacological activity such as antibacterial activity, a method for producing the same, and a formulation containing the same.

Accordingly, one object of this invention is to provide a novel compound MS-7587 substance useful for the treatment and prevention of infectious diseases and others.

Another object of this invention is to provide a method for producing WS-7587 material by fermentation in a nutrient medium of a strain belonging to the genus Streptomyces that produces MS-7587 material.

A further object of this invention is to provide a formulation containing MS-7587 substance as an active ingredient.

“A means to solve problems.

The WS-7587 substance of this invention is Streptomyces
Pyridochromogenes (viridoch)
It can be produced by fermenting an MS-7587 substance-producing strain belonging to the genus Streptomyces, such as P. romoenes) No. 7587, in a nutrient medium.

Details of the microorganisms used to produce the WS-7587 substance are described below.

The microorganisms that can be used to produce the MS-7587 substance on Ruiue are MS-7587 substance-producing strains belonging to the genus Streptomyces, and among them, Streptomyces pyridochromogenes No. 7587 is a strain produced in Atami City, Shizuoka Prefecture. It was newly isolated from a soil sample that had been completely collected.

The newly isolated Streptomyces pyridochromogenes No. 7587 is (305) Ibaraki series southeast of Tsukuba.
It has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Chome 1-3, under deposit number FERM BP-1639 (deposit date: January 8, 1988).

The production of the novel MS-7587 material is not limited to the use of the particular microorganisms described herein, which are mentioned merely for illustrative purposes. -N'-Nitrosoguanidine, 2-aminopurine, etc., by conventional methods such as treatment with MS-7587, including artificial mutants and natural mutants, which can be produced from the microorganisms described. This also includes the use of all mutant strains that can be produced.

Streptomyces pyridochromogenes no.

7587 has the following morphological, cultural, biological and physiological characteristics.

[1] Morphological characteristics: This taxonomic study includes Schirling and Gottlieb (
Sbirling, E. B. and Gottlieb.
, D.: Methods for Characterizing Various Streptomyces Species, International Journal of Battereriology, Tei, 313-340, 1966) was mainly used.

Cultures grown at 30°C for 21 days using yeast-malt extract agar, inorganic salts-starch agar, oatmeal agar, and hiculcose-asparagine agar were morphologically observed using an optical microscope and an electron microscope. Eizen hyphae developed well without fragmentation. The aerial hyphae exhibited axle-shaped branches, with spore chains extending from the sporophyll, and the number of spores per chain was 20 to 60. The shapes of aerial hyphae and spore chains were spiral. Spores are oval (0.5-0.7X
0.8 to t, ba), many thorns were observed on the surface under an electron microscope. No sporangia, flagellated spores, sclerotia, or other special forms could be observed.

[2] Culture properties: See the above Shearing and Gottlieb references, and Waksman, S. A.: The actinofflyetes, Volume 2: Classification, identification and description of genera and species. (The Williams & Wilkins Company, Baltimore, 1961)], culture properties were observed for a total of 10 types of culture media.

Incubation was performed at 30°C for 21 hours. The color names used in this study are from the Methuen Handbook of Colors (Kornerup, A., and Wans
cher, J. H.: Methuen Hand
book of course.

Methuen, London, 1978').

The results are shown in Table 1.

Aerial hyphae were bluish-gray. The underside of the growth is brownish orange with fermented H-malt extract agar, grayish green with oatmeal agar,
Inorganic salt starch agar was dark blue to dark green. The mycelium pigment on the back side is pH sensitive and 0.05N
When hydrochloric acid was added, the color changed from blue or green to red or pink.

Several media produced a bright orange to brown soluble pigment.

Yeast-malt processing G: Good kiss agar A: Abundant, bluish gray R: Brownish orange S: Light orange oatmeal G: Good agar A: Abundant, bluish gray R: Grayish green S: None Inorganic salts-G: Good Starch agar A: Rich, bluish gray R: Dark blue or dark green S: None Glycerin-G: Good asparagine A: Rich, bluish gray agar
R: Olive brown S: Yellowish brown peptone - ○: Good yeast extract - A: None iron agar R: Light brown S; Brown tyrosine - G: Good agar A: Rich, bluish gray R: Dark brown S: Yellowish Brown glucose - ○: Good asparagine agar A: Abundant, bluish gray R: Yellowish brown S: Pale brown shedway agar G: Average A: Sparse, white R: Bright yellow S: None Hennett agar G: Normal A: Normal, bluish gray R: Pale green method yellow S: None Sucrose-G: Good nitrate agar A: Normal, white R: Greenish gray S: Pale orange Abbreviation No. G: Growth A: Aerial mycelium R: Back color S : Soluble pigment cell wall analysis was performed using the method of Becker et al. (Becker, B.
.

