BE547329A - - Google Patents

Description

       

   <Desc / Clms Page number 1>
 



   The present invention relates to a novel antibiotic and to methods of preparing this product. More particularly, it relates to an antibiotic substance produced by strains of microorganisms belonging to a species of Streptomyces not previously described, this antibiotic and its salts being effective against trypanosomes, amoebae, and positive bacteria. and negative on the Gram test.



   The new antibiotic, in its purified form, is a white crystalline material with weakly basic properties. An analytical sample prepared as described below gives, on elemental analysis, the following results: Carbon 34.03 - hydrogen 4.05 - nitrogen 21.60 - sulfur 8.30 - oxygen 32.02 per difference. The compound has a relatively low molecular weight.



  Its empirical formula based on the above analysis corresponds very closely to C11H15-16N6O8S.



   The novel antibiotic is almost completely stable in water at room temperature for 24 hours at pH 3, 7 and 9. At elevated temperatures it exhibits some instability, particularly at 100 ° C and above.



   The infrared absorption spectrum of the antibiotic is shown in Figure 1. This curve is determined on a sample of the material, mixed with KBr crystals, and compressed into a disk. The absorption spectrum in the ultraviolet at pH 7.0 is shown in figure 2.



   The optical rotation of a purified sample of the antibiotic is [a] D24.5 = 33.3 (1052 g in 100 cm3 of a mixture of 50% ethanol and 50% 0.1N hydrochloric acid. Corrected observed rotation = -0.35 in a tube of Idem).



   The new antibiotic is soluble in the following solvents at room temperature according to the approximate values indicated, which are expressed in mg / cm3 of solvent:
 EMI1.1
 
<tb> water, <SEP> pH <SEP> 3.5 <SEP> 5.8 <SEP> (x)
<tb>
<tb> water, <SEP> pH <SEP> 6.5 <SEP> 1.9 <SEP> (x)
<tb>
<tb> water, <SEP> pH <SEP> 9, <SEP> 25 <SEP> 26.8 <SEP> (x)
<tb>
<tb> methanol <SEP> 14.2 <SEP> (x)
<tb>
<tb> acetone <SEP> 4.8
<tb>
<tb> n-butanol <SEP> 0.25
<tb> ethyl acetate <SEP> 0.36
<tb>
<tb> benzene <SEP> 0.137
<tb>
<tb> ether <SEP> 0.031
<tb>
   (x): figures obtained by gravity.
The novel antibiotic prepared by the process of the present invention adsorbs strongly to charcoal over a wide pH range, which is from less than 3 to more than 9.

   It adsorbs weakly to common adsorbents such as activated magnesium silicate, alumina, alfa cellulose and fuller's earth, in acidic, neutral and alkaline pH media. It can adsorb on cationic exchange resins through

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 shooting from neutral or slightly alkaline solutions, and these resins can be eluted with an acidic or basic solution.



   The new antibiotic is very active against trypanosomes,
 EMI2.1
 such as Trypanosoma equiperdum, and amoebae such as Endamoeba histolytica and therefore it is valuable in the treatment of conditions caused by these microorganisms in man and animals, depending on the amounts and mode of administration prescribed by the attending physician or veterinarian.

   It is also active against various bacteria, as shown in the following table in which its bacterial spectrum is illustrated using a 200 micrograms per cm solution of the antibiotic in 20% aqueous methanol, following the common method of diffusion in agar-agar:

