US 3852424 A
The antibiotic ferrimycin A, its components ferrimycin A1 and ferrimycin A2 and the corresponding iron-free compounds desferrimycin A, desferrimycin A1 and desferrimycin A2, and also pharmaceutical preparations which contain these products, and a process for the manufacture of these substances and mixtures containing them. The antibiotic ferrimycin belongs to the sideramycins, a class of iron-containing or iron-binding antibiotics, to which also the antibiotics grisein, albomycin and A 1787 belong. The sideramycins are characterized by their antibiotic effect being antagonized by the ferrioxamines; see Bickel et al., Experientia 16 (1960) 129.
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Description (OCR text may contain errors)
United States Patent [191 Gaeumann, deceased et al.
[4 11 Dec 3, 1974 PURIFIED FERRIMYCIN AND PROCESS FOR OBTAINING SAME  Inventors: Ernst Gaeumann, deceased, late of Zurich, Switzerland; Tino Gaeuman,
legal representaitve, Mont Sur Lausanne; Vladimir Prelog, Zurich; Ernst Vischer, Basel; Hans Bickel, Binningen, all of Switzerland  Assignee: Ciba-Geigg Corporation, Ardsley,
 Filed: Oct. 19, 1966 21 Appl. No.: 626,650
Related US. Application Data . Continuation-impart of Ser. No. 245,349, Dec. 11,
1962, abandoned',,which is a continuation-impart of Ser. No. 32,294, May 27, 1960, abandoned, which is a continuation'in-part of Ser. No. 749,616, July 21, 1958, abandoned.
so Foreign Application Priority Data July 26, 1957 Switzerland 48868/57 July 8, 1958 Switzerland... 61491/58 May 29, 1959 Switzerland..... 73755/59 Mar, l8, 1960 Switzerland 3062/60  U.S. C1. 424/118, 195/80  Int. Cl A6lk 21/00  Field of Search 424/118; 195/80 [5 1 References Cited UNITED STATES PATENTS 3,033,760 5/1962 Gauemann 195/80 OTHER PUBLICATIONS Antibiotics & Chemotherapy, 12, 3445 1962 Experientia, 16, (1960) article commencing page 128.
Helv. Chim. Acta, 43, (1960), pg. 901
Arch. Mikrobiol, 36, (1960) article commencing page 325.
Helv. Chim. Acta, 43, (1960), article commencing page 2105.
Arch. Mikrobiol, 38, (1961) page 326-338;
Primary Examiner-Jerome D. Goldberg Attorney, Agent, or FirmJoseph G. Kolodny 57 ABSTRACT 4 Claims, 8 Drawing Figures I PATENIEL; DEC 31974 SHEET 2 BF 8 v cor PATENTEL EEC 74 SPEET 3 0F 8 PAIENHL WI 31974 T acw 8 com com camp
ucm ooou coma coon
as on in? 2 5 g PURIFHED ,FERRIMYCIN AND PROCESS FOR OBTAINING SAME This is a continuation-in-part of our application Ser. No. 245,349, filed Dec. 11, 1962 (now abandoned), which is itself a continuation-in-part of our application Ser.No. 32,294, filed May27, 1960 (now abandoned), which is itself a continuation-in-part of application Ser. No. 749,616, filed July 21, 1958 (now abandoned), by Vladimir Prelog et al.
The invention relates to a new watersoluble antibiotic which we formerly designated as A 9578 or as pilosomycin and is now called ferrimycin A, its components ferrimycin A and ferrimycin A and the correspondingiron-free compounds desferrimycin A, desferrimycin A and desferrimycin A and also pharmaceutical preparations which contain these products, and a process for the manufacture of these substances and mixtures containing them.
The antibiotic ferrimycin belongs to the sideramycins, a class of iron-bindingantibiotics, to which also the antibiotics grisein, albomycin and A 1787 belong. The sideramycins are characterized by their antiobiotic effect being antagonized by the ferrioxamines; see
Bickel et al., Experientia 16 (1960) 129.
Ferrimycin and its components are red-brown, basic substances which are readily soluble in acids and strongly polar solvents, such as water, methanol, dimethylformamide, glycol,.methyl cellolsolve. They also dissolve in benzyl alcohol, in phenols or in mixtures of phenols and lipoid solvents. They consist of hydrocarbon, hydrogen, oxygen and nitrogen, and also contain iron or are capable of binding iron.-Ferrimycinis a mixture of closely related compounds which consist of two main components, ferrimycin A and ferrimycin B. Ferrimycin A can be separated into two components, ferri mycin A l and ferrimycin A 2. The properties of these various components are described below.
It has not hitherto been possible to obtain ferrimycin or the above-mentioned components in a pure form.
The special difficulty in purifying the ferrimycinsarises principally from the fact that they are present in the culture filtrate in very small proportion in addition to large amounts of inactive substances from the nutrient solution and fermentation products having very similar physical-chemical properties. The culture filtrate contains on an average for every one part of ferrimycin 10,000l5,000 parts by weight of inactive substances which are hydrophilic, like the ferrimycins. The difficulty of enriching and purifying the ferrimycins alsov arises from their instability in a wide range of pH values. The separation of the antibiotic into its individual components is also rendered difficult owing to the fact that these components differ from one another only slightly in their R values even in chromatographically favorable solvent systems.
Antibiotic ferrimycin is obtained by the culture of a new strain of actinomycetes, of the species Streptomyces griseflavus, which has been isolated from a sample of soil collected in Boston, Mass, and which is kept in our laboratories, and also in the Eidg. Technische Hochschule, Institut fur spezielle Botanik under the designation A 9578 and in the U.S. Department of Agriculture, Agricultural Research Service, Northern Utilization Research and Development Division, Peoria, 111., under the designation NRRL 2717.
Streptomyces griseoflavus NRRL 2717 belongs to the species S treptomyces griseoflavus (Krainsky) Waksman and Henrici, and differs from other members of this species in that it forms a new water-soluble antibiotic. Hitherto only one strain of Streptomyc es griseoflavus is known which produces an antibiotic. This is the so-called griseoflavine, which differs from the new antibiotic ferrimycin in its solubility in organic solvents. The air mycelium of Streptomyces griseoflavus NRRL 2717 is ash grey. The spore carriers are branched and form copious spirals generally having 2-5 turns. The spores themselves have a size of 1.0 to 1.3g. X 0.7 to
0.9 1., and have at their surface spines about 0.2 y inlength which arepointed and only slightly widened at their base. Their growth is relatively little dependent on temperature, so that the mould develops well at 18C and also at C, although the optimum temperature is between 25C and 32C.
For the purpose of identification there is described below the growth of Streptomyces griseoflavus NRRL 2717 on various nutrient media. The nutrient media numbers l-7 and also 10 are prepared as described by W. Lindenbein, Arch. Mikrobiol. 17, page 361 (1952).
Growth cloud-like colorless, no air mycellium. Sediment, reddish flocks, Well developed pellicle having cloud-like white-grey air mycellium. Substraturn brown-yellow to deep yellow. Growth initially point-like, wrinkled after 12 days and pale yellow. Growth initially point-like, later cloudy and yellowish.
' Air mycellium velvety,
initially brownish grey, later ash grey. substratum yellow. I Growth cloud-like, white- I yellow, later deep yellow, Air mycellium scanty, 1 covered with flour-likedust, floury white. substratum pale brown.
1 Growth very scanty,
liquefaction very slow (0.5 cm after 30 days). Growth cloud-like, milkwhite, no air mycellium. Hydrolysis 1 cm after 5 days.