Lechevalier, M., p-I Gordo
n+ R, E, and Lechevariar, H,
A.: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole cell hydrolysates:
Appl, Microbiol.

421-423.1964) and Yamaguchi's method (Y
amaguchi, T.: Comparison of cell wall composition of morphologically different actinomycetes: J. Bacteriol.
, 89.444-453, 1965). Analysis of the whole cell hydrolyzate of strain No. 7587 showed the presence of LL-diaminopimelic acid. Therefore, the cell wall of this strain is considered to be of type I.

[3] Biological and physiological properties: No. The physiological properties of strain 7587 were as follows: The growth temperature range was changed to yeast-malt extract agar using a temperature gradient incubator (Atovanchik Toyo Co., Ltd.). I asked for it. The physiological properties are summarized in Table 2. The growth temperature range is 15°C to 38°C, and the optimal temperature range is 29°C to 38°C.
The temperature was 35°C. Positive reactions were shown in gelatin liquefaction, milk peptonization, and starch hydrolysis, and negative reactions were shown in milk coagulation. Melanoid pigment production was observed in peptone-yeast extract-iron agar and tryptone-yeast extract broth.

Growth temperature range: 15-38℃ Optimal growth temperature range: 29-35℃ Gelatin liquefaction
positive milk coagulation
Negative milk peptonization Positive starch hydrolysis Positive melanoid pigment formation
Positive cellulose decomposition The use of negative carbon sources is carried out by the method of Pridham and Gottlieb (
Pridham, T., G., and Gottlieb.
, D: Utilization of carbon compounds by some Actinomycetes to aid in species determination, J. Bactariol, 56.
107-114.1948). 30℃
Results were obtained after 14 days of culture. The results are shown in Table 3.

D-glucose + sucrose
+D-xylose
+D-fructose
+L-rhamnose +raffinose +L-arabinose
+Inositol +Mannitol ++: Usage No. From the morphology and chemical characteristics of strain 7587, this microorganism could be clearly assigned to the genus Streptomyces. No. 7587 strain according to Buchanan, R.E., and Gibb's manual, 8th edition.
ons, N.

E: Bergey's manual of d
eterminative bacteriology,
8th Edition, The Williams & Wilkins Company, Baltimore, 1974) and Shirling's ISF Report (Shirling, E, B.
and Gott, 1ieb, D.: i) Joint description of the reference culture of Streptomyces, part 2.

Species description from the first study, International Journal of Systematic Bacteriology, ■
, 69-189.1968; i) Joint description of the standard culture of Streptomyces, Part 3. Additional species descriptions from the first and second studies. International Journal of Systematic Hacteriology, 18
．． 279-392.1968 and i) Joint description of the reference culture of Streptomyces.

Species descriptions from the fourth, second, third and fourth studies;
International Journal of Systematic Batteriology, 19.391-512, 196
9) as well as the Streptomyces species described in
(Skerman, V.B.D.

McGowan, V., and 5neath, P.
H, A, : Approved 1st of ba
cterial names, International Journal of Systematic Bacteriology,
30.225 to 420.1980).

As a result, strain No. 7587 was found to be extremely low in Streptomyces pyridochromogenes (viridochromogenes). Therefore, No. 7587 shares are L.
Comparison with magazine Yukan a ear prefecture arc 姐朐 IFO3113. As shown in Table 4, S, viridochromo e
It was found that nes IFO3113 was extremely similar to strain No. 7587. The differences observed between both microorganisms were starch hydrolysis and growth temperature range. However, these differences alone do not seem to be sufficient to distinguish the two microorganisms. Therefore, No. 75
87 strains were identified as viridochromo enes No. 7587.
It was named.