     
 EMI2.2
 
<tb> Body <SEP> Width <SEP> of <SEP> zone,
<tb>
 
 EMI2.3
 ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ mmo
 EMI2.4
 
<tb> B. <SEP> cereus <SEP> Waksman <SEP> 8.0
<tb>
<tb> K. <SEP> Pneumoniae <SEP> (Friedlander) <SEP> 2.9
<tb>
<tb> Salmonella <SEP> gallinarum <SEP> pH <SEP> 7, <SEP> 8 <SEP> 7.0
<tb>
<tb> Salmonella <SEP> gallinarum <SEP> pH <SEP> 6.0 <SEP> 6.1
<tb>
<tb> Alcaligenes <SEP> sp. <SEP> ATCC <SEP> 10153 <SEP> insignificant
<tb>
<tb> Staph. <SEP> aureus <SEP> (n <SEP> 209) <SEP> 6.0
<tb>
<tb> B. <SEP> subtilis <SEP> pH <SEP> 7, <SEP> 8 <SEP> 8.9
<tb>
<tb> B. <SEP> subtilis <SEP> pH <SEP> 6, <SEP> 0 <SEP> 9.4
<tb>
<tb> B. <SEP> subtilis <SEP> resistant <SEP> to <SEP> streptothricin <SEP> 3.0
<tb> M. <SEP> ranae <SEP> 0
<tb>
 
 EMI2.5
 M. TUbercu10sis PH 7.8 2.9 M.

   Tubeçcalosis pH 6 7.1
 EMI2.6
 
<tb> Staph. <SEP> àlbus <SEP> 8.0
<tb>
<tb> K. <SEP> Pneumoniae <SEP> 7.5
<tb> E. <SEP> coli <SEP> 7.0
<tb>
 
 EMI2.7
 Streptococcus haemo1yticus NY 5 16.1
 EMI2.8
 
<tb> Corynebacterium <SEP> xerose <SEP> 7.0
<tb>
 
The new antibiotic is produced by a species of Streptomyces which has been assigned the n T 3018 and which apparently has not been previously described. Here is a general description of this new species:
The colors capitalized are those of Ridgeway.



     Aerial mycelium: absent in many environments; present on some media, and then in limited quantity, white to gray (close to "Pale Gull Gray", up to "Light Gull Gray").



   Dense growing vegetative mycelium; whitish to yellow (close to "Ivory Yellow" to "Chamois" and "Honey Yellow").



     Colorat ion upside down whitish to yellow (close to "Ivory Yellow"

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 up to "Honey Yellow" and "Cinnamon-Buff"; on certain media, darkened areas (gray close to "Dab-Gray") appear mixed with yellow areas.



   Soluble pigment: none on most media, light yellow on mannitol / Czapek-Dox agar at 32 and 37 C.



     Morphology: aerial hyphae on starch / Waksman agar-agar are branched; spore-bearing hyphae mainly grow in the form of clusters of lateral branches which terminate in short coils of a few turns, The spores are roughly spherical or elongated (0.57 to 1.14 by 0.57 to 1, 71 micron).



   The characteristics of culture on the usual agar-agar media in Petri dishes are described in Table I.



   Additional culture characteristics are described in Table II.



   Obviously, variations of the above characteristics, of the kind observed when studying cryptogams, will appear with different strains of this new species and under the influence of different environmental conditions.

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    TABLE I Culture characteristics of Streptomyces sp., T-3018, after 14 days of incubation in Petri trays at 28 Co
 EMI4.1
 
<tb> Medium <SEP> Growth <SEP> Mycelium <SEP> aerial <SEP> Mycelium <SEP> Vegetative <SEP> Reverse <SEP> Pigment <SEP> Remarks
<tb> Agar-agar <SEP> CzapekDox <SEP> Good <SEP> light <SEP>;

