US3626055A - Method for producing antibiotic t-2636 - Google Patents

Abstract

Antibiotics T-2636-A, T-2636-B, T-2636-C and T-2636-D, produced by fermentative culturing of Streptomyces rochei var. volubilis or a mutant or variant thereof, are useful, e.g. in topical preparations for the treatment of infections due to Staphylococci, e.g. Staphylococcus aureus.

Description

United States Inventors Appl. No.

Priorities Eiji ll-Iigashide Kawamo, Takarazuka;

Motoo Shihata, Toyonalka, Osaka; Setsuo iiai'ada, Suita, Osaka; Toyoltazu Kishi, Nara, Nara; Komei Mizuno, Settsu, Osaka, all of Japan May 17, 1968 Dec. 7, 1971 Takida Chemical industries, Ltd.

Osaka, Japan May 18, 1967 Japan May 8, 1968, Japan, No. 43/310530 METHOD FOR PRODUCING ANTKBIOTIC T-2636 11 Claims 5 Drawing Figs.

US. Cl

[51] inLCi ..A61h21/00 [50] Field of Search [56] References Cited UNITED STATES PATENTS 2,805,185 9/1957 Hermann et a1. 424/122 3,344,024 9/1967 Whaley et al. 424/120 Primary Examiner-Albert T. Meyers Assistant Examiner Daren M. Stephens Attorney-Wenderoth, Lind & Ponack INVENTORS ATTORNEYS KAZU KISHI l MIZUNO EU HIGASHIDE MOTOO SHIBATA 1ESUO HARADA K8M% FIGS PATENTEU 05c 119m 0 O O O 0 w 8 6 4 2 METHOD FOR PRODUCING ANTIBIOTIC T-2636 This invention relates to antibiotically active novel compounds and to the production thereof.

More particularly, this invention relates to a group of antibiotics T-2636-A, T-2636-B, T-2636-C, T-2636-D and mixtures thereof (hereinafter, including also the claims, each of these antibiotics or a mixture of them is collectively referred to as antibiotic T-2636).

The invention is based on the following findings:

1. that micro-organisms capable of producing the new antibiotics are isolated from soil samples;

2. that the said micro-organisms belong to the genus Streptomyces;

3. that the antibiotics are accumulated in a medium in which the micro-organisms are cultured;

4. that so-accumulated antibiotics can be recovered in a desired purity from the culture broth by taking advantage of the physico-chemical properties of the antibiotics; and

5. that the antibiotics have a strong antimicrobial activity against Gram positive bacteria.

In the method of the present invention, those micro-organisms belonging to the genus Streptomyces and capable of producing the antibiotic T-2636 are employed. These microorganisms include Strain No. T-2636 which has been isolated from a soil sample in Osaka, Japan, and mutants thereof.

The morphological and cultural characteristics of Strain No. T-2636 are as follows:

In the following descriptions, the color names designated Rdg" are based on Ridgeways Color Standard and Nomenclaturez" l. Morphological Characteristics:

Sporophores show monopodial branching and pseudoverticillus, and form loops or spirals with spores arranged in chains.

Spores are oval or ellipsoidal with a smooth surface, ranging from 0.5 to 1.0 p. by 0.9 to 1.5 p. in size.

2. Cultural Characteristics:

Generally the substrate mycelia are colorless. The strain forms brown to brownish gray aerial mycelia, produces no soluble pigments on almost all kinds of media or merely produces faint yellowish pigment, and belongs to the nonchromogenic type.

The temperature for growth is from about 20 to about 45 C. atapHofS to9.

1. Czapeks agar:

Growth: spreading, thin and colorless.

Aerial mycelia: poor, powdery, white to Light Drab (Rdg.

XLVl, l7""-b).

Reverse: colorless.

Soluble pigment: none.

2. Glucose-Czapeks agar:

Growth: thin spreading, colorless.

Aerial mycelia: poor, white to Light Drab (Rdg. XLVl,

l 7 I I r 1 b).

Reverse: colorless.

Soluble pigment: none.

. Glycerin-Czapeks agar:

Growth: thin, spreading, colorless.

Aerial mycelia: poor, powdery, white to Light Cinnamon Drab (Rdg. XLVl, 13""b) to Light Drab (Rdg. XLVl, l7""-b).

Reverse: colorless.

Soluble pigment: none.

4. Glucose-asparagine agar:

Growth: moderate, spreading, colorless.

Aerial mycelia: abundant, powdery and Light Drab (Rdg. XLVl, 17""b) to Drab (Rdg. XLVl, 17"") or Drab Gray (Rdg. XLVl, 17""-d) interspersed with white patches.

Reverse: colorless to pale yellow.

Soluble pigment: none.

. Nutrient agar:

Growth: spreading, fairly good and colorless to pale yellow.

Aerial mycelia: poor, white or sometimes Light Drab (Rdg.

XLVl,17""-b) Reverse: colorless to faint yellow. Soluble pigment: none. 6. Glucose nutrient agar:

Growth: abundant, colorless to pale yellow. Aerial mycelia: poor, white to Drab Gray. (Rdg. XLVl l 7 I I ll d Reverse: faint brown. Soluble pigment: none or sometimes faint brown.

Growth: 7. Glycerin nutrient agar: Growth: abundant, folded, colorless to pale yellowish brown.

Aerial mycelia: fairly good, white.

Reverse: faint brown or dark brown patches.