Table 1 ＼0.7587 strain and S, viridoch
Comparison of taxonomic characteristics of Danbetsu IFO3113 Aerial mycelium color Blueish gray Blueish gray melanoid pigment Positive Positive spore chain Spiral Spiral spore surface Spiny Spiny gelatin liquefaction Positive Positive Milk peptonization Positive Positive Starch hydration decomposition positive
Weakly positive carbon source utilization Sucrose + +D-xylose + +D-fructose + L-rhamnose + +raffinose + joshi-arabinose +
+ Inositol + + Mannitol + + Growth temperature range 15-38℃ 19-BenS-7
The novel MS-7587 substance of the present invention is produced by an MS-7587 substance-producing strain belonging to the genus Streptomyces (eg, Streptomyces pyridochromogenes No.

7587 FERM BP-1639) in a nutrient medium.

Generally, the MS-7587 substance is produced by growing the MS-7587 substance producing strain in an aqueous nutrient medium containing assimilable carbon and nitrogen sources, preferably under aerobic conditions (e.g., shaking culture, deep culture, etc.). It can be produced by culturing.

Preferred carbon sources in the nutrient medium are carbohydrates such as glutenose, xylose, galactose, sucrose, glycerin, starch, dextrin. Other carbon sources that may be included include maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.

Preferred nitrogen sources include yeast extract, peptone, gluten flour, cottonseed meal, soybean flour, cottonseed flour, soybean flour, corn steep liquor, dried yeast, wheat malt, feather meal, peanut flour, etc., as well as ammonium salts ( For example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.)
, urea, amino acids, and other inorganic and organic compounds.

Although carbon and nitrogen sources are advantageously used in combination, less pure materials containing trace amounts of growth factors and significant amounts of inorganic nutrients are also suitable for use;
It is not necessary to use them in pure form; if desired, the medium contains sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, Salts such as copper salts and cobalt salts may be added, if necessary, especially if the medium foams strongly, liquid paraffin, fatty oil, vegetable oil, etc.
Antifoaming agents such as mineral oil and silicone may also be added.

Conditions for mass production of MS-7587 substance:
Deep culture is preferred. For small-scale production, shake culture or surface culture in flasks or bottles is used. In addition, when culturing is carried out in a large tank, in order to avoid growth retardation in the production process of the WS-7587 substance, it is recommended to inoculate the production tank with a culture obtained by culturing the microorganism in advance on a small scale. preferable. The medium therefor shall be substantially the same or different from the medium used for the production of the WS-7587 substance.

Agitating and aerating the culture mixture can be carried out in various ways. Agitation can be caused by a propeller or similar mechanical agitation device, by rotation or shaking of the fermenter, by various pump devices, or by passing sterile air through the medium.1 Aeration is the introduction of sterile air into the fermentation mixture. This can be achieved by passing.

Fermentation is normally carried out at a temperature of about 20°C to 40°C, preferably 25 to 35°C.
C. for about 50 to 150 hours. This time may be varied depending on fermentation conditions and scale.

The MS-7587 substance thus produced can be recovered from the culture medium by conventional means commonly used for the separation and purification of other known biologically active substances. The MS-7587 substance produced is found in the culture filtrate and mycelium, and therefore the MS-7587 substance can be extracted from the filtrate and mycelium obtained by filtration or centrifugation of the culture broth under reduced pressure. Concentration, lyophilization, extraction with conventional solvents,
pH adjustment, treatment with conventional resins (e.g. anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g. activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, re-treatment. It can be separated and purified by conventional methods such as crystallization.

The MS-7587 material produced by the above process has the following physicochemical properties.

(1) Form and color: white powder (2) Molecular weight: 403 (FAB-MS) (3) Elemental analysis: C: 43.20%; H: 7.57X; N: 1
8.97% (4) Color reaction: Positive; Ninhydrin reaction, iodine reaction and potassium permanganate reaction Negative: Ferric chloride reaction, Ehrlich reaction and Molisch reaction (5) Solubility: Soluble 2, slightly soluble in methanol: 2 in ethanol, insoluble in acetone: ethyl acetate, chloroform (6) Specific rotation: [α, g3. ．． -, 7° (C-1, methanol) (7
) Ultraviolet absorption spectrum: ・ Terminal absorption (8) Infrared absorption spectrum; vKB": 3350.2930. 1655.15
90. 1445°ax 1400, 1360.1165 (Cm') (9) ■ Nuclear magnetic resonance spectrum: S (ppm, D O): 1.37 (3H, d
), 1.40 (2H, m).

1.55 (2H, m), 1.78 (2H, m).

2.00 (IH, m), 2.10 (IH, m)
.

2.38 (2Lm>, 3.23 (2) 1.m>
.

3.72 (3H, m), 4.18 (IH, m)
.

