KR840000127B1 - How to prepare Istamycin - Google Patents

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How to prepare Istamycin

1 shows the curve of the infrared absorption spectrum measured in the embrittled potassium tablets of istamycin A carbonate according to the present invention,

2 shows the curves of the infrared absorption spectrum measured in the embrittled potassium tablets of estamycin B carbonate,

3 shows the curve of the infrared absorption spectrum measured in the embrittled potassium tablets of estamycin Ao carbonate according to the present invention,

4 shows the curve of the infrared absorption spectrum measured in the embrittled potassium tablets of Istamycin Bo carbonate.

The present invention relates to a method for producing new antibiotics represented by estamycin A, estamycin B, estamycin Ao and estamycin Bo prepared by culturing microorganisms belonging to the genus Streptomyces, and further, the present invention It relates to a new microorganism useful as istamycin producing bacteria.

Many pathogens, such as bacteria and fungi, have caused diseases in humans, animals and plants, and various antibiotics have been developed, some of which have very high antibacterial activity against one or more pathogens, but more Effective therapies have been required to treat many diseases caused by pathogens in humans, animals and plants.

It is an object of the present invention to provide a myriad of new antibiotics as antimicrobial agents for treating fungal infections or sterilizing surgical materials and instruments in humans and animals, and another object of the present invention is to provide a method for culturing and preparing these new antibiotics. To provide.

In addition, a large amount of expenses have been required to prepare new and useful antibiotics. Therefore, the present inventors have cultured strain No. SS-939 in aerobic bacteria isolated from the seabed soil of Miura Peninsula, Kanagawa City, Japan, under aerobic conditions. It was found that four different substances with broad antimicrobial activity were produced. In the present invention, the isolation of these antimicrobial substances from the culture is continued and purified. By studying these isolated substances chemically, physically and microbiologically, these isolated objects exhibit a stronger antimicrobial activity than known antibiotics and are new, less toxic aminoglycoside antibiotics. Accordingly, in the present invention, these antibiotics were named as isamycin A, isamycin B, isamycin Ao and isamycin Bo, respectively.

The first aspect of the present invention is to provide a new antibiotic, itamicin selected from at least one selected from istamycin A, isamycin B, isamycin Ao, isamycin Bo and acid addition salts thereof.

인 특징을 가지며 ; (b)상기 이스타마이신 B는 실험식이 C₁₇H₃₅N₅O₅이고 ; 이의 분자에 두 N-메틸기와 0-메틸기를 가지고 ; 물에서 이스타마이신B의 자외선 흡수 스펙트럼은 말단흡수만을 나타내고 ; 물과 메탄올에는 용해하나 에탄올이나 기타 일반 유기용매에서 난용이거나 불용이고 ; 닌히드린시약과 리돈-스미스시약과 양성 반응을 나타내고 ; n-부탄올-피리딘-초산-물(용적 6 : 4 : 2 : 4)로 전개한 셀루로즈 박층 크로마토그라피에서 Rf 0.25값을 나타내고 ; 이스타마이신 B 헤미-탄산염은 융점은 명확하지 않으나 112-114℃에서 분해하는 무색분말이고 비선광도가

인 특징을 가지며 ;(a) the istamycin A has an empirical formula of C ₁₇ H ₃₅ N ₅ O ₅ and has two N-methyl groups and one 0-methyl group in its molecule; The estamycin A ultraviolet absorption spectrum in water shows only terminal absorption; Soluble in water and methanol but poorly soluble or insoluble in ethanol or other common organic solvents; Positive reaction with a ninhydrin reagent and a Ridon-Smith reagent and an Rf 0.22 value in cellulose thin layer chromatography using n-butane-pyridine-acetic acid-water (volume 6: 4: 2: 4); Istamycin A Hemi-carbonate is a colorless powder that decomposes at 102-108 ℃ but its melting point is not clear.

Has characteristics of; (b) the istamycin B has an empirical formula of C ₁₇ H ₃₅ N ₅ O ₅ ; It has two N-methyl groups and 0-methyl groups in its molecule; The ultraviolet absorption spectrum of Istamycin B in water shows only terminal absorption; Soluble in water and methanol, but poorly soluble or insoluble in ethanol or other common organic solvents; Positive reaction with ninhydrin reagent and lidone-smith reagent; Rf 0.25 value is shown by cellulose thin layer chromatography developed with n-butanol-pyridine-acetic acid-water (volume 6: 4: 2: 4); Istamycin B hemi-carbonate is a colorless powder that decomposes at 112-114 ℃ although its melting point is not clear.

Has characteristics of;

인 특징을 가지며 ;(c) the istamycin Ao is empirical formula C ₁₅ H ₃₂ N ₄ O ₄ ; It has two N-methyl groups and 0-methyl groups in its molecule; The ultraviolet absorption spectrum of istomycin Ao in water shows only terminal absorption; Soluble in water and methanol, but poorly soluble or insoluble in ethanol and other common organic solvents; Positive reaction with a ninhydrin reagent and a Lidon-Smith reagent; Rf 0.36 value is shown by a silica gel thin layer chromatography developed with a lower mixture of chloroform-methanol-17% aqueous ammonia (volume 2: 1: 1); Istamycin Ao Hemi-carbonate is a colorless crystalline powder that decomposes at 111-114 ℃ but its melting point is not clear.

Has characteristics of;

인 특징을 갖는다.(d) the istamycin Bo has the empirical formula of C ₁₅ H ₃₂ N ₄ O ₄ ; Contains two N-methyl groups and one 0-methyl group in its molecule; The ultraviolet absorption spectrum of Istamycin Bo in water shows only terminal absorption; Soluble in water and methanol but poorly soluble or insoluble in ethanol and other common organic solvents; Positive reaction with ninhydrin reagent and lidone-smith reagent; Rf 0.14 is shown in silica gel thin layer chromatography developed with a lower mixture of chloroform-methanol-17% aqueous ammonia (volume 2: 1: 1); Istamycin Bo Hemi-carbonate is a colorless crystalline powder with no apparent melting point

Has features.

From now on, the term istasycin refers to isamycin A, isamycin B, isamycin Ao, or isamycin Bo, or mixtures thereof, unless otherwise indicated, whose isamycin complex, unless otherwise stated, isamycin A, B, Ao A mixture of two, three or all of and Bo.

In the present invention, isamycin A, isamycin B, isamycin Ao and isamycin Bo materials are alone or a mixture of at least two of these materials, which are free as dilute solutions, crude concentrates, coarse solids, and purified solids. It is present in base form or in inorganic or organic acid and acid addition salt form.