Soluble pigment: none or pale yellow pigment produced.

8. Nutrient broth:

Growth: pellicle or ring, later sediment.

Aerial mycelia: none or poor, white.

Soluble pigment: none.

9. Peptone agar:

Growth: poor, thin, spreading and white. Aerial mycelia: poor and white. Reverse: colorless. Soluble pigment: none. 10. Starch agar:

Growth: poor, colorless.

Aerial mycelia: none or scant, white. Reverse: colorless.

Soluble pigment: none.

1 1. Yeast extract agar:

Growth: abundant, wrinkled, colorless to pale brown.

Aerial mycelia: fairly abundant, white to Drab Gray (Rdg.

XLVl, 17""-d) to Mouse Gray (Rdg. L1, 15"").

Reverse: yellowish brown.

Soluble pigment: none.

12. Whole egg medium (37 C.)

Growth: abundant, lichenoid, colorless or with black patches, later becoming black.

Aerial mycelia: abundant, white to Pale Olive Gray (Rdg. Ll, 23""'-f) or Mouse Gray (Rdg. L], 15"') to Drab Gray (Rdg. XLVl, l7"d).

Soluble pigment: none.

13. Potato plug:

Growth: abundant, lichenoid, colorless.

Aerial mycelia: abundant, white to Drab Gray (Rdg. XLV],

17""d) or Mouse Gray (Rdg. Ll, 15"").

Soluble pigment: none.

14. Carrot plug:

Growth: abundant, lichenoid, colorless.

Aerial mycelia: abundant, powdery, white to Light Drab (Rdg. XLVl 17""-b) to Drab(Rdg.XLVl,17"").

Soluble pigment: none.

15. Litmus milk (37C.):

Growth: ring, colorless to pale brown.

Aerial mycelia: poor, white.

Soluble pigment: none.

Peptonization without coagulation, almost no change in pH value. 16. L'o'fflers serum medium (37 Growth: spreading, wrinkled, colorless.

Aerial mycelia: none or scant, Drab Gray (Rdg. XLVl,

Soluble pigment: none.

No liquefaction or weak liquefaction. 17. Gelatin (24 C. for one month):

Growth: poor, ring-shaped growth, colorless.

Aerial mycelia: poor, white to gale Drab Gray (Rdg. XLVl,

Soluble pigment: none.

No liquefaction or very slow liquefaction. l8. Cellulose:

Growth: none or scant, colorless.

Aerial mycelia: none or scant, Drab Gray (Rdg. XLVl,

l 7llll d).

19. Calcium malate agar:

Growth: moderate, colorless or poor and thin.

TABLE 1 Erythritol Sucrose to +4- Adonitol Lactose +H- D-Sorbitol Raffinose -H- i-lnositol Trehalose +H- D-Mannitol -+H- Salicin -H+- Dulcitol Esculin D-Xylose lnulin L-Arabinose +H- Dextran +-H- L-Sorbose Starch H-l- D-Galactose Sodium ncetate +H- D-Glucose -H-+ Sodium succinate +-H- D-Fructose -H-+ Sodium citrate -Hl- D-Mannose -i-H- Glycerin -l+t- Rhamnosc Control Melibiose Maltese Remarks: abundant growth good growth growth faint growth no With regard to the above morphological properties of the micro-organism, as well as its appearances on various media, reference to S.A. Waksmans Actinomycetes, Vol. 2, 152 (l96l), Bergeys Manual of Determinative Bacteriology, Seventh Edition (I957), Archiv. fu'r Mikrobiologie Vol. 31, pages 326 to 358 (L. Ettlinger et al. 1958) suggests that Strain No. T-2636 is related to Streptomyces rochei.

Streptomyces rochei forms straight or spiral sporophores, produces pale yellow soluble pigments with rapid liquefaction on gelatin, reddish brown pigments on potato plug and shows brown growth on starch medium, while Strain No. T-2636 produces loop or spiral sporophores, shows no soluble pigment nor liquefaction on gelatin and is almost colorless on potato plug, colorless on starch medium.

However, since in other respects, both have many characteristics in common, thus Strain No. T-2636 is considered to be a variant of Streptomyces rochei and has been named Srreptomyces rochei var. volubilis, a culture of which has been deposited at American Type Culture Collection, Rockville, Maryland, USA. under the accession number of ATCC-2l250.

The mycological properties of Actinomycetes, especially those of the genus Streptomyces are not generally fixed and this applies also to the characteristics of the Slreptomyces rochei.

There are many mutants and variants of T-2636-producing organism. Among the mutants and variants of T-2636- producing organism, regardless of whether the variation is caused naturally or artificially, for example, with X-ray, ultraviolet-ray, or by the action of chemical reagents such as nitrogen mustard, nitrosoguanidine or salts of heavy metal, any one can be employed in the method of the present invention, as long as it produces antibiotic T-2636.

The main object of the present invention is to produce a novel antibiotic T-2636.

Another object of the present invention is to provide a method for producing the said antibiotic.

The objects of this invention are realized by incubating a T-2636-Streptomyces in a medium containing assimilable carbon sources, digestible nitrogen sources and other nutrients, and recovering the accumulated antibiotic T-2636 therefrom.

As to the culture medium, this can be liquid or solid, but submerged culture using aerated agitation method is most advantageous.