4.31 (IH, m) (The chart is shown in Figure 1) (10) 13C nuclear magnetic resonance spectrum: 8 (ppm
, 020)' 20.36 (C13), 24.
73 (CH2).

29.95 (CH2), 30.88 (C12).

34.01 (Cm(2), 35.88 (C12)
.

41.93 (C)I2), 45.66 (CH2)
.

53.72 (CH), 56.19 (CI>.

57.01 (CH), 175.88 (CmO).

176.33 (CmO), 177.84 (CmO
), 180.63 (CmO).

182.60 (CmO) (11) Thin layer chromatography: Solid phase Developing solvent Silica gel plate BuOH'EtOH' CH
Cl a (silica gel 60F254: :NH40
H (4:8:2!3 0.5 trademark, manufactured by Merck & Co.)
v/v”)BuOH: Ac0)l:
H2O0,2 (3:1:2 v/v) (12) Properties of the substance: Amphoteric substance 1 Effect of the invention The chiral MS-7587 substance has pharmacological activities such as antibacterial activity. Therefore, it is useful for treating and preventing infectious diseases caused by pathogenic microorganisms.

As an example showing such pharmacological activity, some pharmacological test data are shown below.

Test Example 1 Antibacterial activity: a) Test method Test bacteria (106 live bacteria per 1 mg) were mixed with graded concentrations of M
Mueller-Hinton agar containing S-7587 material (standard concentration of 172) was inoculated by the stamp method. After 18 hours of incubation at 37° C., the minimum inhibitory concentration (MIC) was determined in (/who).

b) Test Results Test Example 2 Acute Toxicity: The acute toxicity of the MS-7587 substance was determined by intravenous injection in ICR mice (female, 4 weeks old).

There were no symptoms of toxicity at the dose of Ig/kg.

The formulations of the invention are in the form of formulations containing the MS-7587 substance as active ingredient together with organic or inorganic carriers or excipients suitable for topical, enteral or parenteral application, e.g. solid, semi-solid Or it can be used in liquid form. The active ingredient may be present, for example, in tablets, pellets,
It can be mixed with conventional pharmaceutically acceptable non-toxic carriers to form capsules, suppositories, solutions, emulsions, suspensions, or any other form suitable for use. Carriers that can be used include water,
Glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, tartar, corn starch, keratin, colloidal silica, potato starch, pure, and other preparations in solid, semisolid or liquid form. Carriers suitable for use in manufacturing.

Additionally, auxiliaries, stabilizers, thickeners, colorants and perfumes may be used. The active compound of interest is included in the formulation in an amount sufficient to exert the desired effect on the disease process or condition.

When applying this formulation to humans, it is preferable to administer it parenterally or enterally. The dosage of therapeutically effective MS-7587 substances will vary and depend on the age and condition of each individual patient being treated, but generally one dose of the active ingredient will be used for the disease or device. Daily dose about 0.01-1000mg, preferably 0
．． 1-500mg, more preferably 0.5-I00m
g is administered, generally with an average daily dose of about 0.5 mg,'
”L 5mgs 10mg, 50mg, 100m
g, 250ITIg or 500mg can be administered.

EXAMPLE 1 The following example is presented to explain the present invention in more detail.

Example technology M 1% soluble starch, 1% sucrose, 1% glucose
, an aqueous seed medium (16
0 m12) into each of six Erlenmeyer flasks, and
It was sterilized at 21°C for 30 minutes. One platinum loop of slant culture of Streptomyces pyridochromogenes No. 7587 was inoculated into each of the media and cultured at 30° C. for 3 days under shaking conditions.

Soluble starch 6%, dry yeast 1%, wheat malt 2%, calcium carbonate 0.3%, sodium iodide 0.001%
and Adekanol (antifoaming agent, trademark, manufactured by Asahi Ichika) 0
．． Aqueous production medium (20ffi) containing 1% 30β
Pour into each jar fermenter and incubate at 121°C for 30
Sterilized for minutes. The previously obtained seed culture solution (32(luQ
) was inoculated into each of the production media, stirred at 250 rpm,
The cells were cultured at 30°C for 3 days with aeration at a rate of 20j2/min.

Separation and Purification The thus obtained culture solution (60j) was filtered using diatomaceous earth (3 kg).The filtrate (2711) was filtered using cation exchange resin 5
xtB (NH4” type, trademark, manufactured by Mitsubishi Kasei) column (52
). After washing the column with water (15i, 0.I
Elution was performed with N ammonium hydroxide.