The physico-chemical properties of istamycin, including those described above, are described in more detail below.

을 나타내고, 원소분석치는 분자식 C₁₇H₃₅N₅O₅·1/2H₂CO₃의 이론치(C 49.99%, H8.63%, N16.65%, O24.73%)와 일치하고, 또한 이 분자식은 고분해질량분석(실측치 : m/e 389.2588 ; C₁₅H₃₅N₅O₅의 이론치 : m/e 389.2635)에 의하여 확인되었다. 취화 칼륨정에서 측정한 이스타마이신 A 헤미-탄산염의 적외선 흡수 스펙트럼은 첨부된 제1도에 도시되었다. 수용액에서 측정한 이스타마이신A 헤미-탄산염의 자외선 흡수 스펙트럼은 말단 흡수만을 나타낸다.Istamycin A is a basic compound, its istamycin A isolated as its carbonate is a colorless powder that decomposes at 102-108 ° C, although its melting point is not clear.

The elemental analysis value is consistent with the theoretical values (C 49.99%, H8.63%, N16.65%, O24.73%) of the molecular formula C ₁₇ H ₃₅ N ₅ O ₅ 1 / 2H ₂ CO ₃ . The molecular formula was confirmed by high resolution mass spectrometry (aspect: m / e 389.2588; theoretical value of C ₁₅ H ₃₅ N ₅ O ₅ : m / e 389.2635). Infrared absorption spectra of istomycin A hemi-carbonate measured in embrittled potassium tablets are shown in FIG. Ultraviolet absorption spectra of estamycin A hemi-carbonate measured in aqueous solution show terminal absorption only.

A Eastar erythromycin A hemi measured in heavy water-proton ⁽¹ H) nuclear magnetic resonance absorption spectrum of the carbonate: In (external standard TMS, pD5.4) δ3.20 (S, N-CH 3), 3.57 (S, N- CH ₃ ), 3.89 (S,)-CH ₃ ), 4.50 (S, CH ₂ ) and 5.80 (d, J = 3.5 Hz, CH). Istamycin A is soluble in water and methanol, but poorly soluble or insoluble in ethanol and other common organic solvents, which is positive for ninhydrin and lidon-Smith reactions. Istamycin A is a cellulose thin layer chromatography (Avicel, Funakoshi Yakahin / Co.) Developed with a mixed solvent of n-butanol-pyridine-acetic acid-water (6: 4: 2: 4 by volume) and a single point at Rf 0.22. This is clearly distinguished from Rf 0.25 of the cellulose thin layer chromatography of istomycin described below. In high-voltage paper electrophoresis (3,300 volts, 15 minutes) using formic acid-acetic acid-water (volumes 25: 75: 900), the concentration of alanine is 1.0 when alanine mobility is 1.0. Mobility is not distinguished by showing 2.15 equally.

을 나타내고, 이의 원소분석치는 분자식 C₁₇H₃₅N₅O₅·1/2 H₂CO₃의 이론치(C49.99%, H8.63%, N16.65%, O24.7%)와 일치하며, 또한 이 분자식은 고분해질량 분석에 의하여 확인되었다(실측치 : m/e 389.2625 ; C₁₇H₃₅N₅O₅의 이론치 : m/e 389.2635). 취화칼륨정에서 측정한 이스타마이신B의 적외선 흡수 스펙트럼은 첨부된 제2도에 도시되어 있다.Istamycin B is very similar to Istamycin A in their properties, Istamycin B is also a basic compound and has no clear melting point with its carbonate, but it is a colorless powder that decomposes at 112-114 ° C.

The elemental analysis thereof is consistent with the theoretical values of C ₁₇ H ₃₅ N ₅ O ₅ 1/2 H ₂ CO ₃ (C49.99%, H8.63%, N16.65%, O24.7%). In addition, this molecular formula was confirmed by high resolution mass spectrometry (a found: m / e 389.2625; a theoretical value of C ₁₇ H ₃₅ N ₅ O ₅ : m / e 389.2635). Infrared absorption spectra of istomycin B measured on potassium embrittlement tablets are shown in FIG.

Ultraviolet absorption spectra of Istamycin B hemi-carbonate measured in aqueous solution show terminal absorption only.

A Eastar hygromycin B hemi measured in heavy water - both of carbonate ⁽¹ H) nuclear magnetic resonance absorption spectrum (external standard: TMS, pD5.4) at _{δ3.26 (S, N-CH 3} ), 3.59 (S, N- CH ₃ ), 3.95 (S, O-CH ₃ ), 4.57 (S, CH ₂ ) and 5.96 (d, J = 3.5 Hz, CH). Similar to Istamycin A, Istamycin B is soluble in water and methanol but poorly soluble or insoluble in ethanol and other common organic solvents, which is positive for ninhydrin and lidon-smus reactions. As mentioned above, isthamycin B can be distinguished from isthamycin A by cellulose rodworm chromatography.

을 나타내고, 이의 원소분석은 분자식 C₁₅H₃₂N₄O₄·1/2H₂CO₃의 이론치(C51.22%, H9.15%, N15.24%)와 일치한다. 취화칼륨정에서 측정한 이스타마이신 Ao 헤미-탄산염의 적외선 흡수 스펙트럼은 첨부된 제3도에 도시되어 있다. 수용액에서 측정한 이스타마이신 Ao 헤드-탄산염의 자외선 흡수 스펙트럼은 말단 흡수만을 나타낸다. 중수에서 측정한 이스타마이신 Ao 혜미-탄산염의 양자(¹H) 핵 자기 공명 흡수 스펙트럼(외부표준 : TMS, pD5.0)에서, δ3.23(S, N-CH₃), 3.28(S, N-CH₃), 3.94(S, O-CH₃)와 5.86(d, J=4Hz, CH)로 특징적 표시를 나타낸다. 이스타마이신 Ao는 물과 메탄올에 용해하나 에탄올과 기타 일반유기용매에 난용이거나 불용이고, 이는 닌히드린반응 및 리돈-스미스반응에서 양성을 나타낸다. 이스타마이신 Ao는 클로로포름-메탄올-17% 암모니아수(용적으로 2 : 1 : 1) 혼합물의 하층으로 전개한 실리카겔 박층 크로마토그라피로 Rf 0.36에서 단일점을 나타낸다. 개미산-초산-물(용적으로 1 : 3 : 36)을 사용한 고전압지 전기영등(3,300볼트, 15분)에서, 알라닌의 이동도가 1.0이면 이스타마이신 Ao의 상대 이동도는 2.35이다.Istamycin Ao is also a basic compound, and isamycin Ao isolated from its hemi-carbonate has no clear melting point but is a colorless crystalline powder that decomposes at 111-114 ° C.