   <SEP> blancjaune ("Cream-Buff" <SEP> Yellow (close <SEP> of <SEP> nil <SEP> growth
<tb> to <SEP> "Chamois") <SEP> "Chamois") <SEP> extensive
<tb> Mannitol <SEP> agar-agar <SEP> good <SEP> very <SEP> light; white <SEP> to <SEP> cream <SEP> light <SEP> to <SEP> yellow <SEP> Yellow <SEP > ("Chamois" <SEP> to <SEP> none <SEP> extensive
<tb> Czapek-Dox <SEP> gray <SEP> light (close <SEP> (close <SEP> of <SEP> "Chamois") <SEP> "Honey <SEP> Yellow")
<tb> of <SEP> "Light <SEP> Mineral
<tb> Gray ")
<tb> Asparagine / dextrose / <SEP> fairly <SEP> Gray ") <SEP> cream <SEP> whitish <SEP> whitish <SEP> to <SEP> yellow <SEP> none
<tb> extract <SEP> from <SEP> meat / <SEP> good <SEP> none <SEP> clear <SEP> clear (close <SEP> to
<tb> agar-agar <SEP> "Cream <SEP> Color")

  
<tb> Asparagine <SEP> acid / dex- <SEP> fairly <SEP> none <SEP> yellow <SEP> whitish <SEP> cream <SEP> whitish <SEP> none
<tb> trose / extract <SEP> from <SEP> vian- <SEP> good
<tb> from / agar-agar
<tb> Starch / agar-agar <SEP> moderate <SEP> rare <SEP> to <SEP> moderate; gray <SEP> whitish <SEP> to <SEP> yellow <SEP> whitish <SEP> to <SEP> yellow <SEP> none <SEP> growth <SEP> denWaks-man <SEP> bluish ("Pale <SEP> Gull (close <SEP> of <SEP>" buff ") <SEP> close <SEP> of <SEP>" cha- <SEP> se; agar-agar,
<tb> Gray "to" Light <SEP> Gull <SEP> month <SEP>; areas <SEP> obscured, <SEP> not <SEP> fully
<tb> Gray ") <SEP> close <SEP> of <SEP>" Drab-Gray "<SEP> liquidated.
<tb>



  Starch / agar-agar <SEP> moderate <SEP> white <SEP> to <SEP> gray (juice- <SEP> whitish <SEP> to <SEP> yellow <SEP> white <SEP> to <SEP> yellow <SEP > none, <SEP> agar-agar <SEP> not
<tb> Czapek-Dox <SEP> only <SEP> near <SEP> of <SEP> (close <SEP> of <SEP> "chamois") <SEP> ("Light <SEP> Buff" <SEP> to <SEP> completely
<tb> "Pale <SEP> Gull <SEP> Gray"); <SEP> "Cream <SEP> Color") <SEP> liquidated.
<tb> rare <SEP> to moderate <SEP>
<tb> Malate <SEP> of <SEP> calcium / <SEP> moderate <SEP> none <SEP> to <SEP> white <SEP> to <SEP> yellow <SEP> ("Cre-banchâtre <SEP> to < SEP> yellow <SEP> none <SEP> liquidation <SEP> of
<tb> agar-agar <SEP> gray <SEP> am-Buff "to" Honey <SEP> (close <SEP> to <SEP> "Honey <SEP> agar-agar
<tb> Yellow ") <SEP> Yellow").
<tb>



  Apple <SEP> dextrose <SEP> moderate <SEP> null <SEP> to <SEP> light; white <SEP> whitish <SEP> to <SEP> yellow <SEP> whitish <SEP> to <SEP> yellow <SEP> none
<tb> from <SEP> earth <SEP> to <SEP> gray (close <SEP> from <SEP> "Ivory <SEP> Yellow" to <SEP> ("Ivory <SEP> Yellow" <SEP> to
<tb> "Light <SEP> Gull <SEP> Gray") <SEP> "Cream-buff") <SEP> "Cream-buff")
<tb> Agar-agar <SEP> Bennett <SEP> good <SEP> rare <SEP> to <SEP> abundant;

   <SEP> whitish <SEP> to <SEP> yellow <SEP> Yellow (close <SEP> to <SEP> none
<tb> white <SEP> "Cinnamon-buff")
<tb>
 

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   TABLE I (continued) T-3018
 EMI5.1
 