Growth initially point-like, pale yellow, later pimply. Air mycellium velvety, white yellow to ashgrey. substratum colored brownish 1) Synthetic agar: 2) Synthetic solution:
5) Calcium malate-agar:
6) Gelatine stab culture 7). Starch plate:
' grey. 9) Carrots: Growth very scanty. Air
mycellium scanty. Substratum not colored. Ring growth and surface skin colorless. Air mycellium white-grey, hydrolysis slow without co-agulation, litmus blue.
l0) Litmus milk:
the use of Streptomyces griseoflavus NRRL 27l7 or other strains corresponding to the description thereof, but also includes the use of variants such, for example, as are obtained by selection or mutation, especially under the action of ultra-violet rays or X-rays or nitrogen mustard oils.
In order to produce antibiotic ferrimycin A or its components a strain of Streptomycetes having the properties of 'Streptomyces griseoflavus NRRL 2717 is incubated aerobically, for example, in an aqueous nutrient solution containing a source of carbon and of nitrogen and inorganic salts until the solution exhibits a substantial antibacterial action, and the antibiotic ferrimycin is then isolated.
As source of carbon there may be used, for example, glucose, saccharose, lactose, mannitol, starches or glycerine. As nitrogenous nutrient substances and, if desired, growth promoting substances there may be mentioned amino-acids, peptides and proteins, and also their degradation products such as peptone or tryptone, and also meat extracts, water-soluble constituents of cereal grains, such as maize or wheat, or of the distillation residues from the manufacture of alcohol, or yeast, or beans, especially soya bean plants, or seeds,
. for example, those of cotton plants etc., and also ammonium salts and nitrates. Among other inorganic salts the nutrient solution may contain chlorides, carbonates, sulfates of alkali metals, alkaline earth metals, magnesium, iron, zinc and manganese.
The incubation is carried out aerobically, for example, in a quiescent surface culture or advantageously submerged while agitating or stirring with air or oxygen in agitated flasks or known fermenters. The. temperature may be within the range of 18C to 40C. The nutrient solution generally exhibits a substantial antibacterial action after 1 to 5 days.
. In order to isolate antibiotic ferrimycin the following methods may be used: The mycelium is separated from the culture filtrate, and the greater part of the antibiotic is-found in the culture filtrate. However, considerable quantities of the antibiotic are adsorbed on the mycelium. It is therefore of advantage to wash to mycelium well. For this purpose water or an aqueous organic solvent may be used, such as an alcohol, for example, aqueous methanol. In order to recover the antibiotic from the culture filtrate and to purify it, various methods may be used which can be employed singly or in combination with one another. It is of advantage during these operations to'main'tain the culture solution at pH value within the range of 35.
1. For isolating the crude ferrimycin from the culture filtrate, and adsorption medium may be used, for example, an active carbon such as Norit, activated earths such as F ullers earth or Floridine(aluminum magnesium silicates) or a resin adsorbant such as Asmit (a meta-phenylenediamine-formaldehyde decolorizing resin). The adsorbate 'is advantageously eluted with a mixture of water and an organic solvent miscible with wateror an aqueous acid, for example, a mixture of water and methanol, water and pyridine, dilute acetic acid and methanol, or water, methanol, glacial acetic acid and butanol. Especially advantageous for the elution of a Norit-adsorbate is a mixture of 2 parts by volume of water, 1 part by volume of methanol, 1 part by volume of glacial acetic acid, and 2 parts by volume of butanol.
2. A second method for separating the antibiotic from the culture filtrate is to adsorb the antibiotic on a cation-exchanger, for which purpose resins containing acid groups, such as Amberlite IRC-5O (a carboxylic acid type ion exchanger) are especially suitable. The latter can be'used either in the acid form or in the sodium form, although a mixture of both fonns in the volumetric ratio 1:2 is particularly advantageous. Elution is advantageously carried outwith a dilute acid, e.g. methanolic hydrochloric acid.
3. The basic antibiotic can also be precipitated directly from the culture filtrate, for example by reaction with an organic acid of the type of picric acid. By treating a precipitate so obtained with a salt of an organic base, for example, with triethyl-ammonium sulfate, or
with a dilute acid, the antibiotic is obtained in the form of the corresponding salt. These operations may be carried out either in an aqueous medium or in a solvent miscible with water, such as methanol or acetone. The conversion of the sparingly soluble salts into the readily soluble salts of the antibiotic is carried out either by means of mineral acids or by treatment with ion exchanging resins, e.g. Amberlite IRA-400 (a polystyrene resin containing quaternary ammonium groups).
4. The antibiotic can be concentrated by adding to an aqueous or alcoholic-aqueous solution of the salt of the antibiotic an excess of an organic solvent miscible with water, such as acetone, dioxane etc., whereby the salt is precipitated in solid form.
5. Another method of enriching the antibiotic consists in extracting an aqueous solution thereof with a solution of phenol in chloroform in which operation both the pH of the aqueous solution and the phenol content of the chloroformic solution can be "varied. As an example, in a distribution between a solution containing in 100 cc of chloroform 100 grams of phenol and an aqueous phase having a pH of l to 6, nearly all of the antibiotic is in the organic phase, whereas, when a solution is used which contains only 33 grams of phenol in 100 cc of chloroform, it can be extracted nearly creases as the phenol content of the organic phase in- 1 creases, and decreases as the pH of the aqueous phase falls. It thus being possible to chose anydesired distribution coefficient of the antibiotic in this system, a large proportion of inactive impurities can be eliminated. by combining a small number of distributing operations. Y
6. There is still another method of enriching the antibiotic, namely, chromatography, such as adsorption chromatography on various materials, e.g. Norit' (activated carbon), alumina, magnesium silicates, silica gel, calcium sulfate, or distribution chromatography with cellulose, starch, silica gel, Celite (an infusorial earth) or the like as carrier substances, or chromatography on ion exchangers, e.g.,' Dowex (a sulfonated polystyrene), AmberlitelRC-SO (a carboxylic acid type cation exchanger) or the like. Good results have been obtained for example with distribution chromatography on cellulose using the solvent system of 4 parts by volume of butanol, 1 part by volume of glacial acetic acid, and 5 parts by volume of water.
7. The antibiotic can also be enriched by the countercurrent distribution according to Craig between two immiscible solvent phases. The following solvent systems have proved particularly successful:
a. secondary butanol 1/10 N-ammonium acetate buffer having a pH of 4.68.
b. 1 N-ammonium acetate buffer having a pH of 46-10% solution of phenol in chloroform. The distribution coefficient of the antibiotic in the system (b), and thus the location of the activity maximum in the distribution can be changed as desired on the one hand by changing the pH of the buffer solution and on the other hand by changing the phenol content of the organic phase.
The following combination of the enriching methods described above gives preparations of considerable purity. From the culture filtrate, the antibiotic is adsorbed on the buffered ion exchanger Amberlite lRC-SO and eluted by means of methanolic hydrochloric acid. At a pH of 5, the eluates are concentrated under reduced pressure, an aqueous concentrate of the antibiotic being obtained in an amount of about 1/ 100 of the volume of the nutrient solution. By method (5 above, the concentrate is distributed several times between phenol-chloroform mixtures and aqueous solutions of varying pH. Oh freeze-drying the resulting active solution there is obtained a preparation the specific activity of which is 500-1000 times higher than that of the lyophilized culture filtrate. A specific embodiment of the present invention consists in further purifying the crude ferrimycin as obtained according to the above mentioned methods by subjecting it to electrophoresis and- /or countercurrent distribution with the use of benzyl alcohol and, if desired, chromatography.
The electrophoresis is carried out in the form of a high voltage electrophoresis at 500 to 4,000 volts, in the form of zonal electrophoresis, and especially countercurrent electrophoresis. V r
In the case of zonal electrophoresis in dilute acetic acid the activity. of ferrimycin A is increased to 7,000 to 8,000 times the activity of the lyophilized culture filtrate.
times) is obtained in the counter-current electrophoresis of ferrimycin A. in this method of separation the antibiotic is present as a locally fixed cation, in that the tendency for movement by an electric field is exactly counteracted by means of an oppositely directed stream of the electrolyte. Substances that undergo electrical movement in a different manner leave the apparatus at either one of the two ends, where the electrodes are fixed.