Its elemental analysis is consistent with the theoretical values (C51.22%, H9.15%, N15.24%) of the molecular formula C ₁₅ H ₃₂ N ₄ O ₄ 1 / 2H ₂ CO ₃ . Infrared absorption spectra of istamycin Ao hemi-carbonate measured on potassium embrittlement tablets are shown in FIG. Ultraviolet absorption spectra of estamycin Ao head-carbonate measured in aqueous solution show only terminal absorption. In the proton ( ¹ H) nuclear magnetic resonance absorption spectrum (external standard: TMS, pD5.0) of estamycin Ao hemi-carbonate measured in heavy water, δ3.23 (S, N-CH ₃ ), 3.28 (S, N -CH ₃ ), 3.94 (S, O-CH ₃ ) and 5.86 (d, J = 4 Hz, CH) to indicate characteristic indications. Istamycin Ao is soluble in water and methanol but poorly soluble or insoluble in ethanol and other common organic solvents, which is positive for ninhydrin and lidon-Smith reactions. Istamycin Ao shows a single point at Rf 0.36 in silica gel thin layer chromatography developed underneath the mixture of chloroform-methanol-17% aqueous ammonia (volume 2: 1: 1) mixture. In a high-voltage paper electrophoresis lamp (3,300 volts, 15 minutes) using formic acid-acetic acid-water (volume 1: 3: 36), the relative mobility of isamycin Ao is 2.35 with alanine mobility of 1.0.

을 나타내고 이의 원소분석은 분자식 C₁₅H₃₂N₄O₄·1/2H₂O의 이론치(C49.98%, H9.20%, N15.04%)와 일치한다. 이 분자식은 고분해질량분석에 의하여 확인되었다(실측치 : m/e 332.2384 ; C₁₅H₃₂N₄O₄의 이론치 : m/e 332.2421). 취화칼륨정에서 측정한 이스타마이신 Bo의 적외선 흡수 스펙트럼은 첨부된 제4도에 도시되어 있다. 수용액에서 측정한 이스타마이신 Bo 헤미-탄산염의 자외선 흡수 스펙트럼은 말단 흡수만을 나타낸다. 중수에서 측정한 이스타마이신 Bo 헤미-탄산염의 양자(1H) 핵자기 공명 흡수 스펙트럼(외부표준 : TMS, p^D5.2)에서 δ3.21(S, N-CH₃), 3.28(S, N-CH₃), 3.91(S, O-CH₃)와 5.92(d, J=3.5Hz, CH)로 특징적 표시를 나타낸다. 이스타마이신 Bo는 물과 메탄올에 용해하나 에탄올과 기타 일반유기용매에서 난용이거나 불용이고, 이는 닌히드린반응 및 리돈-스미스 반응에서 양성을 나타낸다.Istamycin Bo is also a basic compound, Istamycin Bo isolated with hemi-carbonate is a colorless crystalline powder with no clear melting point,

Its elemental analysis is consistent with the theoretical values (C49.98%, H9.20%, N15.04%) of the molecular formula C ₁₅ H ₃₂ N ₄ O ₄ 1 / 2H ₂ O. This molecular formula was confirmed by high resolution mass spectrometry (found: m / e 332.2384; Theoretical value of C ₁₅ H ₃₂ N ₄ O ₄ : m / e 332.2421). Infrared absorption spectra of Istamycin Bo measured on potassium embrittlement tablets are shown in FIG. The ultraviolet absorption spectrum of Istamycin Bo hemi-carbonate measured in aqueous solution shows only terminal absorption. Δ3.21 (S, N-CH ₃ ), 3.28 (S, N-CH) in the proton (1H) nuclear magnetic resonance absorption spectrum (external standard: TMS, p ^D 5.2) of estamycin Bo hemi-carbonate measured in heavy water ₃ ), 3.91 (S, O-CH ₃ ) and 5.92 (d, J = 3.5 Hz, CH) to give a characteristic indication. Istamycin Bo is soluble in water and methanol but is poorly soluble or insoluble in ethanol and other common organic solvents, which is positive in ninhydrin and lidon-Smith reactions.

Istamycin Bo shows a single point at Rf 0.14 with the silica gel thin layer chromatography developed as a lower layer of a mixture of chloroform-methanol-15% aqueous ammonia (volume 2: 1: 1). In the high-voltage paper electrophoresis as described above for Istamycin Ao, Istamycin Bo is difficult to distinguish from Istamycin Ao because their mobility is equal to 2.35.

The structural formula represented by the present invention for istomycin A in structural observation is as follows:

The chemical structural formula for istamycin B is as follows.

The chemical formula for istamycin Ao is as follows.

Further study of the chemical structure of isamycin Ao shows that isamycin Ao is represented by the general formula (III) in its aqueous solution higher than neutral pH, whereas isamycin Ao is lower in its aqueous solution than neutral pH (quantum form). It is represented by the said general formula (IV). The structural formula of Istamycin Bo is as follows.

Istamycin A, Istamycin B, Istamycin Ao, and Istamycin Bo are each commonly obtained as their free base, or as a hydrate or carbonate, which can be reacted with a pharmaceutically acceptable acid in a conventional manner to be pharmaceutically acceptable. Can be converted into acid addition salts. The pharmaceutically acceptable acid salts of isthamycin are obtained by reacting inorganic acids such as hydrochloric acid, brittle acid, sulfuric acid, phosphoric acid, nitric acid, sulfonic acid, and the like, acetic acid, malic acid, citric acid, ascorbic acid, methanesulfonic acid and the like.

Istamycin A and Istamycin B of the present invention have high antimicrobial activity against a wide range of Gram-positive bacteria, as clearly indicated by the antimicrobial spectra of these substances shown in Table 1 below. Istamycin A and Istamycin B strongly inhibit the growth of Gram-negative and Gram-positive bacteria, and the minimum growth inhibitory concentrations (mcg / ml) of Istamycin A and Istamycin B against various bacteria were 37 ° C for 17 hours. Measured by standard serial dilution on nutrient agar plates incubated at temperature.

