US3794721A - Antibiotic steffimycin b and process for the preparation thereof - Google Patents

Antibiotic steffimycin b and process for the preparation thereof Download PDF

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Publication number
US3794721A
US3794721A US00283908A US3794721DA US3794721A US 3794721 A US3794721 A US 3794721A US 00283908 A US00283908 A US 00283908A US 3794721D A US3794721D A US 3794721DA US 3794721 A US3794721 A US 3794721A
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steffimycin
medium
tan
growth
streptomyces
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US00283908A
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T Brodasky
F Reusser
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Pharmacia and Upjohn Co
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Upjohn Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
    • C07G11/00—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • NMR Nuclear Magnetic Resonance
  • Steffimycin B is distinguished from steffimycin by a characteristic papergram R, as shown in FIG. 3 of the accompanying drawings.
  • Matrix Schleicher and Scheull 589 Blue Ribbon paper Detecting Organism: S. lutea Antibacterial Activity Of Stcffimycin B Minimum Inhibitory Con Inhibitory in mcg./ml.
  • BHI Brain Heart Infusion Broth, Difco, Detroit, Mich.
  • Assay tubes 13 mm. X 100 mm.
  • Test organisms grown for 18 hours at 37 C. were used to inoculate the test medium. The assays were read at 17 hours.
  • elgreteus had poor growth on the control (basal medium without added carbon compound), D-xylose, L- arabinose, lactose, raffinose, inulin, dulcitol, D- mannitol, D-sorbitol, salicin, sodium acetate, and sodium citrate; moderate growth on rhamnose, D- fructose, maltose, sucrose, dextrin, and sodium succinate; good growth on D-galac-tose, D- glucose, D-mannose, cellobiose, soluble starch, glycerol, and inositol; no growth on phenol, cresol, sodium formate, sodium oxalate, sodium tartrate, and sodium salicylate.
  • S. elgreteus had good aerial growth on Bennetts agar at 18C-28C and on maltosetryptone agar at 24C28C. The culture had fair vegetative growth on these media at 37C and no growth in 24 hours at 45C and 55C.
  • Antibiotic-producing properties The culture produces steffimycin B and some steffisburgensimycin (U. S. Pat No. 3,309,273), also known as steffimycin.
  • Type culture Streptomyces elgreteus sp. n. UC 5453.
  • a new soil isolate capable of producing the antibiotic steffimycin has been characterized and determined to be a new streptomycete species.
  • the new culture is distinctly different from the steffimycin-producer, Streptomyces steffisburgensis (Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol.
  • the culture has lavender-gray to graypink aerial growth, is melanin negative, solubilizes tyrosine, does not solubilize xanthine, and has straight sporophores bearing long spores with smooth to curly to hairy surface appearance.
  • S. stefflsburgensis has gray aerial growth, is melanin positive, does not solubilize tyrosine, solubilizes xanthine, and has short, straight, open spiral to spiral sporophores bearing short spiny (Dietz, A. 1967. Streptomyces steflisburgensis sp. n. J. Bacteriol.
  • Streptomyces elgreteus Dietz sp. n the culture characterized herein be designated Streptomyces elgreteus Dietz sp. n. It is understood that this type species is also to becqnsigered the type variety in accordance with Rule 7 of the International Code of Nomenclature of Bacteria (INTERNATIONAL CODE OF NOMENCLATURE OF BACTERIA. 1966. Edited by the Editorial Board of the Judicial Commission of the TABLE "2 International Committee on Nomenclature of Bacteria. Intern. J. System. Bacteriol. 162459-490).
  • Streptomyces elgreteus NRRL 5634 The characteristics of Streptomyces elgreteus NRRL 5634, are given in the following tables:
  • the organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material.
  • a carbon source for example, an assimilable carbohydrate
  • a nitrogen source for example, an assimilable nitrogen compound or proteinaceous material.
  • Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like.
  • Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously.
  • Trace metals for example, zinc, magnesium, cobalt, iron, and the like,
  • Production of the compounds of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 40 C., and preferably between 20 and 32 C. Ordinarily, optimum production of the compounds is obtained in about 2 to 10 days.
  • the medium normally remains basic during the fermentation.
  • the final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.
  • Steffimycin B forms salts with alkali metals, alkaline earth metals, and amines.
  • Metal salts can be prepared by dissolving Steffimycin B in methanol, adding a dilute metal base until the pH of the solution is about 7 to 8, and freeze drying the solution to provide a dried resi- .due consisting of the Steffimycin B metal salt.
  • Steffimycin B metal salts include the sodium, potassium, and calcium salts.
  • Amine salts of Steffimycin B, including those with organic bases, such as primary, secondary, and tertiary, mono-, di-, and polyamines also can be formed using the above-described rather commonly employed procedures. Other salts are obtained with therapeutic effective bases which impart additional therapeutic effects thereto.
  • Such bases are, for example, the purine bases such as theophyllin, theobromin, caffeine, or derivatives of such purine bases; antihistaminic bases which are capable of forming salts with weak acids; pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like; phenylalkylamines such as adrenaline, ephedrine, and the like; choline, and others.