For chromatography there are used as adsorbents preferably strong acid ion-exchange resins, such as Dowex SO-WX (a sulfonated polystyrene containing 2 percent of divinylbenzene). As eluting agents there are advantageously used basic buffer solutions of increasing concentrations, such as ammonium acetate buffers of pH value 4.6 in a molecular concentration of 0.2 to
For countercurrent distribution there are used, for
example, the following systems:
Benzyl alcohol (60 parts by weight) methylisobutylketone (44 parts by weight) aqueous sodium chloride solution of 15% strength parts by weight) 0.0 1 N-hydrochloric acid (50 parts by weight); Benzyl alcohol (66 parts by weight) rne thy lisobutylketone (3 30 are ey Weight) '1 aqueous sodium chloride solution of 5% strength (50 parts by weight) 0.01N-hydroch1oric acid (50 parts by weight). Also suitable is the system benzyl alcohol (200 parts by volume)-n-butanol (100 parts by volume) water (300 parts by volume) saturated, aqueous sodium chloride solution parts by volume) N-hydrochloric acid (6 parts by volume). By the use of of these systems the active substance is divided into the two components ferrimycin A and ferrimycin B.
The two componentsA and B ferrimycin are defined by paper-chromatography by a direct comparison of their Rf values with the Rf values of a series'of known antibiotics (2-11) in the systems A G. In the case of system H the figures represent in cm the distance travelled by the antibiotics after 16 hours. The antibiotics are detected autobiographically with Staphylococcus aureus or Bacillus subtilis.
Sy- 1a lb 2 3 4 5 6 7 8 9 10 11 stern A 0 0 0 0 0 0 0 0 0 0.92 0.07 0 B 0.49 0.63 0.05 0.05 0.17 0.02 0.02 0.66 0.55 0.92 0.32 0.22 C 0.34 0.58 0.22 0 0.02 0.22 0.03 0.72 0.62 0.93 0.39 0.11 .D 0.05 0.15 0 0 0 0 0 X X 0.92 -0 E 0.32 0.32 0 0.10 0.22 0 0 X X- 0.86 0.12 F 0.47 0.47 0.22 0.14 0.12 0.04 0.05 0.49 0.42 0.91 0.43 0.36 G 0.74 0.74 0.07 t r 0.02 X X 0.94 0.61 v 0.69 11 2.7 7.6 0 0 0 0 0. (14.5) (8.8) 27 i l X antibiotic distribution over the whole course position unsharp A water-saturated butanol B'butanol-glacial acetic acid-water (421:5) (upper phase) C water-saturated butanol 2% paratoluene-sulfo acid D water-saturated butanol 2% piperidine E butanol-pyridine-water (614:3)
F ethanol 1.5% NaCl. Whalman No. 4 impregnated with 0.95 molar Ni So 0.05 molar NaHSO G butanol-cthanol-water (1:112) H hutanol-butyl acetate-glacial acetic acid-water (l0:3:1.3:14.3) (upper phase) 1:: ferrimyein base A 1b ferrimycin base B 2 Streptomycin 3 Rislocetin A 4 Ristocetin B 5 Neomycin B 6 Viomycin 7 Chlorotetracyclin 8 Oxytetracyclin 9 Actinomycin l 10 Cycloscrin 1 1 Grisein During paper electrophoresis in an 0.1 molar acetate buffer having a pH ,value of 4.6 antibiotic ferrimycin migrates towards the cathode. The speed of migration is about half as great as that of Streptomycin.
For separation into the components A1 and A2 distribution chromatography on cellulose in the system n-butanol-0.5N-a'cetic acid (1:1) is especially suitable.
The resulting preparations have 10 to 20,000 times the acitivity of the lyophilized culture filtrate, whereas the products used as starting material have only 100 to 1,000 times the activity of the lyophilized culture fil-. trate. A ferrimycin A preparation purified in the described manner, a strongly acidic ion exchanger being used in the last purification step, shows, in the form of the dihydrochloride, the following chemical and physical properties:
N:12.95%, Fe 4.56%,
CI: 6.10%, (C)CI-I 1.99% (Roth-Kuhn), Amino-N:
2.33% (Van Slyke). Titration:
pl(*,,, (Helv. 37,1872 (1954)):
Equivalent weight: 1106. Absorption spectrum:
A max:' 228 mu, E 282 max: 319 mu, E
28.2 max: 425 mu, E 22.6
Reduction value according to C. S. Hanes, Biochem. J.23, 99 (1929); 1.7 ml l/lOO N-sodium'thiosulfate. Bound hydroxylamine according to T. Emery and J. B. Neilands, Nature 184, 1632 (1959): 0.83 mol NH OH per atom Fe.
Ferrimycin A is an orange-yellow powder which dissolves very readily in water, methanol and mixtures of phenol and chloroform, dissolves in dimethylformamide, methylcellosolve, benzyl alcohol and glacial acetic acid, dissolves sparingly in ethanol and is practically insoluble in pyrridine, propanol, butanol and the usual organic solvents, especially lipoid solvents. It can be precipitated from an aqueous solution by means of 1 picric acid, picrolonic acid and ammonium reineckate. The orange-red aqueous solutions of the antibiotic change color reversibly through claret red to pale yelalso brought about with sodium hydroxide solution. Neutral solutions do not react with potassium ferrocyanide, and acidified solutions yield colorations or precipitates of Prussian blue. The trivalent iron bound in The componentsA and A are characterized as follows:
A -dihydroch1oride: Elementary analysis (after drying for hours at 25C/ 1 0 mm over P O /KOI-I; average from three determinations) yields: C 46.71%; H 6.80%; N 12.70%; C1 6.76%; Fe (color.) 5.33%; 0 (calc. 21.70%). Titration in 80% methylcellosolve; equivalent weight: 1078; pK 4.11; 7.92; 1 1.4. These values suggest the empirical formula C l-l O N Fe, I-ICl, and the molecular weight 1051. Its ultraviolet spectrum shows maxima at 229 my. (E 336), 319 mp. (E 37) and 425 mu (E 27.6). Its infrared spectrum.(in potassium bromide) is given in FIG. 2. It shows bands at 3.00; 3.44; 6.08; 6.31; 6.90; 7.35; 7.45; 7.95; 8.41; 9.00; 10.25; 11.85; 13.20;/..
A -dihydrochloride: Elementary analysis (after drying) as above) gives: C =45.78%; H 6.77%; N 12.75%; C1 6.23%; Fe (color.) 5.29%; titration in 80% methyl-cellosolve; molecular weight: 1086; pK 4.04; 7.91; 11.05. These values suggest the empirical formula C, H O N Fe, l-lcllts ultraviolet spectrum shows maxima at 227 mp (E 332), 319 mp. (E 37) and 425 mp. (B 32 25). Its infra-red spectrum (in potassium bromide) is shown in FIG. 3. It shows bands at the same positions as the spectrum of component A In FIG. 5 are shown the ultraviolet spectra (in water: c= 5 X 10*) of purified ferrimycin A (Curve 1, Extinction E A), of the component A (Curve 2, E A 0.1) and of the component A, (Curve 3, E =A 0.2).