TABLE 1

The antimicrobial activity of Istamycin Ao and Istamycin Bo is measured by standard serial dilution on nutrient agar plates in the same way as Istamycin A and Istamycin B, and Istamycin Ao and Istamycin Bo show very weak activity. When using Bacillus subtilis as a test bacterium, the antimicrobial activity of Istamycin Ao is about 1/200 times as high as that of Istamycin A, and the antibacterial activity of Istamycin Bo is about 1/200 times as high as that of Istamycin B. have.

Tolerance was given when 100 mg / μg of isthamycin A, isamycin B, isamycin Ao, or isamycin Bo was administered intravenously in rats.

In view of the properties of Istamycin A and B described above, Istamycin A and B are formicin A (antibiotics such as eggs and Okachi, vol. 30, p541 (1977)) and sporarisin (T. Antibiotics, vol. 32, p173 (1979)].

However, because itamicins do not contain C-methyl groups in their molecules but contain N-methyl groups, these known antibiotics and itamycins A and B are completely distinguished, so isimacin A and B and isamycin Ao and Bo are new antibiotics. It can be seen that.

According to a second aspect of the present invention, there is provided a method for preparing an antibiotic istamycin complex, which belongs to the genus Streptomyces under aerobic conditions in a culture solution containing a carbon and nitrogen source until an actual amount of istamycin is produced and accumulated in the culture medium. It refers to a method for producing istamycin by culturing the production bacteria of itamycin, this method of the second aspect of the present invention refers to the step of collecting the itamycin complex from the culture. Herein, the imatomycin complex refers to a mixture of two, three or four isamycins A, B, Ao and Bo.

In detail, the second aspect of the present invention, the production of istamycin A belonging to the genus Streptomyces under aerobic conditions from the culture medium containing carbon and nitrogen until it is produced and accumulated by the production bacteria mentioned in the culture medium. Istamycin A is a method for producing istamycin A, which is cultured, and isstar belonging to the genus Streptomyces under aerobic conditions from a culture solution containing carbon and nitrogen sources until it is produced and accumulated by the production bacteria mentioned in the medium. It is a method for preparing istamycin B by culturing the production of mycin B, and streptomyces under aerobic conditions from a culture medium containing carbon and nitrogen until it is produced and accumulated by the production bacteria mentioned in the culture medium. Ise by culturing the bacteria producing istamycin Ao A method for producing mycin Ao, and further, the production of streptomycin istamycin Bo under aerobic control from a culture medium containing carbon and nitrogen until it is produced and accumulated by the production bacteria mentioned in the medium. It provides a method for producing istamycin B by culturing. The method according to the second aspect of the present invention provides a process such as pure istamycin A, pure istamycin B, pure istamycin Ao, pure istamycin Bo, or a crude product or a solution or a solid thereof, or these istamycin A, B, Ao Harvesting istamycin, such as a mixture of at least two of and Bo.

As an example of istamycin-producing bacteria, actinomycetes collected by the method of the present invention by seabed soil collected from the seabed of the Miura Peninsula in Kanagawa City, Japan, in August 1978, which is represented by the strain number SS-939. It has the same bacteriological properties as

(a) microscopic form

SS-939, grown well on agar plate, simply branched from well-developed basal mycelium to elongate straight or slightly curved basal mycelia, and mature basal mycelia are 10-50. Spore chain comes from the cutting edge. Spores are cylindrical (1 micron wide x 4-5 micron long). No spirals or axle branches are observed. Electron microscopy shows that the surface of the spores is smooth and has no needle-like or hair-like structure. Since neither braided or spore sac is visible, SS-939 strain is a typical streptomycete.

(b) Characteristics on various media

(1) Sudang-nitrate agar medium (cultivated at 27 ° C), which produces white mycelia on weak, colorless growth, gradually becoming turquoise gray ("Color Harmony Manuelle, published by US Container Corp. 17ec, blue liquid: unless otherwise stated, the same color standard will be described later).

No significant diffuse pigment is produced in the medium.

(2) Glycerin asparagine agar medium (incubated at 27 ° C): Almost the same as Sudang-nitrate agar medium mentioned in (1) above.

(3) Starch agar medium (cultivated at 27 ° C): Almost the same as Sudang-nitrate agar medium mentioned in (1) above, the mycelia are engrafted, and the starch around the fungus is transparent.

(4) Tyrosine agar medium (cultivated at 37 ° C): Almost the same as the medium shown in (1) and (2) above, melanin pigment is produced.

(5) Nourishing agar medium (cultivated at 27 ℃): colorless growth, white white mycelia are cultivated, medium is dark brown.

(6) Yeast extract-malt extract agar medium (cultivated at 27 ° C). Colorless development gives white mycelia of mycelia, white mycelium becomes white, and then greenish gray (19dc, green solution). Medium is dark brown.

(7) Oat-corn agar medium (cultivated at 27 ° C). The mycelia of the colorless color develop white-blue grey-grey (17ec, blue liquid).

(c) physiological properties

(1) Growth temperature: grow at 20-41 ℃

(2) Hydrolysis of Starch: Starch is hydrolyzed in starch medium.

(3) Coagulation and peptonization of skim milk are hardly seen.

(4) Formation of melanin pigment: produced in tyrosine agar medium and peptone-yeast extract-iron agar medium

(d) assimilation of carbon sources for growth (evaluated in Friedham-Gottoliv medium)

Only glucose and inositol can assimilate. Arabinose, D-xylose, sudang, rhamnose, raffinose and D-mannitol do not assimilate, and assimilation of D-fructose is almost difficult.

Summarizing the above-mentioned characteristics of SS-939 strain, these strains belong to Streptomyces, have no helix in the mycelia and have chromosome characteristics. In the above-mentioned nature, the SS-939 strain is compared with the International Streptomycer Protection (ISP) and Bergi's Bacterial Assay, known streptomyrs mentioned in 1974. The characteristics of the SS-939 strain are almost similar to that of Streptomyces viridochromogen. However, SS-939 strain cannot make use of xylose, arabinose, rhamnose, fructose, raffinose and mannitol without making spiral yarns. It is different from viridochromogen. Another discernible difference between them is that the surface of the spores produced by the SS-939 strain is not needle-like, and then Streptomyces viridispaciens has no color development and is similar to that of the SS-939 strain. Make a mycelia of color. However, they can be distinguished from each other because SS-939 mycelium cannot equalize fructose and sucrose as a growth carbon source without making spiral yarns from the mycelia. There is no known type of streptomyces that exhibits the ability to exploit the characteristic carbon source assimilation of other SS-939 strains.

Therefore, SS-939 strain is named as Streptomyces tenjimariensis as a new kind.