The culture conditions such as temperature, culture period and pH of the medium are determined so that the strain of micro-organism being used grows luxuriantly and the output of the antibiotic T-2636 is maximum.

By way of example, when the T-2636-producing strain Streptomyces rochei var. volubilis is used, 2 to 7 day culture is generally of advantage.

The medium is preferably maintained at a pH of 6 to 8 and the optimum incubating temperature lies between about 24 and 40 C. and, for better results, between about 28 and about 37 C.

By changing these conditions, the antibiotics T-2636-A, -B, C and D are accumulated in the medium in various proportions.

While T-2636-A and -C, for instance, are more preferably produced by incubating the T-2636-producing strain in a medium comprising 2 percent of glucose, 3 percent of soluble starch, 1 percent of corn steep liquor, 1 percent of soy bean flour, 0.5 percent of polypeptone, 0.3 percent of sodium chloride and 0.5 percent of calcium carbonate (pH 7.0) for about 42 hours, T-2636-B and -D are luxuriantly obtained when the same strain is incubated in a medium including 5 percent of soluble starch, 2 percent of corn steep liquor, 0.5 percent of polypeptone, 0.2 percent of K,HPO,, 0.5 percent of calcium carbonate (pH 7.0) for about 78 to about hours.

Antibiotic T-2636 thus produced is contained mostly in the liquid part of the culture broth, but also in the mycelia. These antibiotics are weakly basic or neutral fat-soluble substances, and therefore can be extracted en bloc from the culture broth or its filtrate or the mycelia by the use of a suitable organic solvent. The respective antibiotics can be divided into the several antibiotic T-2636-A, B, -C or -D, utilizing, for example, different adsorbances between the antibiotics.

As antibiotic T-2636 exists mostly in the liquid part of the culture broth, it is advantageous to separate at first the liquid part of the culture broth from the culture broth. For example, the culture broth is filtered with or without adding a filter aid at a pH between about 3 and about 8 to obtain culture filtrate. The filtrate contains most of the objective antibiotic T-2636 showing antimicrobial activity. The filtrate is extracted at a pH between about 2 and about 9 with a water-immiscible organic solvent such as lower fatty acid ester (e.g. ethyl acetate, amyl acetate), aromatic hydrocarbon (e.g. benzene), chlorinated hydrocarbon (e.g. methylene chloride, chloroform), ketone (e.g. methyl isopropyl ketone, methyl isobutyl ketone), alcohol (e.g. n-butanol, isoamyl alcohol), ether (e.g. diethyl ether), and the like. Thus obtained extract is washed with an acidic aqueous solution of pH about 2 to 5 and an alkaline aqueous solution of pH about 8 to 10, respectively, followed by concentration. To the concentrate is added petroleum ether, n-hexane, benzene, diethyl ether or a mixture thereof to,

precipitate crude powder. From this crude powder, there can be separated the desired respective antibiotics T-2636-A, B, -C and -D. As for the means of the isolation, for example, the crude powder is dissolved in a suitable organic solvent such as ethyl acetate, chloroform or a mixture thereof, and is charged on a column packed with silica gel or alumina, followed by elution with benzene, diethyl ether, ethyl acetate, acetone, chloroform, methanol or a mixture thereof, to obtain eluates containing the active components -A, -B, -C and -D in this order.

When an eluate thus obtained contains plural components among the four, such components can be respectively isolated. Namely, the eluate obtained by the above procedure is concentrated, and the concentrate is again treated with chromatography (e.g. thin layer chromatography or, more industrially, column chromatography) on silica gel or alumina. Thus the objective antibiotics are obtained as pure crystals respectively.

The separation of the active ingredients in mycelia is advantageously achieved, for example as follows: The wet mycelia are subjected to extraction with a 70 percent aqueous acetone or a 70 percent aqueous methanol and the extract is concentrated. The concentrate is extracted with such a solvent as benzene, ethyl acetate, methylene chloride and the like. The extract contains antibiotic T-2636 and respective components can be recovered, for example, by means of chromatography as mentioned above. The aqueous residue is extracted with n-butanol, followed by adding ether, whereby there is obtained a powdery substance showing significant maximum ultraviolet absorptions at the wavelengths of 308, 321, 338 and 355 millimicrons when measured in methanol.

The substance has antimold activity and is presumed to be a pentaene.

As the result of the treatments as mentioned above, there are obtained from the culture broth of the T-2636-producing strain the four antibiotics: T-2636-A, T-2636-B, T2636C and T-2636-D.

PHYSICOCHEMICAL PROPERTIES a. T-2636-A T-2636-A forms white plate crystals melting at 200 to 205 C. (recrystallized from diethyl ether), and its elementary analysis is as follows:

C: 64.73:l.0%, H: 7.00- 0.5%, N: 2.80:*:O.S%, OCH;,: OAc: about 11.56%. Specific rotation in methanol is [a],,= 22l.2;t20(C=0.5).

The molecular weight measured by osmotic pressure in ethyl acetate is about 490, while according to mass spectrometry measurements, its highest mass number is 441. Thus its molecular weight is about 500, and it has one acetyl group. Ultraviolet absorption spectrum in methanol solution is shown in FIG. and shows a maximum at 22812 mu (E =lO50;*:l00). Infrared absorption spectrum by KBr disc method is shown in FIG. 1 and has the following absorption bands: 3350(M), 3400(M), 2960(W), 2900(W), 2850(W), 1755(8), 1725(sh), 1720(vs), 1700(8), 1690(8), 1650(W), 1500(M), 1450(W), 1440(W), 1360(M), 1320(M), 1260(vs), 1140(M), 1100(W), 1010(M), 960(M), 950(M), 920(W), 860(W), 810(W), 800(W), 740(W), 680(W), 650(W),620(W),580(W)cm.