The ferrimycins can be definitely distinguished by paper chromatography from Grisein (F. A. Kuehl, M. N. Bishop, L. Chaiet and K. Folkers, Am. Soc. 73, 1770 (1951)), Albomycin [G. F. Gause, Brit. Med. J. 1955, 1177] and A 1787 [H. Thrum, Naturwiss. 44, 561 (1957)]. In the system n-butanol-glacial acetic acidwater 4:1:5 (see FIG. 4) these antibiotics are still almost at the starting point aftera running time of 10 hours. The test is carried out bio-autographically with Staphylococus aureus.
low on'the addition of mineral acid. Decoloration is 5 complex union is liberated when the pH value is de- 50 creased.
Ferric chloride causes a claret red coloration and ferric chloride plus potassium ferricyanide give a blue coloration. The following tests are negative: Molish, Anthron, Folin-Ciocalteu and Sakaguchi. Its ninhydrin reaction in a mixture of butanol and pyridine is only slightly positive after heating for a long time. Hydrolysis with 6N-hydrochloric acid yields a mixture of about 15 substances detectable by paper chromatography. Among these compounds the following can be identified: succinic acid, 1amino-Shydroxyaminmpentane, S-aminovaleric acid, cadaverine, ammonia, proline, a crystalline substance (C H O N.HCI) with max 227 and 323 my. and ferric chloride.
The purified ferrimycin A and its components A and A respectively react with 8-oxyquinoline in solution in methanol with the separation of a black colored crystalline precipitate of iron oxyquinoline. The iron-free ferrimycin A can be isolated from the solution as a yellowish powder. It is also obtained when ferrimycin A is treated with alkali or with strong mineralic acids. Desferrimycin A possesses one half of the activity in vitro of the starting material, and exhibits, apart from the absence of the iron band at 425 mu, the same absorption spectrum as ferrimycin A. By the addition of ferric chloride its specific activity in vitro is doubled and the found in desferrioxamine B occur also in desferrimycin The purified base A can be split up by chromatography into components A and A As an adsorbent it is of advantage to use cellulose, and as the flow agent tertiary butanol/0.001 N-hydrochloric acid/saturated aqueous NaCl solution 2:12].
A Additionally desferrimycin A contains a phenolic constituent. Upon hydrolysis of desferrimycin A with 6-n. hydrochloric acid at room temperature there is obtained this phenolic compound bound to one 1 amino-Shydroxylamino-pentane residue, besides two moles of N-(5-hydroxylaminopentyl)-succinimide hydrochloride and one mol of acetic acid. The formula of the phenolic hydrolysis product is C H O N 3 l'lCl. It yields, on hydrolysis with l-n. hydrochloric acid at l C, 1-amino-5-hydroxylamino-pentanedihydrochloride, ammonium chloride and a phenolic aminocarboxylic acid hydrochloride of the formula c gHzlogNg, l-lCl. The latter yields, on hydrolysis with 6-n. hydrochloric acid at 1 10C, the crystalline hydrochloride of 3-amino-5-hydroxybenzoic acid, C H O N, l-lCl. From the different hydrolysis products and their spectra the following formula can be deduced for ferrimycin A -dihydrochloride:
ular weight 998 for the dihydrochloride of desferrimycin A Desferrimycin A shows in the UV-spectrum in ethanol maxima at )t 210 (log 5 4.57); 233 (log e =4.38) and 322 (log 6 =3.22') mu. In 0.01 N alcoholic sodium hydroxide there are maxima at 230 my. (log 6 2.61) and 335 mu (10g 1'm 1.54) and a weak shoulder at 245 my. (log e q 2.48). The IR- spectrum in liquid petrolatum shows bands at 845; 930 (shoulder); 975; 1002; 1022; 1057; 1125; 1161; 1195; 1225; 1259; 1305 (shoulder); 1378; 1466; 1550; 1612; 1677 (shoulder); 1715 cm (cf. FIG. 7).
The NMR-spectrum in trifluoracetic acid is given in FIG. 8.
The antibiotic ferrimycin A and its components ferrimycin A and ferrimycin A and the corresponding iron-free compounds can easily be obtained in the form of the free base from a salt thereof, for example, from the sulfate, for example, by reaction in an aqueous medium with barium hydroxide, neutralization of the ex cess of baryta with carbon dioxide, separation of the precipitate of barium carbonate and barium sulfate, and isolation of the free base by freeze drying. A simple method of producing the base from its salts is to use a strongly basic anion-exchanger, for example, the OH- form of the product known in commerce as Dowex-2 (a polystyrene resin containing dimethylethanolamine).
One of the most remarkable properties of ferrimycin A and components is its pronounced stability minimum between pH values of 7 and 1 1. Solutions of the antibiotic at 20C and at a pH value of 8-10 lose their activity in the course of 48 hours, whereas at the same tempera ture and at pH values of 1-5 they remain fully and at pH values 6-7 or greater than 111 partially active.
Salts of antibiotic ferrimycin A and its components and the corresponding iron-free compounds can be obtained from the known inorganic and organic acids, for example, hydrochloric acid, sulfuric acids and phosphoric acids, acetic acid, propionic acid, valeric acid, palmitic acid, or oleic acid, succinic acid, citric acid, mandelic acid, pantothenic acid, glutamic acid or other amino acids. They are neutral or acid salts. They can be prepared by the action of the corresponding acids on the free base or by the double decomposition of salts, for example, of ferrimycin-sulfate with calcium pantothenate.
Antibiotic ferrimycin A, ferrimycin A and ferrimycin A and the corresponding iron-free compounds have a very high antibiotic activity against various test organisms. In the so-called agar cross streak test they are active against the following test organisms: Micrococcus .pyogenes var. aureus, Streptococcus pyogenes, Streptococcus vin'dans, Streptococcus faecalis, Corynebacterium diphteriae, Escherichia coli, Bacillus megatherium and Bacillus subtilis.
The bacteriostatic activity of the ferrimycins, for in stance againstStaphylococcus aureus, can be used as a measure for the activity of preparations of different degrees'of purity. One unit is defined as the activity which in 20 ml of meat extract agar (contained in a Petri-plate of 9 cm diameter) inoculated' with about'200 germs of Staph. aureus ATCC 6538 and incubated for 24 hours at 37C suppresses 50% of the growth. The culture filtrate has an activity of 2,0005,000 units/m1. The activity of the purest preparations of ferrimycin A was found to be 900,000 units/mg; that of ferrimycin A 1,000,000 1 100,000 units/mg and that of ferrimycin A 1,100,000 r 100,000 units/mg.
1n the following Table are given the activities in vitro of purified ferrimycin products. There are given in mil- Strain Ferrimycin A Ferrimycin A Ferrimycin A,
Staph. aur. 26 27 26.5 Strepto. face. 8 9 9.5 Esch. coll 10.5 10.5 11 Shigella sonnei 13.5 14 l 14 Klebs.typ.A 23* 22.5" 22.5 Past.pestis 12 12 12 Bac.megatherium 20 20 20 Ustilaga sphaemgena 28 Uslilago scabt'osae 24 Cloudy zones of inhibition Table I Infection with Staph. aureus Strept. haemol. Pneumococc. lll
s.c. p.o. s.c. p.o. s.e. p.o.
Ferrimycin A, 0.1 5 0.1 3.3 0.33 3.3 1- Penicillin-G 1 3.3 5 3.3 Erythromycin 5 100' 50 or corresponding iron-free compound Table I: Doses (in mg/kg) of different antibiotics which cure 75l00%-of the infected animals with live subcutaneous or oral doses within 30 hours (observation period at least 10 days)v lnthepaper disc fest the diameter s of'the zones of i'n From the above Table it is clear that ofthTtlir ee antibiotics investigated the ferrimycins or desferrimycins respectively are the most effective against Staphylococcus aureus and Streptococcus haemolyticus when administered subcutaneously, being quantitatively 10 to 50 times superior to penicillin and 50 to 100 times superior to erythromycin. When administered orally, the
new antibiotics have about the same effect as penicillin, but are definitely superior to erythromycin.