The eluate (271) was concentrated under reduced pressure.
Mixed with Q and placed on a silica gel column (11), n-
It was developed with a butanol-ethanol-chloroform-28% aqueous ammonia (2:4:1:1) solution. enemy S-75
87 substances were eluted in fractions 3.61 to 5.5, which were concentrated under reduced pressure. The residue was diluted with n-butanol.
Dissolved in acetic acid-water (3:1:2) solution (5 Makoto),
Chromatography was performed on a column packed with silica gel (200 m ) using the same solvent system. MS-75
Fractions containing 87 substances were collected and concentrated to 200 fractions under reduced pressure. The resulting aqueous solution was mixed with C-tocephadex C-25 (
NH4 type, trademark, manufactured by Pharmacia Co., Ltd.) column (xoo
mm) to remove the silica gel. The column was washed with water (300 mi) and eluted with 0.02N ammonium hydroxide. The eluate (200+19) was concentrated under reduced pressure to obtain a white powder (410 mg) of Qi-7587 substance.

[Brief explanation of the drawing]

FIG. 1 shows the 1H nuclear magnetic resonance spectrum of the MS-7587 material of the present invention. May 23, 1989 1. Display of the case 1999 Patent Application No. 15796 2 Name of the invention WS-7587 substance, its manufacturing method and preparations containing it 3 Person making the amendment Relationship to the case Patent applicant Doshomachi, Chuo-ku, Osaka City 3-4-7 (Address change on February 13, 1989) (524) Fujisawa Pharmaceutical Co., Ltd. Representative Fujisawa Tomoyoshibe 4, Agent 2-1-6 Kashima, Yodoyou-ku, Osaka 532 Fujisawa Yakuhin Kogyo Co., Ltd. Osaka Factory March 31, 1989 (all date of delivery: April 25, 1989) 6. Drawings subject to correction 7. Contents of correction First drawing clearly drawn in sufficiently rich black Submit the diagram as attached (no changes to the content). that's all

Claims (1)

[Claims] 1) WS-7587 substance with the following physicochemical properties: (1) Form and color: white powder (2) Molecular weight: 403 (FAB-MS) (3) Elemental analysis: C: 43 .20%; H: 7.57%; N: 18.97%
(4) Color reaction: Positive: Ninhydrin reaction, iodine reaction and potassium permanganate reaction Negative: Ferric chloride reaction, Ehrlich reaction and Molisch reaction (5) Solubility: Soluble: Slightly soluble in water, methanol: Ethanol, acetone insoluble: ethyl acetate, chloroform (6) Specific rotation: ▲Mathematical formulas, chemical formulas, tables, etc.▼=-5.7° (C=
1. Methanol) (7) Ultraviolet absorption spectrum: Terminal absorption (8) Infrared absorption spectrum: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼: 3350, 293
0, 1655, 1590, 1445, 1400, 136
0, 1165 (cm^-^1) (9)^1H nuclear magnetic resonance spectrum: δ (ppm, D_2O): 1.37 (3H, d), 1.
40 (2H, m), 1.55 (2H, m), 1.78 (
2H, m), 2.00 (1H, m), 2.10 (1H,
m), 2.38 (2H, m), 3.23 (2H, m),
3.72 (3H, m), 4.18 (1H, m), 4.3
1(1H,m) (10)^1^3C nuclear magnetic resonance spectrum: δ(ppm
, D_2O): 20.36(CH_3), 24.73(
CH_2), 29.95 (CH_2), 30.88 (C
H_2), 34.01 (CH_2), 35.88 (CH
_2), 41.93 (CH_2), 45.66 (CH_
2), 53.72 (CH), 56.19 (CH), 57
．． 01 (CH), 175.88 (C=0), 176.3
3 (C=0), 177.84 (C=0), 180.63 (C=0), 182.60 (C=0) (11) Thin layer chromatography: ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (12) Properties of the substance: Amphoteric substance 2) WS-7587 substance producing strain belonging to the genus Streptomyces is cultured in a nutrient medium, and the WS-7587 substance is isolated and purified. Manufacturing method. 3) A formulation containing the WS-7587 substance as an active ingredient together with a pharmaceutically acceptable substantially non-toxic carrier or excipient. 4) Microorganism Streptomyces viridochromogenes N
o. Biologically pure culture of 7587.