<tb> Medium <SEP> Growth <SEP> Mycelium <SEP> aerial <SEP> Mycelium <SEP> Vegetative <SEP> Backing <SEP> pigment <SEP> soluble <SEP> Remarks
<tb> Liquor <SEP> from <SEP> mac- <SEP> moderate <SEP> rare <SEP> to <SEP> moderate <SEP>;

   <SEP> clear <SEP> yellow (close <SEP> of <SEP> none <SEP> --ration <SEP> of <SEP> but / <SEP> white <SEP> dull <SEP> "Chamois" <SEP> to <SEP> "Hoagar-agar <SEP> ney <SEP> Yellow")
<tb> Dextrose / agar- <SEP> fairly <SEP> none <SEP> yellow (close <SEP> to <SEP> Yellow (Ivory <SEP> Yellow "<SEP> none <SEP> --agar <SEP> Krainsky <SEP> good <SEP> "Ivory <SEP> Yellow") <SEP> to <SEP> "Cream-Buff")
<tb> Agar-agar <SEP> nutritious <SEP> moderate <SEP> none <SEP> yellow <SEP> light <SEP> dull <SEP> Yellow (close <SEP> of <SEP> none <SEP> ---
<tb> "Cream-Buff")
<tb> Maltose / agar-agar <SEP> fairly <SEP> none <SEP> whitish <SEP> to <SEP> whitish <SEP> to <SEP> yellow <SEP> none <SEP> --Sabouraud <SEP> good <SEP> "Cream-Buff" <SEP> (close <SEP> of <SEP> "Honey
<tb> Yellow ")
<tb> Glucose / agar-agar <SEP> moderate <SEP> none <SEP> to <SEP> light <SEP>;

   <SEP> Whitish yellow <SEP> Whitish yellow <SEP> <SEP> none <SEP>. <SEP> --white <SEP> to <SEP> brown-yellow, light <SEP> to <SEP> yellow <SEP> ("Chamois"
<tb> dull <SEP> to <SEP> "Yellow <SEP> Honey")
<tb> Agar-agar <SEP> Emerson <SEP> moderate <SEP> light; white <SEP> yellow <SEP> light <SEP> dull <SEP> Yellow <SEP> (close <SEP> to <SEP> none. <SEP> ----
<tb> "Honey <SEP> Yellow")
<tb>
 

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   TABLE II Culture characteristics of Streptomyces sp., 6-3018 after 14 days of incubation at 28 C.
 EMI6.1
 
<tb>



  <SEP> growth medium <SEP> Mycelium <SEP> aerial <SEP> Vegetative <SEP> mycelium <SEP> Pigment <SEP> soluble <SEP> Remarks
<tb> dice <SEP> of <SEP> carrot <SEP> mediocre <SEP> none <SEP> very <SEP> light; whitish <SEP> - <SEP> -
<tb> to <SEP> yellow <SEP> light
<tb> dice <SEP> of <SEP> apple <SEP> of <SEP> earth <SEP> moderate <SEP> light;

   <SEP> white <SEP> yellow <SEP> (close <SEP> of <SEP> "Pin- <SEP> - <SEP> de <SEP> no <SEP> colored
<tb> kish <SEP> Cinnamon ") <SEP> to <SEP> yellow
<tb> olive <SEP> (close <SEP> to <SEP> "Buffy
<tb> Brown ")
<tb> gelatin <SEP> moderate <SEP> none <SEP> whitish <SEP> to <SEP> yellow <SEP> none <SEP> liquefaction
<tb> sunflower <SEP> milk <SEP> <SEP> light <SEP> to <SEP> none <SEP> yellow <SEP> whitish <SEP> none <SEP> liquefaction <SEP>
<tb> good <SEP> thousand <SEP> to <SEP> complete
<tb> broth <SEP> to <SEP> nitrate <SEP> from <SEP> light <SEP> to <SEP> none <SEP> whitish <SEP> to <SEP> yellow <SEP> clear <SEP> none <SEP > <SEP> reduction of <SEP>
<tb> dextrose <SEP> good <SEP> nitrate
<tb> cellulose <SEP> (filter paper <SEP> light <SEP> none <SEP> yellow <SEP> light <SEP> none <SEP> paper <SEP> no <SEP>
<tb> in <SEP> solution

  <SEP> Czapek) <SEP> decomposed <SEP> at
<tb> end <SEP> of <SEP> 21
<tb> days
<tb>
 

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THE FERMENTATION PROCESS.