Even when administered onlyonce, the ferrimycins or desferrimycins respectively show a superior effect.
if rimes Staph. aureus Strept. haemol Pneumococc. lll s.c. p.o. s.c. p.o. s.c. p.o.
Ferrimycin A, A A, 0.33 l0 l0 l0 10 33 Penicillin 10 33 50 Erythromycin 100 100 25 100 or corresponding iron-free compound Table 2: Doses (in mg/kg) of different antibiotics which effect a 75-100% cure when administered once.
in vivo. Mice were infected with virulent strains of Staphylococcus aureus, Streptococcus haemolyticus and Diplococcus pneumoniae Type III which is chemotherapeutically difiicult 'to attack. As infection dose approximately 100 lethal doses were administered intraperitoneally in each case; in the untreated control animals this led in 100% to sepsis followed by death within 24-48 hours. Treatment was carried out a. five times within hours (first administration k hour before infection, then 3, 5, 21 and 30 hours after infection), b. with single doses which were given after infection. 7 In both series of experiments the preparation was administered both subcutaneously and per os. In general, at least 10 animals were used in each group. The result of the treatment was checked on the'tenth day after infection and expressed as a percentage per group of the surviving animals. The results are put together in Tables l and 2.
In experiments on at least ten animals per group the immediately difference between a treatment effect of -100% su rvival and a control group mortality of (no survivors) fulfils the requirements of the 2 X 2 test for a significance of P 0,01 (Mainland & Murray, 1952, Science 1161591-594). Thus, in both series of the above experiments, statistically significant data were obtained at the 1% level (P z 0.01).
The toxicity Ifinarea myans and desferrimycin's'is low. Thus, for example, mice tolerated the subcutaneous administration of 1000 mg or the oral administration of 3,000 mg per kilogram of body weight without suffering harm. Higher doses have not been tested.
Because of their good antibacterial activity and their low toxicity ferrimycin A, ferrimycin A and ferrimycin A can be used as medicaments against infections caused by the above-mentioned microorganisms, especially infections caused by Staphylococci, Streptococci or Pheumococci. The compounds can be applied orally or parenterally, for instance subcutaneously or intramuscularly. Antibiotic ferrimycin A, its components ferrimycin A, and and ferrimycin A its salts and derivatives are useful as medicaments, for example, in the form of pharmaceutical preparations. They can also be used as additives for animal feedstuffs or as disinfectants or preserving agents, The pharmaceutical preparations contain the active compounds in admixture with a pharmaceutical organic or inorganic carrier suitable for enteral, parenteral or local administration. For making the carrier there are used substances which do not react with the new compounds, for example, gelatine, lactose,starches, magnesium stearate, talc, vegetable oils, benzyl alcohols, gums, polyalkylene glycols, white petroleum jelly, cholesterol or other known carrier for medicaments. The pharmaceutical preparations per volume of solution per minute. After incubating for i3 may be, for example, in the form of tablets, dragees, powders, salves, creams, suppositories or in liquid form as solutions, suspensions or emulsions. If desired, they may be sterilized and/or may contain auxiliary substances, such as preserving, stabilizing, wetting or emulsifying agents. They may also contain other therapeutically valuable substances.
FlG. II shows the IR-spectrum in potassium bromide of crude ferrimycin A monohydrochloride;
FIG. 2 shows the lR-spectrum in potassium bromide of ferrimycin A dihydrochloride;
FIG. 3 shows the IR-spectrum in potassium bromide of ferrimycin A dihydrochloride;
FIG. i shows paper chromatography of various enriched ferrimycin products (1 product of Ex. 7; 2 product of Example 10; 3 product of Example 13; 4 ferrimycin A 5 ferrimycin A in the systems I butanol-glacial acetic acid-water 4:1 :5; 10 hours II butanoI-butyl acetate-glacial acetic acid-waterlid By using instead of the above nutrient solution, solutions which contain, per liter of tap water, the following nutrient substances culture filtrates of similarly high antibiotic activity are obtained by incubation and working up in an analogous manner.
a) Glucose 10 grams Soya bean meal 10 grams Sodium chloride 5 grams Sodium nitrate 1 gram b) Glycerine grams Soya bean meal 10 grams Sodium chloride 5 grams Sodium nitrate 1 gram Calcium carbonate 10 grams c) Glucose 10 grams Soya bean meal 10 grams Corn steep liquor 20 grams Sodium chloride 5 grams Sodium nitrate I gram Calcium carbonate 10 grams d) Lactose 20 grams Distillers solubles 20 grams Sodium chloride 5 grams Sodium nitrate I gram 1001131143; 24 hours III butanol-butyl acetate-glacial acetic acid-water 100:301131143; 60 hours; FIG. 5 shows the UV-spectra (in water, c 5.10 of 25 ferrimycin A (Curve 1, Extinction E A) ferrimycin A (Curve 2, Extinction E A0. I )and ferrimycin A (Curve 3, Extinction E A-0.3); FIG. 6 shows the countercurrent distribution of crude ferrimycin'(containing ferrimycin A and ferrimycin B) over 115 stages according to Craig. Extinctions at 425 my. (iron colorfcurve I) and at 570 mu (ninhydrin color, curve 2) and in vitro activity (against Staph. aureus, curve 3) are indicated;
FIG. 7 shows the llR-spectrum in liquid petrolatum of desferrimycin A I FIG. 0 shows the NMR-spectrum in trifluoracetic acid of desferrimycin A The following examples illustrate the invention:
or. shaking well at 27C, the cultures in the fermenters being aerated with about 2 parts by volume of sterile air 48-120 hours the culture solution has a high inhibiting value against test organisms (B. subtilis, B.megatherium, micrococcus pyogenes, var.aureus). The culture is interrupted and the pH value is adjusted to 4.5 60 by the addition of dilute sulfuric acid, and the mycelium and'any other solid material is separated from the main body of the solution containing the antibiotic by filtration or centrifuging, 1% of a filtration assistant, for example, Hyflo Supercel, being added if desired to 5 the culturesolution before filtration. The filter residue is washed with water and with aqueous methanol, and the washings are united with the culture filtrate.
EXAMPLE 2 3 Liters of a culture filtrate obtained as described in Example I, are adjusted to a pH value of 7.5 by the addition of a dilute solution of caustic soda, and gramstered, the carbon residue being extracted once more in the same manner. The eluate contains the whole antibiotic activity.
EXAMPLE 3 3 Liters of a culture filtrate obtained as described in Example I are adjusted to a pH value of 7.5 by the addition of a dilute solution of caustic soda, and then the solution is immediately suppliedat the rate of 0.5 liter 0 per hour to a column of Amberlite IRC-SO (I-I-form) having a length of 30 centimeters and a diameter. of 5 centimeters. The antibiotic is completely adsorbed by the column. The column is washed withfi3 liters ofwater. For the purpose of elution there is used one liter of 5 0.4N-hydrochloric acid, the first 500 cc of the'eluate are antibiotically inactive, whereas the second 500 cc contain the whole activity. In order to remove theexcess of hydrochloric acid this active eluate is percolated through a column of Amberlite IR-4B (a weakly basic polystyrene resin containing polyamine exchange groups). By freeze drying the percolate' the enriched antibiotic ferrimycin is obtained in the form of ahighly active amorphous powder.