Streptomyces Tenjimariensis SS-939 strain is the cultivation research organization of Japan's official organization "Culture Research Organization," an industrial science and technology company of Chuguba-Ibaragi, Japan, with the number FERM-P4932 since April 21, 1979. Washington, D. with number 31603. Seed. It is located in the US culture collection center.

Mutations of degenerative bacteria often occur under artificial or natural conditions. Therefore, the present invention uses the SS-939 strain and its mutant as long as they produce istamycin.

Istamycin can be obtained by aerobic culturing of spores or bacteria of Streptomyces, eg, Streptomyces tenjimariensis (FERM-P 4932 or ATCC No. 31603). In carrying out the production process of the present invention, the itamicin-producing spores or fungi are cultured in a suitable medium which is a nutrient source capable of assimilation with fungi and then itamycin, i.e. at least one isamycin A, B, Ao and Bo It is cultured under aerobic conditions until a culture solution containing is obtained. In general, the nutrients in the medium usually used to culture actinomycetes can be used in the present invention. For example, commercially available soy flour, peanut powder, cotton seed powder, dry yeast, peptone, meat extract, casein, corn soaked liquid, sodium nitrate, ammonium sulfate, etc. are useful as nitrogen sources, and carbon, glucose, starch, glycerin Carbohydrates such as maltose, dextrin, sucrose and lactose, and soybean oil and fat are useful.

In addition, inorganic salts such as sodium chloride, calcium carbonate, magnesium sulfate, and manganese chloride superphosphate may be used as well as various amino acids. Nutrients that can be used to cultivate actinomycetes can be used in the present invention as long as they can assimilate with the itamycin-producing bacteria when producing istamycin.

The liquid aeration culture is preferable for mass production of estamycin, and the culture temperature is applicable in the range which can develop and produce the estamycin producing bacteria, and the preferable temperature is 25-30 degreeC. Incubation is usually continued for a sufficient period until a sufficient amount of estamycin has accumulated. Incubation time is usually 2-7 days.

The quantification of Istamycin A and Istamycin B can be performed using Bacillus subtilis PCI 219 as a test bacterium by a cylindrical plate method commonly used for quantifying known antibiotics. The sample of purified istamycin A obtained in Example 3 described later was used as a standard as 1000 mcg (unit) per mg. And the sample of the purified estamycin B obtained from Example 3 shows the titer of 3170mcg (unit) / mg.

In addition, quantitative determination of isamycin Ao and isamycin Bo is carried out by a cylindrical plate method which is commonly used for quantifying antibiotics using Bacillus subtilis PCI 219 as a test bacterium. The sample of purified istasycin Ao obtained in the present invention has a titer of 5 mcg (unit) / mg, the sample of purified istasycin Bo has a titer of 10 mcg (unit) / mg, and the sample of purified itamycin A is 1000 mcg (unit It can be seen that it has a titer of) / mg.

Istamycin A, Istamycin B, Istamycin Ao and Istamycin Bo and their acid addition salts are readily soluble in water, and the Istamycin complex is mainly present in solution in the cultures of estamycin producing bacteria. Since the aqueous solutions of estamycin A, estamycin B, and estamycin Ao and Bo are not extracted with organic solvents such as butanol, butyl acetate and chloroform, these organic solvents can be used to remove impurities from the culture, if necessary. The estamycin complex in the culture solution or the solution of estamycin can be collected using various adsorbents. When activated carbon is used as an adsorbent, istasycin adsorbed by carbon is eluted with weakly acidic water, weakly acidic methanol water, weakly acidic propanol water, weakly acidic acetone water and the like.

Istamycin is eluted by cation-exchange resin with good yield because they are basic. This method is the best method for harvesting istamycin from a large amount of culture. Examples of cation-exchangers that can be used include Amberlite IRC-50, CG-50 (manufactured by Rohm and Haas, USA), Rewarite CNP (manufactured by West Germany Bayer), and CM-Separdex. H type, Na type, NH ₄ type, and mixtures thereof, such as (Sweden, Palmacia products) are used, and the adsorbed antibiotics are eluted with good yield by treating with an aqueous solution of acidic water, dilute ammonia water or inorganic salt. Usually, 0.2N-1N hydrochloric acid and 0.2N-1N ammonia water are used to elute. Istamycin A, Istamycin B, and Istamycin Ao and Istamycin Bo cannot be adsorbed with an anion-exchange resin, so they can be suitably used to neutralize the acid solution of isamycin or to remove acidic impurities.

Silica gel thin layer developed with chloroform-methanol-17% ammonia water (2: 1: 1) in a mixed solution to obtain istasycin A, istasycin B, istasycin Ao, and istasycin Bo from the thus obtained mytasycin complex. Chromatography is characterized by istomycin A with a single point at Rf 0.17, istamycin B with a single point at Rf 0.11, istamycin Ao at Rf 0.36 and isamycin Bo at Rf 0.14. These can be separated from each other by silica gel column chromatography with a lower layer of chloroform-methanol-17% aqueous ammonia (volume 2: 1: 1) developed with a mixed solvent or some similar mixed solvent. In cellulose thin layer chromatography developed with a mixed solvent of n-butanol-pyridine-acetic acid-water (6: 2: 2: 4) to separate istamycin A from istamycin B, isamycin A is a single point at Rf 0.22. On the other hand, Istamycin B is a mixed solvent of n-butanol-pyridine-acetic acid-water (volume 6: 4: 2: 4) by using the characteristics of Istamycin A and B which show a single point at Rf 0.25. Isolate by chromatography on the developed cellulose column. The chromatographic isolation method can be used for the isolation and purification of estamycin and for the extraction of estamycin. Istamycin A, B, Ao, or Bo can be isolated in a purified state by using the above-mentioned extraction method and chromatographic isolation method by repeating or using a suitable combination.

The following methods are preferred for the collection of istasycin complexes and for the isolation and purification of each istasycin: pH 2.0 was adjusted by adding hydrochloric acid to the culture medium of the itamycin-producing bacteria, and then the filtrate was filtered out of Amberlite IRC-50. Continuous passage through a column of a cation-exchange resin such as (NH ₄ type) adsorbs isamycin to the resin. The resin is washed with water and then eluted with 1N ammonia water. The active eluates are pooled together, concentrated under reduced pressure, and the concentrate is passed through a column of cation-exchange resins such as Amberlite CG-50 to adsorb mystamycin to the resin. The resin was washed with water, washed with 0.2N ammonia water and then eluted with 0.4N ammonia water. The active portions containing the actual amount of estamycin B are combined with each other and concentrated to dryness in vacuo to obtain the biopowder of estamycin B. This raw powder was subjected to repeated column chromatography on silica gel to obtain a colorless powder of purified istomycin B.