These absorption bands show the existence of macrolactone and acetyl group in the carbonyl region.

lts Rf values of thin layer chromatography on silica gel with different solvent systems are as follows:

Solvent (ratio by volume) Rf value Chlurol'orm/Mcthanol (93:7) 0.573105 Methyl ethyl ketone/diethyl ether l :3) 0.82:0.05 Ethyl acetate/diethyl ether l:3) 0.62:0.05

The molecular weight measured by osmotic pressure method in ethyl acetate is about 851, while according to mass spectrometry measurements, the highest mass number is 850. Thus its molecular weight is about 850.

Ultraviolet absorption spectrum of a solution of 1.169 mg.

of T 2636-B in the 20 ml. of methanol exhibits no characteristic absorption. Infrared absorption spectrum by KBr disc method is shown in FIG. 2, and has the following absorption bands: 3500(8), 2960(8), 2930(M), 1750(vs), 1730(vs), 1710(5), 1630(W), 1460(M), 1380(5), 1340(M), 1250(5), 1220(vs), 1100(5), 1150(8), 1110(8), 1090(5), 1050(8), 1030(5), 1000(vs), 060(M), 940(W), 910(W), 900(W), 000(W), 520(W) cm."-

The above infrared absorption spectrum and N.M.R. spectrum show the existence of macrolactone, acetyl and methoxyl groups in its molecule.

lts Rf values on thin layer chromatogram on silica gel with different solvent systems are as follows:

Solvent (ration by volume) Rf value Chloroform/Methanol (93:7) 0.321003 Methyl ethyl ketone/diethyl ether l:3) 0.6021105 Diethyl ether/Ethyl acetate (3:1) 0.491005 T-2636-B is positive to Molischs reaction and to the Erythromycin Test, but is negative to the Carbomycin Test. T-2636-solvents as those of T-2636-A. c. T2636C Solvent (ratio by volume) Rf value chloroform/Methanol (93:7) 0.24:1).05 Ethylacetate/Diethyl ether l:3) 0.25:0.05 Methyl ethyl ketone/diethyl ether 1:3) 0.431005 Specification rotation in ethanol shows [a] =-237fl0 1.0).

T-2636C is soluble in ethylacetate, acetone, ethanol, methanol, hardly soluble in diethylether, insoluble in n-hexane. Ultraviolet absorption spectrum in ethanol solution is shown in FIG. 5 and shows a maximum at 226 mu (E,,,,, =l0l3il00). T-2636-C is positive to Molischs reaction, but is negative to the ferric chloride reaction, and to the Erythromycin and Carbomycin Tests. Infrared absorption spectrum by RB! disc method is shown in FIG. 3, and has the following absorption bands: 3450(5), 3400(5), 2940(w), 2000(5), 1755(s), 1725(sh), 1710(vs), 1680(vs), 1520(M), 1450(M), 1402(M), 1360(M), 1325(M), 1310(M), 1260(s),1220(M), 1100(M),1137(M),1100(W),1055(M),1048(M),1005(s), 962(5), 075(W), 850(W), 830(W), 016(W), 800(W), "742(W), 680(W). d. T-2636-D I T-2636-D forms colorless needles melting at to l9 1 C. (recrystallized from a mixture of methanol and ethylacetate), and its elementary analysis is as follows: C: 63.95:l%, H: 7.34i0.5%, N: 3.ll:0.5%, OCH ,0%, OAc: l0.38%. Specific rotation in methanol is [u],, =220' *20 (C=O.5).

The molecular weight measured by osmotic pressure in ethyl acetate is about 509, while by mass spectrometry measurements, its mass number is 459. Thus its molecular weight is about 500. Ultraviolet absorption in methanol solution in methanol solution is shown in FIG. 5 and shows a maximum at 228511012 mp. (E, =l070itoil).

Infrared absorption spectrum of T-2636-D by KBr disc method is shown in FIG. 4 and has the following absorption bands: 3350(s), 3320(vs), 2960(M), 2930(W), 1740(8), 1725(vs), 1705(vs), 1650(vs), 1560(5), 1450(W), 1370(M), 1320(M), 1240(vs), 1200(M), 1170(W), 1130(s), 1080(M), 1040(M), 1020(s), 1010(M), 960(8), 920(W), 880(W), 710(W) cm. T-2636-D is positive to the Carbomycin Test, Molishs reaction, but is negative to the Erythromycin Test, ninhydrin reagent and benzidine reagent.

T-2636-D is hardly soluble in diethyl ether, chloroform, ethylacetate, slightly soluble in acetone, easily soluble in methanol or ethanol.

Its Rf values of thin layer chromatogram on silica gel in different solvent systems (Silica gel HF 254, Merck & Co.) are as follows:

Solvent (ratio by volume) Rf value Chloroform/Methanol (93:7) 0.191005 Methyl ethyl ketone/diethyl ether l :3) 0.35 20.05 Ethyl acetateldiethyl ether (l:3) 0.l7 10.05

BIOLOGICAL ACTIVITIES Antibiotics T-2636 show a fairly strong antimicrobial activity even in a crude state, especially against Gram-positive bacteria. The antimicrobial activities are observed not only in vitro but also in vivo when the test animals are administered the antibiotic T-2636 per 05 or parenterally.