. EXAMPLE 4 6 Liters of a culture filtrate obtained as described in Example 1 are adjustedto a pH of 7.5 by the addition of dilute caustic soda solution, and then immediately percolated through a column of Asmit 173, 14 cm long and having a diameter of 4.5 centimeters. The antibiotic is completely adsorbed. The adsorbant resin is then washed with 3 liters of water and the antibiotic is eluted, with 1 liter of a mixture of methanol and 1N- acetic acid lzl The eluate, which contains the whole of the active substance, is concentrated in vacuo at 35C to 15 cc. 75 cc of a 0.25N-solution of hydrogen chloride in methanol is then added to the solution, and the mixture is poured into 10 liters of acetone, whereby the hydrochloride of the antibiotic is precipitated. The antibiotic is filtered off and washed with acetone. For the purpose of further purification it is dissolved in 150 cc of methanol, and the somewhat turbid solution is filtered with the addition of Celite (an infusorial earth). By evaporating the filtrate in vacuo at 25C, the hydrochloride of antibiotic ferrimycin is obtained in the form of an amorphous powder.
EXAMPLE 5 30 Liters of a culture filtrate obtained as described in Example 1 are adjusted to a pH value of 4.5, and then concentrated to 2 liters in a thin-layer evaporator. The concentrate is adjusted to a pH value of 8 by the addition of dilute caustic soda solution, and the mixture is then filtered with the addition of Celite. The clear filtrate is adjusted to a pH value of 5, and 1.5 liters of a hot aqueous solution of picric acid of 5 percent strength are added while stirring. The precipitate so formed, after being allowed to stand for several hours is filtered off at C with the addition of 50 grams of Celite. The filtrate has only a very slight antibiotic activity. The filter residue is then stirred three times with 800 cc of cold acetone each time and filtered. The filtr, ate is concentrated in vacuo to 80 cc, whereupon the picrate of the antibiotic and excess of picric acid precipitateout. After separation there are obtained 8.5 grams of dry substance.
In order to isolate the antibiotic in the form of its hydrochloride 2 grams of the aforesaid dry substance are dissolved in 30 cc of methanol. There are then added first a mixture of 1 cc of concentrated hydrochloric acid and cc of acetone, and subsequently 500 cc of ether, whereupon'the hydrochloride precipitates out quantitatively. By repeated dissolution of the precipitate in methanol acidified withhydrochloric acid and precipitation with ether the last residues of picric acid are removed. Finally, the hydrochloride is dissolved in as small a quantity as possible of methanol, andthe solution is filtered and evaporated in vacuo. There are obtained 714 mg of the hydrochloride of antibiotic ferrimycin.
EXAMPLE 6 .600 liters of a culture filtrate obtained as described in Example 1 are stirred with 5.5 kilograms of Hyflo .Supercel, adjusted to a pH value of 4 with 2.5 liters of 2N-HCl and then filtered. The filter residue is washed with 100 liters of water. The clear filtrate is stirred with 7 kilograms of pretreated Norit for 45 minutes. The pretreatment of the Norit is carried out by stirring several times with lN-HCl and then washing adsorbate twice for one hour with a mixture of nbutanol-methanol-glacial acetic acid and water (2:1: 1:2), the active charcoal being separated by filtration. The combined eluates (140 liters 46 liters) are mixed well with 96 liters of butyl acetate. The aqueous phase is separated and the organic phase is washed with 1.2 liters of water. The combined aqueous phase (65.5
liters) are washed in succession by stirring with 72 liters EXAMPLE 7 For the purpose of isolating antibiotic ferrimycin from 300 liters of a culture filtrate obtained as described in Example 1, 1 part by volume of Amberlite [RC 50 in the Hform is mixed mechanically with 2 parts by volume of Amberlite IRC 50 in the Na-for m. 6.3 liters of this mixture are poured into a glass column. The ratio of the height to diameter of the resin filing is 8:1. The culture filtrate is adjusted to pH 4 with 2N HCl and percolated through the resin at a rate of flow of 0.2
liter per minute per literof resin, an orange-brown zone being formed in the upper two-thirds of the column. The resin is then washed with 30 liters of water and with 60 liters of methanol of 80% strength. The runnings and the washings contain only little antibiotic activity. Elution is carried out in two portions with a total amount of 37 liters of a mixture of 8 parts by volume of methanol and 2 parts by volume of 1N HCl. Both portions are adjusted to pH 5 with 5N NaOH, combined and then concentrated to 2 liters at a temperature of at the most 30C in a circulation evaporator. The aqueous concentrate is adjusted to pH 5.6 and filtered through Hyflo Supercel for the purpose of removing any insoluble material. The filtrate (2.3 liters) contains approximately the whole antibiotic activity of the chloroform and extracted three times with 500 ml of 1/10 N-ammonium acetate buffer of pH 4.60 each time, colored antibiotically inactive impurities thus being removed from the organic phase. The chloroform phase is then extracted first with 300 ml and then twice with ml of l/ ION HCl each time. The deep red colored acid extract containing the antibiotic is adjusted to pH 3.5 with potassium bicarbonate and re-extracted with four 50 ml portions of a phenol-chloroform mixture (100 grams 100 ml). The phenol-chloroform extract is filtered through Celite. To the clear, redcolored filtrate (200 ml) 50 ml of water,'500 ml of ether and 300 ml of petroleum ether are added with vigorous agitation. After separating the aqueous phase,
the organic phase is washed twice with 50 ml of water each time. The combined aqueous extracts are extracted twice with 500 ml of ether each time and once with 500 ml of benzene and then lyophilized. There are obtained 2.60 grams of an antibiotically highly active orange-brown colored powder. This material shows 5001 ,000 times more specific antibiotic activity compared with the starting material (activity per weight unit of dry substance). Paper-chromatography of this material on Whatman No. l paper in n-butanol: n-butyl acetate: glacial acetic acidrwater (:3:1. 3:143) system reveals two spots after autobiographic development with Staphylococcus aureus. The slowly travelling antibiotic substance is designated Base A, the substance travelling 2.5 times more quickly Base B. Base A gives a blue color reaction on paper on being sprayed with ferric chloride and potassium ferricyanide.
EXAMPLE 8 338 Grams of an antibiotic preparation obtained as described in Example 6 are dissolved in 1.5 liters of water. 150 Grams of crystalline ammonium sulfate are added and the solution is extracted first with 1 liter and then twice with 500 ml each time of a phenolchloroform mixture containing 100 grams of phenol in 100 ml of chloroform. The combined phenol chloroform extracts are extracted with 750 ml of in hydrochloric acid and then filtered through a layer of Celite. To the clear red-brown filtrate there are added 600 ml of water, 4 liters of ether and finally 4 liters of petroleum ether with stirring. The aqueous phase is separated and the organic phase extracted with 200 ml of water twice. The combined aqueous phases l liter) are washed twice with 1 liter of ether and then lyophilized, 136 grams of a brown powder are obtained which has a specific antibiotic activity twice as high as the starting material.
EXAMPLE 9 550 mg of highly active antibiotic preparation (Base A) obtained as described in Example 11 are dissolved in 55 ml of a 1/ ION-ammonium acetate buffer having a PH value of 4.6 and extracted 4 times with 20 ml of a phenol-chloroform mixture containing '100 grams of phenol in 400 ml of chloroform. The organic extracts are re-washed twice with 15 ml buffer solution. The organic extract (80 ml) containing the antibiotic is diluted with 40 ml of chloroform, washed once again with 60 ml of buffer solution and then filtered through a double pleated filter. The deep red-colored filtrate is extracted successively with 30, 20, and 10 ml of 0.2N- hydrochloric acid. The acid solution containing the antibiotic is diluted with 50 ml of water and exhaustively extracted twice with and then with 10 ml of a mixture of phenol and chloroform containing 100 grams of phenol in 100 ml of chloroform. The combined phenolchloroform extracts are filtered through a double pleated filter and agitated with 20 ml of water, 200 ml of ether and 100 ml of petroleum ether. After separating the aqueous phase, the organic phase is reextracted twice with 15 ml of water. The combined aqueous extracts are washed twice with 50 ml of ether and once with 50 ml of benzene and then lyophilized. There are obtained 1 17 mg of a brown-red powder which has approximately five times the amount of antibiotic activity compared with the starting material.