The active portions containing the actual amount of Istamycin A and the appropriate amount of Istamycin Ao and Bo are mixed with each other and concentrated to dryness in vacuo to obtain a crude powder of Istamycin A, Ao and Bo. This crude powder was developed with a mixed solvent of chloroform-methanol-8.5% aqueous ammonia (volume 2: 1: 1) and subjected to silica gel column chromatography.

These active portions of the eluate containing only istamycin Ao are mixed with each other and concentrated in vacuo, the concentrate is passed through a column of cation-exchange resins such as Amberlite CG-50 (NH ₄ type) and the resin is poured into 0.5N ammonia water. Elute. The eluted active portions containing only istomycin Ao are mixed with each other, and concentrated to dryness in vacuo to give a colorless crystalline powder of purified isamycin A.

These active portions eluted from the silica gel column chromatography containing istomycin A are mixed with each other and concentrated in vacuo. The concentrate thus obtained is subjected to column chromatography of silica gel again, and the active portions containing only istasycin A are mixed with each other and concentrated to dryness to obtain a colorless powder of purified istasycin A, from the silica gel column chromatography containing only istasycin Bo. These eluted active portions are mixed with each other and concentrated in vacuo, and the resulting concentrate is developed with 0.5 N aqueous ammonia and column chromatography of a cation-exchange resin such as Amberlite CG-50 (NH ₄ type). The active eluates containing only istomycin B are mixed with each other and concentrated to dryness in vacuo to give a colorless crystalline powder of purified isamycin Bo.

Structural studies of Istamycin A, B, Ao and Bo indicate that Istamycin Ao is a di-glysyl derivative of Istamycin A, and Istamycin Bo is a di-glycile derivative of Istamycin B (described above). Comparison of general formulas (III), (IV), (V) with general formulas (I) and (II)), therefore, the present invention isatomycin Ao or isamycin Bo by the known method. The amino group and glycine molecules present in the methyl amino group at the 4-position of may be condensed and converted into istomycin A and istomycin B, respectively. This conversion is valuable because Istamycin A and Istamycin B have higher antimicrobial activity than Istamycin Ao and Bo and are more useful as chemotherapeutic agents.

In addition, Istamycin Ao and Istamycin Bo can be prepared by hydrolyzing Istamycin A and Istamadin B, respectively, under alkaline conditions. A third aspect of the present invention provides a process for preparing istasycin Ao from istamycin Ao or isamycin B from isamycin B, which hydrolyzes isamycin A or isamycin B with a solution in water under alkaline conditions to give itamycin Ao. Or to prepare Istamycin Bo. Alkaline hydrolysis of Istamycin A or Istamycin B according to the present invention is performed in the presence of alkali metals or alkali earth metal hydroxides or carbonates such as sodium hydroxide, potassium hydroxide and barium hydroxide. It is effective to heat an aqueous solution of at a temperature of 50-110 ° C. Alkali, such as alkali hydroxide metal, is present in a concentration of 0.1-4 M and the hydrolysis can be completed in 1-3 hours. For example, hydrolysis of Istamycin A or Istamycin B can be achieved by heating it at 100 ° C. for 1 hour in water containing 4M sodium hydroxide or at 100 ° C. for 3 hours in water containing 0.5M barium hydroxide. have. Under these reaction conditions, the glycosidic linkages of isthamycin Ao or isamycin Bo are not broken. Alkaline hydrolysis may also be performed in a filtrate or culture medium containing isthamycin A or isamycin B.

Istamycin A and Istamycin B of the present invention have high antibacterial activity and low toxicity to animals. Thus, istamycin A and B have similar utility as antibiotics known as antibiotics, and are formulated into known pharmaceutical forms and administered in the same manner as known antibiotics. Another aspect of the present invention is to provide a pharmaceutical composition obtained by mixing a safe and effective antimicrobial amount of at least one istamycin A, istamycin B, and acid addition salts thereof with a pharmaceutically acceptable carrier. Moreover, the present invention provides a method for inhibiting bacterial growth by administering at least one istamycin A, istamycin B and acid addition salt thereof to an animal that is prone to bacterial growth. Many factors apply medication, depending on the age, weight, sex, diet, time of administration, the ratio of excretion, composition, response and disease state. The optimal adaptation ratio for the listed conditions can be selected by the expert using a common dosing assay.

As described above, Streptomyces tenzimariens strain SS-939 (FERM-P 4932, ATCC No. 31603) is a novel bacterium useful for producing a new antibiotic, istomycin, and has the above-mentioned bacteriological properties. In addition, according to the present invention, the color tone of the mycelia is gradually changed from white to cyan-grey, and the mycelium is not spiral, and the spore surface is smooth to develop color, and as a carbon source, glucose and inositol are assimilated, but arabinos, D-xylose, sudang, rhamnose, raffinose and D-mannitol do not assimilate and are characterized by the ability to produce istomycin A, B, Ao and Bo, which are substantially purely isolated FERM-P 4932 or It is found in Streptomyces tenzimariens itself, indicated by ATCC number 31603.

The strain SS-939 was obtained from MY medium containing 10 mcg / mg of kanamycin A (1% maltose, 0.4% yeast extract, 1.5% agar pH7.2) on the coast of Miura Peninsula, Kanagawa, Japan. The collected undersea soil was incubated at 27 ° C. for 3 days to collect the actinomycetes, and again, 1% glucose, xylose, arabinose, rhamnose, raffinose or inositol were added to the carbohydrate assimilation test medium (friram-gotliv medium). Each of them, and by assimilating the inositol in the medium containing this nositol, the actinomycetes were isolated. Actinomyces such as Tejimarises can be obtained.

Those skilled in the art can use the present invention to its fullest extent without difficulty using the methods described above. The present invention will be described in detail with reference to Examples, which are not intended to limit the present invention.