   The process by which the new antibiotic is formed is aerobic fermentation of an aqueous nutrient medium inoculated with the new organism described above. The constituents of the fermentation medium and the conditions of the fermentation are generally the same as in other fermentation processes in which cryptogams are employed to produce antibiotics.



   Sources of carbon include starch, hydrolyzed starch, sugars such as lactose, maltose, dextrose, sucrose, or sources of sugar such as molasses; alcohols such as glycerol and mannitol; organic acids such as citric acid and acetic acid; and various natural products which may contain, in addition to carbonaceous substances, various other nutrients.

   Nitrogen sources include proteins such as casein, zein, lactalbumin; protein hydrolysates, proteoses, peptones, peptides, and commercial materials such as "N-2-amine" which must be a casein hydrolyzate; also, corn maceration liquor, soybean meal, gluten, cottonseed meal, fish meal, meat extracts, animal juices, liver cake, extracts. yeast, soluble distillation materials etc ...; amino acids, urea, ammonium salts and nitrates, etc.



  Advantageously used, for the fermentation of inorganic cations such as sodium, potassium, calcium, magnesium, etc .; and anions such as chloride, sulfate, phosphate, and various combinations of these anions and cations in the form of inorganic salts. So-called "trace elements" such as boron, cobalt, iron, copper, zinc, manganese, chromium, molybdenum, and others can be used with advantage. Usually, the antibiotic's sulfur and trace elements are found in sufficient quantities either in the carbonaceous and nitrogenous constituents of the medium, especially if they are obtained from natural sources, or in tap water, and it it may be unnecessary to add additional amounts of these bodies.



   The fermentation is aerated in the usual manner by forcing sterile air through the fermenting mixture, generally at a rate of about 1 volume of air per volume of fermentation medium per minute.



  To minimize contamination by foreign microorganisms, the fermentation vessels should be closed and the vessel pressure 0.14 to 1.05 Kg / cm higher than atmospheric pressure must be maintained. Mechanical agitation is generally advisable, in addition to agitation provided by aeration. Antifoaming agents such as 1% octadecanol in lard oil can be added from time to time, as needed, to prevent too much foaming.



   The fermentation is carried out at a temperature preferably of the order of 26-30 C, but this can also go down to 1700 or go up to 42 C.



   The time required for maximum antibiotic production varies considerably depending on the other fermentation conditions. Usually, it takes about 48 hours for appreciable amounts of antibiotic to be detected in the medium. The production of the antibiotic increases over time, and fermentation can take up to 120 hours.



  PH conditions normally vary from about 6 to 8.0, although deviations from these values are permissible.

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   EXAMPLE I. -
A suitable inoculum is prepared as follows for initiating large scale fermentation.



   5 to 10 cm3 of aseptic water are used for the suspension of the surface vegetation of an agar-agar culture in an inclined test tube. The resulting suspension of spores and mycelium fragments was used to inoculate two 100 cm3 batches of aseptic medium in 500 cm3 Erlenmeyer flasks. After inoculation, both vials are incubated on an alternating shaker at about 28 ° C for about two days, after which the 200 cm3 of primary inoculum is used to inoculate 6 liters of medium into a 9-liter glass carboy, and incubate again, with aeration, usually for about a day, at about 28 ° C.