EXAMPLE 10 v 700 mg of the antibiotic preparation obtained as described in Example 7 are chromatographed over 127 grams of Whatman No. 1 cellulose powder. For the purpose of elution there is used the upper phase of a mixture of 4 parts of butanol, 1 part of glacial acetic acid and 5 parts of water, to which are added 10% by volume of butanol. The substance is triturated with ten times the quantity of cellulose powder and put on the column as powder. The rate of running through is 15-20 ml per hour. Fractions of 40 ml are collected. The separate fractions are agitated with 50 ml of petroleum ether. The separated aqueous phase is washed with benzene and lyophilized. The major portion of the antibiotic activity is in the fractions 7-8 (121 mg) and l0-l3 (142 mg). On being examined by paperchromatography it is found that the fractions 7-8 contain chiefly Base B and fractions 110-13 primarily Base A.
EXAMPLE 1 1 3 Grams of an antibiotic preparation obtained as described in Example 8 are distributed over a hundred stages in a Craigs distributing apparatus in the secondary butanol 0.lN-ammonium acetate buffer system having a pH value of 4.68, each unit contains ml upper phase and 100 ml lower phase and the substance being charged into unit No. 3. After distribution the content of each unit is worked up by adding double the volume of petroleum ether to the mixture of the two phases and freeze-drying the aqueous phase; The dark- .colored fractions 3-ll show only little antibiotic activity. The orange-yellow colored fractions l2-20 (631 mg) contain the major portion of the antibiotic activity (chiefly Base A). The yellow-colored fractions 21-40 are less active and contain a mixture of Base A and Base B in which the latter predominates.
EXAMPLE 12 220 mg of an antibiotic preparation (Base A) obtained as described in Example 10' are distributed over 29 stages in a Craigs distributing apparatus in the 1 1ONammonium acetate buffer (pH value 4.58) 10% phenol in chloroform. Each stage contains 10 ml each of upper and lower phase. The major portion of the antibiotic activity is in fractions 6 15. The latter ,are combined (about 200 ml), 400' ml of ether and-300 ml of petroleum ether are added and the whole agitated. The separated, orange-red colored aqueous phase is washed with chloroform and extracted successively with 20 and three times with 10 ml of a'mixture of 100 grams of phenol in 100 ml of chloroform; 20 ml of water, 300 ml of ether and 200 ml of petroleum ether are added to the phenol-chloroform extracts with agitation. The red-colored aqueous phase is separated, washed with much ether and-benzene and lyophilized. There are obtained 80.4 mg of Base A in the form of a yellow water-soluble powder. Color reactions: FeCl brown-red; FeCl K Fe(;CN) blue;
Ninhydrin: weakly positive.
Negative: Sakaguchi, Maltol, Elson-Morgan.
EXAMPLE l3 8 grams portions of an enriched ferrimycin product (main component A: activity in relation to the lyophilized culture filtrate 2,000 to 3,000) such as is ob tained for example, according to Example 10 were subjected in a vertical glass column having a length of 1 meter and a diamter of 6 centimeters which was filled with cellulose powder and provided with a cooling jacket, to zonal electrophoresis by the method of J. Porath [Biochimica et Biophysica Acta, Vol. 22 page 151 (1956)]. As an electrolyte solution there was used 54; N-acetic acid. The product was dissolved in 160 ml of water, and the brown-red solution was poured on to the upper anode end of the column. At a voltage of 1,000 and a current of 100 milliamperes the orange antibiotic zone about 20 cm wide migrated towards the cathode at a speed of 3.3 cm per hour. In order to increase the separating action of the column this electric movement was exactly compensated by a stream of the electrolyte moving the opposite direction at the rate of 81 ml per hour, and in this way the antibiotic was held stationary in the same place in the column. Brownish colored inactive accompanying substances having higher or lower electrical migration speeds than that of the antibiotic were moved to the cathode zone or anode zone, and wahsed away from the zone either continuously or periodically. After a period of electrophoresis lasting -6 days the antibiotic had moved through a liquid column 4-5 meters long. The substance on the column was then eluted with electrolyte solution, and the eluate fractions collected in an automatic fraction receiver were tested biologically. The deep. red colored biologically active fractions were combined (1.3 liters). From the acetic acid solution the antibiotic could be extracted after the addition of 75 ml of a saturated aqueous solution of sodium chloride, with 250 ml of a mixture of phenol and chloroform (lgzlml). After filtering the extract through Celite, the antiobiotic could be precipitated as an orange colored precipitate on 16 grams of Hyflo Supercel by the addition of 1 liter of ether and 500 ml of petroleum ether. The mixture of the precipitate and filtration assistant was washed well with acetone and finally extracted with a small amount of cold methanol. From the methanol extract the crude ferrimycin A monohydrochloride was precipitated with acetone in the form of an orangecolored powder. After being dried at room temperature under 0.001 mm pressure for two days the product contained approximately 100% of the original activity in a form enriched four to six times. Analysis: C 50.68%; H 6.99%; N =.13.45%; Fe (gravimetric) 3.55%; Fe (colorimetric) 3.66%; Cl =2.75%; 2.46% drying loss at 120C under 0.01 mm pressure.
Infra-red spectrum in potassium bromide; see FlGl. It shows bands at 2.97; 3,43; 6.05; 6.30; 6.92; 7.25; 7.98; 8.25; 8.40; 8.95; 13.20 pt.
Ultra-violet-spectrum in water; maximum at 318 mp. (E 47.2); inflexions at 229 mp. and at 400 mu. Solubility: dissolves very well in water, methanol and mixtures of phenol and chloroform, soluble in dimethylformamide, methyl-cellosolve, benzyl alcohol and glacial acetic acid, sparingly soluble in ethanol and practically insoluble in pyridine, propanol, butanol and the usual organic solvents, especially lipoid solvents.
Precipitation reactions: precipitatable from aqueous solution by means of picric acid, picrolonic acid, and ammonium reineckate.
Color reactions: orange-red aqueous solutions change color upon the addition of mineral acid reversibly through claret red to pale yellow. Decoloration is likewise caused by caustic soda solution. Neutral solutions do not react with potassium ferrocyanide. Acidified solutions give colorations or precipitations of Prussian blue. Trivalent iron bound in complex union is liberated as the pH-value decreases.
Ferric chloride produces a claret red coloration and a mixture of iron chloride and potassium ferricyanide gives a blue coloration. The following tests are negative: Molish, Anthron, Folin Ciocalteu, Sakaguchi. The ninhydrin reaction in butanol-pyridine is weakly positive after heating for a long time. Hydrolysis with 6N- hydrochloric acid yields a mixture of about 15 substances detectable by paper chromatography.
lron-free ferrimycin A:
mg of the purified ferrimycin A so obtained were dissolved in 1 ml of methanol. 334 mg of ooxyquinoline in 2ml of methanol were added and the whole was allowed to stand at room temperature for 8 hours. After allowing the whole to stand for a further 15 hours at 0C the precipitated black-green crystals of iron 8-oxyquinoline were separated. The solution was diluted with a small amount of water and extracted by thorough agitation with chloroform and benzene to remove the excess of oxyquinoline. The remaining pale yellow aqueous phase, which contains the iron-free antibiotic was lyophilised: 90 mg of a beige powder. The iron-free antibiotic exhibited the same absorption spectrum in the ultraviolet region as the starting material, but in the visible region it lacked the flat band at 400-430 mu. It had only /i of the activity in vitro of the starting material. A colorless solution of the iron-free antibiotic is instantaneously colored deep red on the addition of ferric chloride, whereupon the iron absorption at 400-430 my. in the spectrum returns and the specific antibiotic activity is increased.