Example 1

Streptomyces Tenjimarienssis SS-939 strain (FERM-P 4932, identical to ATCC No. 31603) cultured in agar slope medium was prepared with 1.0% starch, 0.2% glucose, 1.0% soy flour, 0.3% sodium chloride, 0.1% phosphoric acid. Inoculate in a liquid medium (110 ml, 500 ml Erlenmeyer flask) consisting of dipotassium and 0.1% magnesium sulfate (7H ₂ O), then incubate with rotation and shaking at 27 ° C. for 48 hours to obtain culture seeds. This culture seed (220 ml) contains 30 liters of liquid medium consisting of 2.0% starch, 0.2% glucose, 2.0% soy flour, 0.3% sodium chloride, 0.1% dicalcium phosphate and 0.1% magnesium sulfate (7H ₂ O). Inoculate in liter of Jar. Fermentors, incubate for 72 hours at 27 ° C. with air (15 liters of air per minute) and stir (300 r. Pm). The cultures collected from the six characters are pooled together, adjusted to pH 2.0 with hydrochloric acid and filtered to yield 99 liters of filtrate (2.5 mcg / mg). The filtrate was passed through a column filled with 12 liters of Amberlite IRC-50 (Type NH ₄ ) (product of Rohm and Haas, USA), washed with 24 liters of water and eluted with 1N ammonia water. . When the active eluate (3 liters) is mixed with each other and concentrated to dryness in vacuo, 3.51 g of crude powder (titer 61 mcg / mg) is obtained.

The crude powder was dissolved in 100 ml of water, and the resulting solution was passed through a tower (40 mm inner diameter) filled with 300 ml of anion-exchange resin, Dowex 1-X4 (type OH) (manufactured by Dow Chemical Company, USA). Deploy the tower with water, discard the first effluent (200 ml) that flowed out of the tower, collect the next effluent (880 ml), and charge the tower with 150 ml of Amberlite CG-50 (type NH ₄ ) to adsorb istomycin. Inside diameter 25mm). The cation-exchange resin tower was washed with water (300 ml), eluted with 900 ml of 0.2N ammonia water and then eluted with 900 ml of 0.4N ammonia water. Collect the eluate in 18 ml oil. Active oil Nos. 66-80 were mixed and concentrated to dryness under reduced pressure to obtain 210 mg of a white powder of istomycin A and B (titer 460 mcg / mg) Fraction Nos. Preparation of 28-65 in a similar manner elutes several antibiotics other than Istamycin A, B, Ao and Bo.

Example 2

The filtrate obtained by culturing Streptomycer tenzimariensis in the same manner as in Example 1 was passed through a tower filled with 20 liters of Amberlite IRC-50 (type NH ₄ ). After adsorbing, washing with water (40 liters), eluting with 1N ammonia water, and drying the active eluate under reduced pressure, 4.1 g of crude powder (titer of 240 mcg / mg) is obtained.

This crude powder is dissolved in 20 ml of water, and this aqueous solution is placed in a tower (inner diameter 30 mm) filled with 200 ml of Dowex 1-X4 (type OH), and the tower is developed with the next water. Discard the first effluent (270 ml), collect the next effluent (270 ml), pass it through a tower of 150 ml Amberlite CG-50 (type NH ₄ ), adsorb, wash with 150 ml water, and then Elution with 0.2N ammonia water and 9000 ml of 0.4N ammonia water. The eluate was fractionated by 18 ml and fraction Nos. 55-90 are mixed together and concentrated to dryness under reduced pressure to yield 980 mg of a white powder (titer 940 mcg / mg) of Istamycin A and B.

Example 3

980 ml of white powder (titer 940 mcg / mg) containing Istamycin A and B obtained in Example 2 was added to a mixture of 15 ml of n-butanol-pyridine-acetic acid-water (6: 4: 2: 2 by volume). The resulting solution was taken up in a tower (25 mm inner diameter) filled with 60 g of cellulose powder (Avisel, manufactured by Furakoshi Yaguin Co., Ltd.), and the cellulose tower was first subjected to 1200 ml of n-butanol-pyridine-acetic acid-water. (Volume 6: 4: 2: 2) followed by 600 ml of n-butanol-pyridine-acetic acid-water (volume 6: 4: 2: 4). The eluate was fractionated by 10 ml, and fraction Nos. 45-74 only elastamycin B elutes, fraction Nos. Only elastamycin A is eluted at 114-154 and fraction Nos. At 75-113 elastamycin A plus B are eluted into the mixture.

Fraction Nos. 45-74 are mixed and concentrated to dryness under reduced pressure to give a powder which is dissolved in 5 ml of water. The aqueous solution of isthamycin B thus obtained is passed through a tower filled with 25 ml of Amberlite CG-50 (NH ₄ type), adsorbed, washed with 25 ml of water, and eluted with 0.4 N ammonia water. The active eluate (40 ml) was concentrated to dryness under reduced pressure to give 15.3 mg of purified powder, istomycin B (titer 3170 mcg / mg).

Fraction Nos. 114-154 is mixed and concentrated to dryness under reduced pressure to give a powder which is dissolved in 5 ml of water. The aqueous solution of estamycin A thus obtained is passed through a tower filled with 25 ml of Amberlite CG-50 (NH ₄ type), adsorbed, washed with 25 ml of water, and eluted with 0.4 N ammonia water. The active eluate (45 ml) was concentrated to dryness under reduced pressure to yield 50 mg of purified powder (titer 1000 mcg / mg).

Example 4

Streptomyces Tenjimarienssis SS-939 strain (FERM-P 4932, identical to ATCC No. 31603) cultured in agar slope medium was prepared with 1.0% starch, 0.2% glucose, 1.0% soybean powder, 0.3% sodium chloride, 0.1% Inoculate a liquid medium (110 ml in 500 ml Erlenmeyer flask) with dipotassium phosphate and 0.1% magnesium sulfate (7H ₂ O), and incubate at 27 ° C. for 48 hours with shaking and rotation to obtain culture seeds. This culture seed (220 ml) was weighed in 15 liters of 2.0% starch, 0.2% glucose, 2.0% soybean powder, 0.2% sodium palmitate, 0.3% sodium chloride, 0.1% dipotassium phosphate and 0.1% magnesium sulfate (7H ₂ O). It is inoculated into a 30 liter jar permeator containing a liquid medium, and incubated at 27 DEG C for 72 hours while injecting air (15 liters of air per minute) and stirring (300 r. Pm).

The resulting cultures are collected from 10 jar fermentors, adjusted to pH 2.0 with hydrochloric acid and filtered to yield 150 ml of filtrate (titer 6 mcg / mg). The filtrate is passed through a tower packed with 12 liters of Amberlite IRC-50 (NH ₄ type), adsorbed, washed with 24 liters of water, and eluted with 1N ammonia water. The active eluents (5.6 liters) are mixed together and concentrated under reduced pressure. The resulting concentrated solution was poured into a tower filled with 200 ml Amberlite CG-50 (NH ₄ type), washed with water, washed with 1200 ml of 0.2 N ammonia water, and eluted with 1200 ml of 0.4 N ammonia water. Fractions of 18 ml.