  • the purine bases such as theophyllin, theobromin, caffeine, or derivatives of such purine bases
  • antihistaminic bases which are capable of forming salts with weak acids
  • pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like
  • phenylalkylamines such as adrenaline, ephedrine, and the like
  • choline and others.
  • Steffimycin B and its salts are active against Staphylococcus aureus and Streptococcus faecalis and used as disinfectants on various dental and medical equipment contaminated with Staphylococcus aureus. Further, since steffimycin B and its salts are active against Streptococcus hemolyticus, they can be used to disinfect instruments, utensils or surfaces where the inactivation of this microorganism is desirable.
  • the new compound of the invention is an acidic chemical compound. it is soluble in halogenated hydrocarbons and lower alcohols. it is relatively insoluble in hydrocarbons and water.
  • steffimycin B A variety of procedures can be employed in the isolation and purification of steffimycin B, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, absorption on resins, and crystallization from solvents. Solvent extraction procedures are preferred for commercial recovery inasmuch as they are less time consuming and less expensive.
  • steffimycin B is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation.
  • the antibiotic is then removed from the filtered or centrifuged broth by extraction.
  • water-immiscible organic solvents in which it is soluble for example, l-butanol, methyl ethyl ketone, benzene, and methylene chloride (preferred) can be used.
  • the extraction is carried on after the filtered beer is adjusted to a pH of about 2 to 4 with a mineral acid.
  • the methylene chloride extractions are combined and evaporated to dryness under vacuum.
  • Steffimycin B can be recovered in the pure form from a methylene chloride extract, as described above, by use of silica gel chromatography.
  • steffimycin B can be recovered from fermentation filtered broth by use of a resin comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene.
  • Suitable resins are Amberlite XAD-l and XAD-2 disclosed in U. S. Pat. No. 3,515,717. The resin is eluted with an organic or aqueous organic solvent in which the sorbed antibiotic is soluble.
  • An alternative purification procedure involves subjecting a crude preparation of steffimycin B to successive crystallizations from lower alcohols, for example, methanol, ethanol, propanol, and the like; chlorinated hydrocarbons, for example, chloroform, methylene chloride, and the like; hydrocarbon solvents, for example, pentane, hexane, 'heptane, and the like; or admixtures of any of these.
  • lower alcohols for example, methanol, ethanol, propanol, and the like
  • chlorinated hydrocarbons for example, chloroform, methylene chloride, and the like
  • hydrocarbon solvents for example, pentane, hexane, 'heptane, and the like
  • admixtures of any of these for example, pentane, hexane, 'heptane, and the like.
  • the presterilization of the seed medium is 7.2
  • the seed is grown for three days at 28 C. on a Gump rotary shaker operating at 250 r.p.m.
  • the assay was an agar disc-plate assay against the microorganism Sarcina lutea.
  • the assay against Sarcina lutea is conducted on agar buffered to pH 7.4 with pH 7.4 phosphate buffer.
  • a unit volume (0.08 ml.) of solution containing the substance to be assayed is placed on a 12.7 mm. paper disc which is then placed on a Penassay Seed Agar (Difco), l mm.'deep, plate seeded with the assay organism.
  • the agar plate is then incubated for 16-18 hours at 32 C.
  • a biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm.
  • the potency of such beer is BU per ml.
  • Part B. Recovery 7 Whole fermentation beer, obtained as described above, is filtered using diatomaceous earth as a filter aid. The filter cake is washed with water. The wash is combined with the clear beer and the combined solution is extracted twice with an equal volume of methylene chloride. The methylene chloride extracts are combined and evaporated to dryness at less than 40 C. under vacuum. The potencies of the clear broth, spent broth and extracts are determined by dip spotting (12 mm. discs) on S. lutea seeded agar. (The potency of the preparation is determined by preparing a 50; meg/ml.-
  • composition of the preparation is determined by thin layer chromotography (tlc) on silica gel using a mobile phase consisting of the following solvents:
  • This fraction is cut so that none of the second red-orange band which contains steffimycin is collected.
  • the purity of steffimycin B is checked by tlc on silica gel using the above mobile phase.
  • the purified steffimycin B, thus obtained, does not contain any other colored (or fluorescent) material by tlc.
  • Steffimycin B is obtained in its pure crystalline form by subjecting the fraction containing steffimycin B, as described above, to successive crystallization procedures from methanol.
  • Steffimycin B a compound which: a. is effective in inhibiting the growth of various Gram-positive bacteria;
  • f. has a characteristic nuclear magnetic resonance spectrum as shown in FIG. 2 of the drawings;
  • g. has a characteristic R; in the chromatographic system, as shown in FIG. 3, which differentiates it from steffimycin; and
  • aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.