Results of tests are shown by way of examples as follows:

The respective acute toxicities of T-2636-A, -B, -C and -D are observed on 4-weeks old mice by intraperitoneal injection.

LD tmgJkg.)

T-Z636-A 400 (i.p.) T-2636-B about 400 (i.p.) T-2636-C 400 (i.p.) T-Z636-D 400 (i.p.)

The respective medium effective does (ED of T-2636-A and -C are observed on 4-weeks old mice infected with Slaphlococcus pyogenes by subcutaneous injection.

ED, (mg./kg.)

T-2636-A 5.04 T-2636-C L56 Triacetyloleundomycin (control) 30 Antimicrobial spectra:

Common bacteria as the test organisms were cultured on bouillon agar at 37 C. for 18 hours. Acid fast bacteria were cultivated on glycerin bouillon agar at 37C. for 40 hours. In case of fungi or yeast, glucose bouillon agar was used as assay medium and the incubation was carried out 40 hours at 28 C.

The respective minimum inhibitory concentrations of antibiotics T-2636 are shown in the following table:

Minimum inhibitory concentration (kg/ml.)

Test organisms A B C D M Bacillus ccreus 50 2O 100 100 Bacillus brevia...... 100 20 5 100 100 Mycobacterium aviumc 100 50 100 100 20 M ycabacteriu'm phlei. 100 20 50 100 20 M ycobacterz'um sp. ATCC-607. 100 100 100 100 20 Piricularia ory/zae 100 100 100 100 1-2. 0 Penicillium chrysogenum. 100 100 100 100 2.0 Aspergillas niger 100 100 100 100 2.0 Saccharomyces ccrcvisiae. 100 100 100 100 2. 0 Candida albicans 1 100 100 100 100 2.0

M: Mycelia extract.

Synergistic efi'ecl in antimicrobical activities:

Dilution method on bouillon agar Minimum inhibitory It is concluded from the above-mentioned physicochemical properties that each of antibiotics T-2636A, B, -C and D is a new antibiotic different from any known antibiotics. While T-2636-A has some resemblance to Bundlin A, it has a different melting point, specific rotation. infrared absorption spectrum, and solvent solubility from those of Bundlin A (Shinichi Kondo et al., Japanese Patent Publication No. 4848/1962; J.M.J. Sakamoto et al., Journal of Antibiotics lS-A 98, (1962)). T-2636-B can be regarded as an antibiotic bearing resemblance to Lankamycin (E. G'a'umann et al., Japanese Patent Publication No. l6700/l962, Helvetica Chimica Acta 43 60l (1960)), but has a melting point, molecular weight, ultraviolet spectrum, infrared spectrum, especially 600-800 cm. value, different from those of the known Lankamycin. T-2636-C can be regarded as an antibiotic bearing resemblance to Lankacidin (E. Gat'imann et al., Japanese Patent Publication Number 16700/1962, Helvetica Chimica Acta 43 601 (1960)). But the melting point, specific rotation, elementary analysis and color reaction with l FeCl, in ethanol are different from those of the known Lankacidin, and also the antibacterial activity of T-2636-C against Gram positive bacteria is higher than that of Lankacidin. T-2636D bears some resemblance to Bundlin B (Sinichi Kondo et al., supra and Sakamoto et al., supra), but has an elementary analysis and infrared spectrum different from those of Bundlin B.

Staphylococci are pyogenic or pus-forming bacteria. They tend to produce circumscribed lesions, e.g. in the form of abscesses and the like, which often occur in the skin. Staphylococci are the cause of furuncles and of carbuncles and other common wound infections. The new products of the present invention are useful in topical preparations for the treatment of this type ofinfection in mammals (dogs, cats, humans, etc.). Thus, a useful preparation for topical application to an infection due to Staphylococcus aureaus is as follows:

into i gram of wool fat is uniformly incorporated one of the following itemized components:

(l) 100 mg. .of T-2636-A; (2) 20 mg. of T2636C; (3) 25 mg. 0fT2636-A and 25 mg. of T-2636B; or (4) 100 mg. of T-2636-l00 mg. of T-2636-D, and the mixture is then admixed uniformly with sufficient white petrolatum to make 10 grams of ointment.

This is topically applied in amount sufficient to cover the wound being treated, with gentle rubbing in, the application being made at least once daily and being repeated several times daily, if necessary or desired.

Due to the disclosed bactericidal and bacteriostatic properties of the new products of the invention, they are useful e.g. to disinfect hospital apparatus, etc. which are generally exposed to pathogenic Gram-positive bacteria of the type which are sensitive to such products, as aforementioned. Disinfection is effected by application or spraying of a solution (e.g. methanolic or ethanolic, etc.) containing one of the following itemized components: (1) 200 pg/ml. of T-2636A; (2) 20 ig/ml. of T-2636-C; (3) 50 ugJml. of T-2636-A and 50 g/ml. or (4) 200 pig/ml. of T2636-B and 200 pg/ml. of T-2636-D.

The following are examples of presently preferred embodiments.