   The 6 liters of culture in the cylinder are then used to inoculate 100-200 liters or more of aseptic medium into a fermentation tank. For larger batches of 500 liters or more, usually larger inocula are obtained by pooling two or more inocula into 6 liter cylinders.



   A fermentation medium is prepared having the following composition:
 EMI8.1
 
<tb> liqueur <SEP> from <SEP> maceration <SEP> from <SEP> but <SEP> 12.5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb> mannitol <SEP> 10.0 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 2.0 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> (NH4) 2 <SEP> HPO4 <SEP> 2,0 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> KH2PO4 <SEP> 1.5 <SEP> g
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> MgSO4. <SEP> 7H2O <SEP> 0.25
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> solution <SEP> of trace elements <SEP> Winsonsin <SEP> A-Z <SEP> 1.0 <SEP> cm3
<tb>
 and the mixture is made up to 1000 cm 3 with water, then adjusted to pH 7-7.2 with aqueous sodium hydroxide.



   1500 liters of this medium are placed in a 2000 liter fermentation tank, sterilized for about 60 minutes under a vapor pressure of 1.05 Kg / cm2 (120 C) and inoculated with 12 liters of a good culture. good 23 hour old, as described above. The pH is 6.96 before sterilization and 6.68 after sterilization. The mixture is closed for 112.5 hours at 26-29 C (mostly 27-28 C).



   In addition to the above fermentation, a number of similar fermentations are carried out under varying conditions, as shown in the following table. The results of the titrations were obtained using as test organism the Streptoooocus haemolyticus strain C-203, and as growth medium a brain-heart infusion "Difco-Bacto" reconstituted with water and 1.5%. agar-agar.

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 EMI9.1
 



  ¯¯¯¯¯¯¯Inoculum¯¯¯¯¯¯¯¯¯¯¯ i
 EMI9.2
 
<tb> Exem- <SEP> Capacity <SEP> liters <SEP> liters <SEP> of <SEP> Age <SEP> of <SEP> the <SEP> ass- <SEP> Tempera- <SEP> Report <SEP> Time <SEP> of <SEP> Titration,
<tb> ple <SEP> of <SEP> the <SEP> tank, <SEP> of <SEP> culture <SEP> in <SEP> ture <SEP> in <SEP> bobon- <SEP> ture <SEP> d 'aeration:

   <SEP> fermented. <SEP> pH <SEP> width
<tb> in <SEP> liters <SEP> medium <SEP> man <SEP> ries <SEP> hours <SEP> C <SEP> vol.d'air / <SEP> tion, <SEP> in <SEP> of
<tb> vol.de <SEP> mid- <SEP> hours <SEP> zones
<tb> location
<tb>
 
 EMI9.3
 1! 1900 1,500 12 23 26 - 29 0.85 0 X- 6.96
 EMI9.4
 
<tb> 0 <SEP> 6.68
<tb> 96 <SEP> 6.22 <SEP> 29.0
<tb> 112.5 <SEP> 6.54 <SEP> 31.2
<tb> II <SEP> 380 <SEP> 200 <SEP> 6 <SEP> 28.5 <SEP> 28 <SEP> 0.95 <SEP> 0 <SEP> X- <SEP> 7.02
<tb> 0 <SEP> 6.72
<tb>
 
 EMI9.5
 48 6.52 23.3
 EMI9.6
 
<tb> 72 <SEP> 7.13 <SEP> 27.7
<tb> 89 <SEP> 7.22 <SEP> 31.3
<tb> III <SEP> 380 <SEP> 200 <SEP> 6 <SEP> 26 <SEP> 28 <SEP> 0.95 <SEP> 0 <SEP> - <SEP> 7.26
<tb> 0 <SEP> 6.72
<tb> 74.5 <SEP> 6.43 <SEP> 31.3
<tb> 91.5 <SEP> 6.70 <SEP> 32.7
<tb>
 