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  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Communicable Diseases (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Compounds Of Unknown Constitution (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
US00283908A 1972-08-28 1972-08-28 Antibiotic steffimycin b and process for the preparation thereof Expired - Lifetime US3794721A (en)

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US (1) US3794721A (zh)
JP (1) JPS5641231B2 (zh)
DE (1) DE2339136A1 (zh)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4209611A (en) * 1979-04-06 1980-06-24 The Upjohn Company Dihydrosteffimycin compounds
US4264726A (en) * 1979-04-06 1981-04-28 The Upjohn Company Process for producing 10═dihydrosteffimycin and 10═dihydrosteffimycin B and microorganisms for producing same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2354978A1 (fr) * 1976-06-17 1978-01-13 Saint Gobain Procede pour le revetement d'un support, notamment d'un vitrage, avec une mince couche d'oxyde metallique, et applications de ce procede

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3309273A (en) * 1965-12-29 1967-03-14 Upjohn Co Antibiotic steffisburgensimycin and method of producing

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3309273A (en) * 1965-12-29 1967-03-14 Upjohn Co Antibiotic steffisburgensimycin and method of producing

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4209611A (en) * 1979-04-06 1980-06-24 The Upjohn Company Dihydrosteffimycin compounds
US4264726A (en) * 1979-04-06 1981-04-28 The Upjohn Company Process for producing 10═dihydrosteffimycin and 10═dihydrosteffimycin B and microorganisms for producing same

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NL7311551A (zh) 1974-03-04
JPS4942895A (zh) 1974-04-22
GB1387080A (en) 1975-03-12
DE2339136A1 (de) 1974-03-14
FR2197574A1 (zh) 1974-03-29
JPS5641231B2 (zh) 1981-09-26
FR2197574B1 (zh) 1977-09-09

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