EXAMPLE 1 A 2-liter Erlenmyer flask containing 0.5 liter of an aqueous culture medium (pH 7.0) containing 2 percent of glucose, 3 percent of soluble starch, 1 percent of corn steep liquor, l percent of soy bean meal, 0.5 percent of polypeptone, 0.3 percent of sodium chloride, 0.5 percent of calcium carbonate is inoculated with 1 loop of a slant culture of Slreptomyces rochei var. volubilis l.F.O. 12507 ATCC-21250 and incubated at 37 C. on a rotary shaker for about 40 hours. (Note-Percentages in the examples are by weight).

1.5 liters of the culture broth is transferred to a SO-liters tank which contains 30 liters ofa culture medium of the same composition as mentioned above plus 30 grams of soy bean oil as an antifoaming agent, and incubation is carried out at 37 C. under an aeration of an equal volume per minute to the culture medium and an agitation of 180 rotations per minute for 20 hours. l liters of thus-obtained culture broth is used as a seed for the following fermentation.

A 200-liters tank containing 100 liters of a culture medium of the same composition and soy bean oil as an antifoaming agent is inoculated under submerged aerobic condition (aeration: 100 liters/minute, and agitation: 200 r.p.m.

The proceeding of the fermentation is as follows:

*du: Dilution unit by agar dilution method.

100 liters of thus-obtained culture broth is adjusted at pH 5 to 6 and filtered in the presence ofa filter aid (e.g. 5 to 10 percent of Hyplo Supercel). 72 liters of the filtrate is adjusted to pH 7, and extracted three times with 24 liters each time of ethyl acetate. 57 liters of the combined extract is washed with water, dried and concentrated below 40 C. in vacuo to obtain 2.25 liters ofa concentrate.

The concentrate is successively washed twice with one sixth the volume each time of hydrochloric acid (pH 3), twice with one-sixth the volume each time of water, twice with one sixth the volume each time ofsodium hydroxide (pH 8.5) and twice with one sixth the volume each time of water, and then dried with sodium sulfate overnight.

The concentrate thus treated is further concentrated, and thereto is added 1 liter of a mixture of diethyl ether and petroleum ether (1:20) or 1 liter of n-hexane to give 12 grams ofcrude substance.

EXAMPLE 2 10 grams of the crude substance is dissolved in 100 ml. of a mixture of chloroform and ethyl acetate (1:1), and is chromatographed on 300 grams of silica gel (0.05 to 0.2 mm.). Elution with 1 liter of diethyl ether or benzene affords antimicrobially inactive yellow substance. Subsequent running of diethyl ether of (a) 1 liter of a mixture of diethyl ether and ethyl acetate 1:1 1 liter of ethyl acetate, 1 liter of a mixture of ethyl acetate and acetone 1:1) or (b) 1 liter of chloroform and 2 liters of a mixture of chloroform and methanol (95:5) in that order, results in elution of most of the antibiotically effective fractions.

The combined effective fractions are concentrated to give about 5.5 grams of antibiotic T-2636 as yellow powder. When 200 grams of alumina in place of silica gel is employed as the carrier, the same procedure gives about 3.5 grams of the same yellow powder.

5 grams of the yellow powder obtained in example 11 is subjected to thin layer chromatography with 1.3 kg. of silica gel as a carrier and a combined solvent of diethyl ether and ethyl acetate (3:1) as a developer to separate T-2636-A, -B, -C and -D. When each fraction is unable to separate clearly, they are again chromatographed on the same carrier and as a developer with another mixture consisting of diethyl ether and ethyl acetate (1 to 4:1), chloroform and methanol (95 to 98:5 to 2), methyl ethyl ketone and diethyl ether 1:2 to 3), and the effective fractions are extracted each with ethylacetate and a small amount of acetone, or ethylacetate and water, washed, dried and concentrated, followed by recrystallization to give T2636-300 mg.), T-2636-B mg), T-2636-C (40 mg.) and T-2636D mg.).

EXAMPLE 3 10 grams of the crude yellow powder obtained in the manner as in example 1 is subjected to chromatography utilizing 500 grams of silica gel (0.05 to 0.20 mm.), and washed with 1 liter of benzene to remove impurities, then successively eluted with 1 liter of a series of solvents of benzene and ethyl acetate (8:2),(7:3), (6:4), (5:5), (4:6),(3z7), (2:8) and 1 liter of ethyl acetate, in that order whereupon T-2636A, B, C and -D are obtained in the fractions eluted by the above-mentioned solvent (8:2) and (7:3), (5:5), (4:6) and (3:7), (2:8) and ethyl acetate, respectively.

The procedure is repeated again to obtain pure T-2636A (500 mg.), -B (700 mg), C (20 mg.) and -D (800 mg). Almost the same result can be attained when diethyl ether is employed as a developer instead of benzene.

EXAMPLE 4 2 liters ofa seed culture broth obtained in the same manner as in example 1 is transferred to a 200-liter tank which con tains 100 liters of an aqueous medium containing 5 percent of soluble starch, 2 percent of corn steep liquor, 0.5 percent of polypeptone, 0.2 percent of potassium monohydrogenphosphate, 0.5 percent of calcium carbonate (pH 7.0) and 100 grams of silicone as an antifoaming agent. Incubation is conducted at 37 C. under an aeration of an equal volume per minute to the culture medium and an agitation of 200 rotation per minute for 90 hours, while supplying additional 300 grams ofsilicone.