 EMI9.7
 IV 760 400 6 30.5 27-29 1.00 0 r 7.1 6.55 6.55
 EMI9.8
 
<tb> 72.5 <SEP> 6.80 <SEP> 29,

  2
<tb> 89 <SEP> 7.24 <SEP> 30.6
<tb>
   (@ -): before sterilization. -
 EMI9.9
 5ais: S-): "0" indicates after sterilization. 4

 <Desc / Clms Page number 10>

 
Isolation operations.-
Several methods have been developed for recovering the antibiotic from the fermentation liquor, which are based on the physical and chemical properties of the antibiotic. In general, it is preferable to filter the fermentation liquor to remove mycelium and other insoluble fermentation constituents by filtration at harvest pH. A filter aid such as diatomaceous earth is used. The antibiotic can then be adsorbed onto and eluted from an adsorbent.

   Solvent extraction, chromatography and release methods are also used, as will be seen in the specific examples which follow.
A preferred isolation method consists of adsorbing the antibiotic from the filtered fermentation liquor onto activated charcoal and then eluting the active ingredients by means of a polar solvent. The active ingredients are adsorbed on charcoal at the normal pH of the fermentation liquor, which may be approximately 6.5 to 8.0. The eluent is preferably a water-miscible polar solvent, such as a 95% acetone and 5% water mixture.



  No particular adjustment of the pH of the eluent is required. Instead of aqueous acetone, methanol acidified to pH 2 or 3, or pH greater than 9, can be used. Other alcohols, such as ethyl alcohol, propanols, butanols, etc. .., can also be used as eluents. They can be cut off from water by 50% or more if desired. Other water-miscible hydroxylated polar solvents include cellosolves such as ethoxyethanol, etc. Dilute acids such as 0.05 N hydrochloric acid and acetic acid can serve as eluent to recover. the antibiotic adsorbed on the charcoal.

   Other polar solvents which can be used to recover the active ingredients from the charcoal include pyridine, aqueous solutions of phenol, and others of a similar type.



   The adsorption and elution can be conducted in various ways, familiar to those skilled in the art. One can simply stir the charcoal and the filtered fermentation liquor, then refilter it and recover the active ingredients in the cake by passing the eluent through it, or by stirring it towards the eluent. Another common method would be to pass the fermentation liquor through a column packed with charcoal, and then to pass the eluent through the column. Prior to elution, one can wash with water to remove water soluble impurities which are not strongly adsorbed on the charcoal.



   After having recovered the active ingredients adsorbed on the charcoal, it is generally desirable to concentrate the eluent. If desired, the concentration can go to dryness, and the material can be kept until it is ready for further purification. Due to the very high antitrypanosomal activity of the material, it may be suitable for the treatment of animals at this stage of the process. This is because the antibiotic is so active that the fermentation liquor itself has been found to be effective, in vivo, against trypanosoma equiperdum.



   Further purification can be carried out by activated carbon chromatography. In this method, a column of activated charcoal is prepared, the concentrated antibiotic is dissolved in a suitable solvent such as a 50/50 mixture of acetone and water, and passed through the colon. - born. The column is then developed by continuously passing a similar solvent through it until impurities weakly adsorbed on the column are eluted and removed. When the antibiotic active ingredients begin to pass through the column, as determined by simple activity tests of the type described later, then pass through.

 <Desc / Clms Page number 11>

 the carbon layer gives a stronger eluent of the type described above, and the active ingredients are thus recovered.



   The eluent can be concentrated or dried by lyophilization. Frequently, crystals of the antibiotic are recovered as a result of the concentration process.



   Further purification of the antibiotic can be achieved by several methods, some of which will be illustrated below. A preferred method is to use normal butanol and a fractional column of diatomaceous earth, as described in Example II. It will be understood, of course, that these methods serve merely as an example and are not intended to restrict the present invention to any particular method of purification.
EXAMPLE V.-
Preliminary purification - Adsorption column -
The fermented liquor is combined from several vats as described above, so as to have a fermented mass volume of 1450 liters at pH 7.1.