EXAMPLE 14 800 mg of a preparation of ferrimycin A obtained as described in Example 13 were chromatographed over a cellulose-column measuring 3 X 65 cm (198 grams) at 12C. As flowing agent there was used the system tertiary butanol/0.001N hydrochloric acid/saturated aqueous NaCl-solution (2:1 :1 The fractions collected in an automatic fractionreceiver amounted to 30-40 ml and were examined. biologically, spectroscopically and by paper chromatography. The fractions 60-104 contained 191 mg of ferrimycin A fractions -200 contained 92 mg of ferrimycin A The correspondingly unified fractions were agitatedwith an equal volume of petroleum ether and with about 10% by volume of water, whereby the antibiotic substance was driven into the aqueous phase. From the latter it was worked up by the method described in' Example 13 with a mixture of phenol and chloroform and was obtained in the form of an orange powder. After being dried at 25C under 0.001 mm pressure for 50 hours over P O /KQH the product had the following properties: Ferrimycin A dihydrochloride: Analysis (average from three determinations): C 46.71%; H 6.80%; Fe 5.33%; N
12.70%; C1 6.76%; O (calc.) 21.70%. Titration in 80% methylcellosolve; equivalent weight: 1078; pK.
4.11; 7.92 and 11.4. Ultra-violet spectrumzltmax 229' mp.(E 336), 319 my. (E 37) and 425 mp. (E 27.6). Infra-red spectrum potassium bromide: see FIG. 2. It shows bands at 3.00; 3.44; 3.51; 6.08; 6.31; 6.90; 7.10; 7.35; 7.45; 7.95; 8.41; 9.00; 10.25; 11.85; 13.20 1L.
Ferrimycin A -dihydrochloride: Analysis: C 45.78%; H 6.77%; N 12.75%; C1 6.23%; Fe 5.29%; titration in 80% methyl-cellosolve; molecular weight: 1086; pK: 4.04, 7.91 and 11.05. Ultra-violet spectrumzA max. 227 mp. (E 332), 319 mp. (E 37) and 425 mp. (E 25). Infra-red spectrum (in potassium bromide): see FIG. 3. The paper chromatography of various enriched and purified ferrimycin products on Whatman No. l-paper is shown in FIG. 4. The test was made bio-autographically with Staphylococcus aureus.
In FIG. 4 the symbols have the meanings: 1: System butanol glacial acetic acid-water 411:5; 10
hours 11: System butanol-butyl acetate-glacial acetic acidwater 100:30z13z143; 24 hours 111: System butanol-butyl acetate-glacial acetic acidwater 100:30:l3:l43; 60 hours. 1: Antibiotic ferrimycin, base A-l-B, according to Example 7 (1 pg) 25 Antibiotic ferrimycin B, according to Example 1 Mg) 3: Antibiotic ferrimycin A according to Example 13 (0.1 pg) 4: Antibiotic ferrimycin Al, according to this Example (0.05 ,ug) 5: Antibiotic ferrimycin A2, according to this Example (0.05 ug).
EXAMPLE l5 6 grams of ferrimycin product having about 1,000 times the antibiotic activity of the lyophilized culture filtrate and containing ferrimycin A as well as ferrimycin B, are distributed by countercurrent over 115 stages according to Craig. The apparatus consists of 120 units. It is filled per unit with 100 cc of upper phase and 100 cc of lower phase of a mixture, equilibrium at 19C, of benzyl alcohol (200 parts by volume), nbutanol (100 parts by volume), N-hydrochloric acid (6 parts by volume), water (300 parts by volume) and aqueous sodium chloride solution (60 parts by volume) saturated at 19C. The first two units merely contain solvent. In each of the following three units 2 grams of substance are introduced and the whole distributed 1 15 times at 19C. The number of shakes per distribution is 30, the duration of intervals 15 minutes.
When the distribution is complete, the resulting 118 fractions are kept at C. From every third fraction there are taken for test purposes 10 cc of the upper and 10 cc of the lower phase which are agitated with 50 cc of petroleum ether. The separated aqueous phase (10 cc) which now contains all the hydrophilic material is freed from any adhering organic solvent by brief evacuation. The resulting test solutions are used on the one hand for colorimetric evaluation (extinction at 425 my. in 1 cc cuvette. Compare FIG. 6, curve 1) and for the ninhydrin color reaction. To carryout the latter 0.5 cc of test solution and 0.5 cc of ninhydrin reagent, prepared is described by S. Moore and W. H. Stein, J. Biol. Chem. 211, 907 (1954), are mixed, heated at 100 C for minutes, diluted with 5 cc of a mixture of alcohol and water (1:1) and then measured in a spectrophotometer at 570 mu (curve 2). For biological tests the test solutions are diluted 1:50. In curve 3 there are shwon the in vitro activities against Staphylococcus aureus in the plate diffusion test in relation to an arbitrary standard. The active fractions 40-70 contain, as can be shown by paper chromatography, ferrimycin A, whilst ferrimycin B is in fractions 75-100. A considerable enrichrnent can be achieved. The antibiotically active fractions 25-39, 40-48, 49-55, 56-70 and 71-95 are combined and agitated with the same volume of petroleum ether. Thered colored substancesare driven into Fraction Quantity Enrichment Yield of Ferrimycin in mg factor activity The starting material is obtained as follows:
30 liters of an aqueous eluate concentrate obtained as described in Example 7 by elution of ferrimycin from Amberlite IRC 50 and subsequent removal of the methanol in vacuo, are mixed with 1 kg of Hyflo with stirring and then in the course of 2 hours with 1.8 liters of a mixture of 11 parts of phenol and 1 part of water. Stirring is then continued for half an hour, and the mixture is then suction-filtered. The filtrate contains about 2-5 percent of the activity and is discarded. The well squeezed filter cake is stirred twice with 4 liters of ether each time and once with a mixture of 8 liters of acetone and 2 liters ofv ether and suction-filtered on each occasion, whereupon the residue is washed with succession on the filter with 5 liters of acetone and 4 liters of chloroform. All the washings are practically inactive. The chloroform-moist filter-cake is then introduced into 3 liters of a mixture of phenol and chloroform 1:1
(weight/volume) and stirred for 1 hour. In the course.
of 1 hour 15 liters of chloroform are added in a uniform current. The Hyflo is suction-filtered and washed twice with 2 liters of phenol-chloroform 1:11 (weightlvolu me) and once with 2 liters oi chloroform. The residue contains 5-10% of the activity.
The combined active filtrate (22 liters) are concentrated to 4 liters at 25C and the concentrate added dropwise in the course of 30 minutes to a mixture of 4 liters of ether, 8 liters of petroleum ether and 400 grams of Hyflo. After another 30 minutes the mixture is suction-filtered. The filter-cake is washed with 2 liters of ether and twice with 1 liter of acetone. All the filtrates contain only traces of activity.
The filter-cake is stirred three times for 10 minutes with 1.5 liters of methanol each time, suction-filtered and finally washed with 0.5 liter of methanol onthe filter. The washed Hyflo does not contain any activity.
The dark brown filtrates (4 4.5 liters) are evaporated to dryness cautiously at 20-30C in a water-jet vacuum. The still sticky residue is dried in a high vacuum for 20 hours. 75-88 grams of a red-brown strongly active ferrimycin product are obtained. The yield of activity calculated on the starting material is EXAMPLE 16 2 grams of a ferrimycin A product (fractions 49-55) obtained as described in Example 115 which has about 9,000 times the antibiotic activity of the lyophilized culture filtrate, are chromatographed on a 70 cm X 7.14 cm column of a strongly acid ion exchanger Dowex 50 WX (/200 mesh). The ion exchanger is first purified according to Hirs et: al., J.Bi0l.Chem 219,