The course of the fermentation is shown as follows:

In the same manner as in example 1,T-2636-B (200 mg.) and -D (300 (mg.) are recovered from 100 liters of the culture broth obtained above.

What is claimed is:

l. A method for preparing at least one antibiotic from the group of T-2636-A, T-2636-B, T-2636-C and T-2636-D as defined in claims 9, l0, l1 and 12, respectively, which comprises culturing Streptomyces rochei var. volubilis in a culture medium containing assimilable carbon source and digestible nitrogen source under aerobic conditions until at least one of said antibiotics is substantially accumulated in the culture broth, and recovering at least one of the said accumulated antibiotics therefrom.

2. A method according to claim 1, wherein the antibiotic is T-2636-A.

3. A method according to claim l, wherein the antibiotic is T-2636-B.

4. A method according to claim 1 wherein the antibiotic is T-2636-C.

5. A method according to claim 1, wherein the antibiotic is T-2636D.

6. A method according to claim 1, wherein the antibiotic is substantially a mixture of at least two members selected from the group consisting of T-2636-A, T-2636-B, T-2636-C and T-2636-D.

7. T-2636-A, characterized by thefollowing properties:

1. it forms white plate crystals melting at 200 to 205 C.

(recrystallized from diethyl ether);

2. its molecular weight is about 500;

3. its elementary analytical values are 64.73110 percent of carbon, 7.00105 percent of hydrogen and 2.80105 percent of nitrogen;

4. it is insoluble or not easily soluble in water, n-hexane,

' petroleum ether or isopropylether, but easily soluble in diethyl ether, ethyl acetate, acetone, methanol, ethanol, chloroform or methylene chloride;

5. it is negative to the Erythromycin Test and the ferric "'chloride reaction, but positive to the Carbomycin Test and Molischs reaction;

. it shows infrared absorption bands in KBr disc at the wave numbers, in cm", of 3550(M), 3400(M), 2960(W), 2900(W), 2850(W), 1755(8), 1725 (sh), 1720(vs),

1700(8), 1690(8), 1650(W), 1500(M), 1450(W), 1440(W), 1360(M), 1320(M), 1260(vs), 1140(M), 1100(W), 1010(M), 960(M), 950(M), 920(W),

860(W), 810(W), 800(W), 740(W), 680(W), 650(W), 620(W) and 580(W); its ultraviolet absorption: h =22812 mu;

its specific rotation: [a],,""=22 1120 (C=0.5, in methanol). T-2636-B, characterized by the following properties: it forms colorless plate crystals melting at 205 to 207 C.

(recrystallized from diethyl ether);

. its molecular weight is about 850;

3. its elementary analytical values are 59.27110 percent of carbon, 8.60105 percent of hydrogen, percent of nitrogen;

4. it is negative to the Carbomycin Test, but positive to the Erythromycin Test and Molisch's reaction;

5. it shows infrared absorption bands in KBr disc at the wave to -00 pow numbers, in cm, of 3500(8), 2960(8), 2930(M), 1750(vs), 1730(vs), 1710(8), 1630(W), 1460(M), 1380(8), 1340(M), 1250(S), 1220(vs), 1180(8), 1150(8), 1110(8), 1090(8), 1050(8), 1030(8),

ble in n-hexane'; 5. it is negative to the ferric chloride reaction, the Erythromycin Test and the Carbomycin Test, but is positive to Molischs reaction; 6. it shows infrared absorption bands in KBr disc at the wave numbers, in cm", of 3450(8), 3400(8), 2940(W), 2880(W), 1755(8), 1730(sh), 1710(vs), 1680(vs), 1520(M), 1450(M), 1402(M), 1360(M), 1325(M), 1310(M), 1260(8), 1220(M), 1160(M), 1137(M), 1100(W), 1055(M), 1048(M), 1005(8), 962(8),

875(W), 850(W), 830(W), 816(W), 800(W), 742(W), and 680( W 7. it shows ultraviolet absorption: A =22612 mu;

8. it shows specific rotation: [a] =237120 (C=l.0,

in ethanol).

10. T-2636-D, characterized by the following properties:

1. it forms colorless needles melting at to 191 C. (recrystallized from a mixture of methanol and ethyl acetate) 2. its molecular weight is about 500;

3. its elementary analytical values are 63.95110 percent of carbon, 7.34105 percent of hydrogen and 3.1 110.5 percent of nitrogen;

4. it is hardly soluble in diethyl ether, chloroform or ethyl acetate, slightly soluble in acetone, and soluble in methanol or ethanol;

5. it is negative to the Erythromycin Test, ninhydrin reagent and Benzidine reagent, but is positive to the Carbomycin Test and Molisch's reaction;

6. it shows evident infrared absorption bands at the wave numbers, in cm, of 3350(8), 3320(vs), 2960(M), 2930(W), 1740(5), 1725(vs), 1705(vs), 1650(vs), 1560(S), 1450(W), 1370(M), 1320(M), 1240(vs), 1200(M), 1170(W), 1130(8), 1080(M), 1040(M), 1020(8), 1010(M), 960(8), 920(W), 880(W) and 710(W);

Claims (35)