Fraction Nos. 80-166 is mixed and concentrated to dryness under reduced pressure to give 240 mg of isthamycin crude powder titer) (380 mcg / mg). The silica gel column chromatography is repeated, developing this crude powder underneath the mixture of chloroform-methanol-8.5% aqueous ammonia (2: 1: 1) until a white powder of purified and titer of 1350mcg / mg istasycin B is obtained. Yield 25 mg.

Fraction Nos. 107-124 was mixed and concentrated to dryness under reduced pressure to yield 380 mg of crude powder (titer 180 mcg / mg) containing Istamycin A, Ao and Bo. The crude powder was column chromatographed using 38 g of silica gel (60, manufactured by E.Merck, Germany) and developed using a mixed solvent as a lower layer of chloroform-methanol-8.5% ammonia water (volume 2: 1: 1). do. The eluate from the silica gel column is separated by 5 ml.

Fraction Nos. 54-61 were mixed, concentrated under reduced pressure, and then adsorbed onto a tower of 3 ml of Amberlite (NH ₄ type), eluted with 0.5N ammonia water, and the active eluate was concentrated to dryness under reduced pressure to give colorless crystals of purified istomycin Ao. Obtain a powder (titer 5 mcg / mg). Yield 25 mg.

Fraction Nos. 65-76% was mixed, concentrated under reduced pressure, purified by silica gel (10 g) chromatography, and developed into a mixed solvent lower layer of chloroform-methanol-8.5% ammonia water (2: 1: 1). In this manner, a colorless powder (titer 1000 mcg / mg) of istamycin A purified in a yield of 23 mg is obtained.

Fraction Nos. 77-90 were mixed and concentrated under reduced pressure, then adsorbed by passing through a tower filled with 3 ml of Amberlite CG-50 (NH ₄ type), eluted with 0.5N ammonia water, and the active eluate was concentrated to dryness under reduced pressure. Obtain a colorless crystalline powder (titer 10 mcg / mg) of purified Istamycin Bo. Yield 18 mg.

Example 5

This example and the following examples exemplify the preparation of isamycin Ao or Bo from itamycin A or itamycin B by alkaline hydrolysis.

3 the mixture to a solution obtained by dissolving the Eastar azithromycin A of 20mg to 300mg in 2ml water effectively to the barium hydroxide _{(Ba (OH) 2 .8H 2} O) were mixed, and alkaline hydrolysis of Eastar azithromycin A in the sealed tube Heat at 100 ° C. for hours. The reaction mixture was neutralized by addition of solid carbon dioxide and then filtered, and the filtrate was adsorbed through 3 ml of Amberlite CG-50 (NH ₄ type) tower and eluted with 0.5N ammonia water. Concentration and drying of the active eluate under reduced pressure yields 16 mg of colorless crystalline powder, purified Istamycin A.

Alkaline hydrolysis of Istamycin B (20 mg) in the same manner as described above yields 15 mg of a colorless crystalline powder of purified Istamycin Bo.

Example 6

Incubated in agar slope in liquid medium (pH 7.0, 50 liters) of 1.0% starch, 0.2% glucose, 1.0% soybean powder, 0.3% sodium chloride, 0.1% dipotassium phosphate and 0.1% magnesium sulfate (7H ₂ O) Inoculate one strain of strain T. marihensis SS-939 (FERM-P 4932), incubate for 24 hours at 28 ° C (50 liters of air per minute) and incubate with stirring (200r. Pm). Seeds are produced. 20 liters of this culture seed was a two-ton stainless steel culture tank containing 1000 liters of liquid medium (pH 7.0) of 4.0% starch, 0.4% glucose, 5.0% wheat germ, 0.6% calcium carbonate and 0.3% sodium chloride. Inoculate and infuse air at 28 ° C. for 108 hours (800 liters of air per minute) and incubate with stirring (280 r. Pm).

The culture solution (pH7.3) thus obtained was adjusted to pH 2.0 with hydrochloric acid, filtered, and neutralized by addition of aqueous sodium hydroxide solution to the filtrate to obtain 850 liters of culture solution (pH 6.2, titer 105 mg / ml). The culture solution was passed through a 55 liter Amberlite IRC-50 (NH ₄ type) tower and adsorbed, washed with 300 liters of water, eluted with 1.1 N ammonia water, and the first eluate (38 liters) was discarded. The eluate (188 liters) is collected and then concentrated to 2.8 liters under reduced pressure.

475.2g of barium hydroxide to the concentrate _{(Ba (OH) 2. 8H} 2 O) was added to, and then heated to 110 ℃ for 3 hours under reflux, and 2N sulfuric acid was added to neutralize the reaction mixture to 1960ml, and then filtered, and the filtrate Was adsorbed through 15 liters of Amberlite CG-50 (NH ₄ type) tower, washed with 45 liters of water, eluted sequentially with ammonia water at each concentration shown below, and the eluate was fractionated by 33 liters. .

Fraction Nos. 82-104 is collected and concentrated to dryness under reduced pressure to give 38.05 g of crude powder containing isamycin Ao. In addition, fraction Nos. 105-134 was collected and concentrated to dryness under reduced pressure to obtain 19.19 g of crude powder containing isamycin Bo.

Example 7

Crude powder of Istamycin Ao (2.0 g) obtained in Example 6 was subjected to column chromatography using a column of 180 g silica gel, and a lower layer of chloroform-methanol-8.5% ammonia water (2: 1: 1 in volume) was developed. Purify by silica gel (180 g) column chromatography, and fractionate the eluate by 25-ml.

Concentration and drying of the fraction Nos. 65-104 under reduced pressure yields a colorless crystalline powder of purified isamycin Ao. Yield 566 mg.

The crude powder of istasycin Bo (2.0 g) obtained in Example 6 was purified by silica gel column chromatography in the same manner as described above, and the eluate was separated by 23 ml. Fraction Nos. Concentration-drying 73-170 under reduced pressure yields a colorless crystalline powder of purified isamycin Bo. Yield 1369 mg.

Claims (1)

Strains of Streptomyces tenzimarihensis SS-939 were cultured in a culture solution containing a carbon and nitrogen source under aerobic conditions to accumulate an actual amount of estamycin in the culture medium. From this culture, estamycin A, istomycin B, and itamycin Ao or estamycin Bo and a method for producing estamycin, characterized in that a mixture of at least two of these estamycin A, B, Ao and Bo.

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