1. 2. its molecular weight is about 850;
2. 2. A method according to claim 1, wherein the antibiotic is T-2636-A.
3. 2. its molecular weight is about 500;
4. 2. its molecular weight is about 500;
5. 3. its elementary analytical values are 63.95 + or - 1.0 percent of carbon, 7.34 + or - 0.5 percent of hydrogen and 3.11 + or - 0.5 percent of nitrogen;
6. 3. its elementary analytical values are 65.30 + or - 1.0 percent of carbon, 7.2 + or - 0.5 percent of hydrogen and 3.13 + or - 0.5 percent of nitrogen;
7. 3. its elementary analytical values are 64.73 + or - 1.0 percent of carbon, 7.00 + or - 0.5 percent of hydrogen and 2.80 + or - 0.5 percent of nitrogen;
8. 3. A method according to claim 1, wherein the antibiotic T-2636-B.
9. 3. its elementary analytical values are 59.27 + or - 1.0 percent of carbon, 8.60 + or - 0.5 percent of hydrogen, 0 percent of nitrogen;
10. 4. it is negative to the Carbomycin Test, but positive to the Erythromycin Test and Molisch''s reaction;
11. 4. A method according to claim 1, wherein the antibiotic is T-2636-C.
12. 4. it is insoluble or not easily soluble in water, n-hexane, petroleum ether or isopropylether, but easily soluble in diethyl ether, ethyl acetate, acetone, methanol, ethanol, chloroform or methylene chloride;
13. 4. it is soluble in ethyl acetate, acetone, ethanol or methanol, but hardly soluble in diethyl ether and insoluble in n-hexane;
14. 4. it is hardly soluble in diethyl ether, chloroform or ethyl acetate, slightly soluble in acetone, and soluble in methanol or ethanol;
15. 5. it is negative to the ferric chloride reaction, the Erythromycin Test and the Carbomycin Test, but is positive to Molisch''s reaction;
16. 5. it is negative to the Erythromycin Test, ninhydrin reagent and Benzidine reagent, but is positive to the Carbomycin Test and Molisch''s reaction;
17. 5. it is negative to the Erythromycin Test and the ferric chloride reaction, but positive to the Carbomycin Test and Molisch''s reaction;
18. 5. A method according to claim 1, wherein the antibiotic is T-2636-D.
19. 5. it shows infrared absorption bands in KBr disc at the wave numbers, in cm 1, of 3500(S), 2960(S), 2930(M), 1750(vs), 1730(vs), 1710(S), 1630(W), 1460(M), 1380(S), 1340(M), 1250(S), 1220(vs), 1180(S), 1150(S), 1110(S), 1090(S), 1050(S), 1030(S), 1000(vs), 960(M), 940(W), 910(W), 900(W), 880(W), 800(W) and 520(W);
20. 6. it shows specific rotation: ( Alpha )D24 -92 + or - 10* (C 1.0, in ethanol);
21. 6. A method according to claim 1, wherein the antibiotic is substantially a mixture of at least two members selected from the group consisting of T-2636-A, T-2636-B, T-2636-C and T-2636-D.
22. 6. it shows infrared absorption bands in KBr disc at the wave numbers, in cm 1, of 3550(M), 3400(M), 2960(W), 2900(W), 2850(W), 1755(S), 1725(sh), 1720(vs), 1700(S), 1690(S), 1650(W), 1500(M), 1450(W), 1440(W), 1360(M), 1320(M), 1260(vs), 1140(M), 1100(W), 1010(M), 960(M), 950(M), 920(W), 860(W), 810(W), 800(W), 740(W), 680(W), 650(W), 620(W) and 580(W);
23. 6. it shows evident infrared absorption bands at the wave numbers, in cm 1, of 3350(S), 3320(vs), 2960(M), 2930(W), 1740(S), 1725(vs), 1705(vs), 1650(vs), 1560(S), 1450(W), 1370(M), 1320(M), 1240(vs), 1200(M), 1170(W), 1130(S), 1080(M), 1040(M), 1020(S), 1010(M), 960(S), 920(W), 880(W) and 710(W);
24. 6. it shows infrared absorption bands in KBr disc at the wave numbers, in cm 1, of 3450(S), 3400(S), 2940(W), 2880(W), 1755(S), 1730(sh), 1710(vs), 1680(vs), 1520(M), 1450(M), 1402(M), 1360(M), 1325(M), 1310(M), 1260(S), 1220(M), 1160(M), 1137(M), 1100(W), 1055(M), 1048(M), 1005(S), 962(S), 875(W), 850(W), 830(W), 816(W), 800(W), 742(W), and 680(W);
25. 7. it shows ultraviolet absorption: lambda maxEtOH 226 + or -2 m Mu ;
26. 7. it shows ultraviolet absorption: lambda maxMeOH 8. 2 m Mu ;
27. 7. its ultraviolet absorption: lambda maxMeOH 228 + or - 2 m Mu ;
28. 7. T-2636-A, characterized by the following properties:
29. 8. its specific rotation: ( Alpha )D25 -221 + or - 20* (C 0.5, in methanol).
30. 8. T-2636-B, characterized by the following properties:
31. 8. its specific rotation: ( Alpha )D25 -220 + or - 20* (C 0.5 in methanol).
32. 8. it shows specific rotation: ( Alpha )D2525-237 + or - 20* (C 1.0, in ethanol).
33. 9. T-2636-C, characterized by the following properties:
34. 10. T-2636-D, characterized by the following properties:
35. 11. An antibiotic substance which is substantially a mixture of at least two members selected from the group consisting of T-2636-A, T-2636-B, T-2636-C and T-2636-D as defined in claims 9, 10, 11 and 12, respectively.

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