  The mass is filtered using "Hyflo Super-Cel" (diatomaceous earth) to give 1300 liters of filtrate. The titration indicates a total of 5.64 g of pure antibiotic in the 1300 liter solution. 1950 gr of "Darco G-60" (activated charcoal) are added, and the mixture is stirred for 1/2 hour. 5850 g of "Celite 545" (diatomaceous earth) are added, and the mixture has become homogeneous by stirring, it is filtered. The charcoal and diatomaceous earth cake is stirred in 40 liters of water for 5 minutes and filtered. The washed cake was mixed with 20 liters of water to make a thick homogeneous slurry which was poured into a 23 cm inside diameter glass column. The resulting column was about 60 cm high.

   This column was developed with a 95% acetone and 5% water solution. A total of 68.5 liters of this solution was used to develop the column.



   The first 65 liters of percolation product are collected, the first ten liters form a first fraction and the following 55 liters a second fraction. These fractions contain 17% and 46% respectively of the 5.64 g of primary active ingredients. The first fraction is concentrated under reduced pressure to give an aqueous solution of 2 liters, and the second fraction is similarly concentrated to give 3.5 liters of aqueous solution.

   Each concentrate is then lyophilized separately, and the following is obtained Fraction n 1: 46 g, the titration of which shows that they contain the equivalent of 0.94 g of crystalline antibiotic; Fraction n 2: 59.5 g, the titration of which shows that they contain the equivalent of 1.92 g of crystalline antibiotic EXAMPLE VI.-
Preliminary purification - Adsorption-elution method -
350 liters of fermentation mass at pH 7.4 were taken, and filtered with "Hyflo Super-Cel" to give 300 liters of filtrate. Adjust the pH to 6-7, and add 600 g of "Darco-G-60". The mixture is stirred for 1/2 hour, about 1200 g of "Hyflo Super-Cel" is added, and

 <Desc / Clms Page number 12>

 stir the slurry until homogeneous and filter.

   The cake is stirred in 10 liters of water for 5 minutes and filtered. The washed cake is stirred in a solution of 5700 cm3 of acetone and 300 cm3 for 20 minutes, and filtered. The volume of this first elution product is 5.5 liters.



    The residual cake was eluted two more times in the same manner, and a second and a third elution product of 5.4 liters and 4.6 liters respectively was obtained.



   The three elution products were reduced to obtain a 15.5 liter solution. The pH is adjusted to 6-6.5 and the solution is concentrated in vacuo to obtain an aqueous solution of 1.5 liters. The pH of the concentrate is adjusted to 6.5 and the solution is lyophilized. The yield is 25.5 g.



     EXAMPLE VII Preliminary purification - Solvent extraction The fermentation mass of tank 60 (prepared essentially as in Examples I-IV) is treated exactly as described in Example VI, except that no does not freeze-dry the final aqueous concentrate. The volume of the concentrate is 2 liters, and this solution is adjusted to pH 2 (with hydrochloric acid) and extracted twice, each time with 1 liter of n-butanol. The aqueous residue is adjusted to pH 7 with aqueous sodium hydroxide, and saturated with approximately 1200 g of ammonium sulfate. The resulting mixture is extracted three times, each time with 1 liter of butanol.



    A certain amount of insoluble material which forms at the interface during extraction is removed. The following table shows the results of these extractions:
 EMI12.1
 
<tb> Solution <SEP> Solids <SEP> to- <SEP> Antibiotic
<tb>
<tb> rate <SEP> in <SEP> pure <SEP> total, <SEP> in
<tb>
<tb> grams <SEP> mg, <SEP> (calculated)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> concentrated <SEP> primitive <SEP> 57.4 <SEP> 976
<tb>
<tb>
<tb>
<tb> first <SEP> washing <SEP> with <SEP> butanol <SEP> pH <SEP> 2 <SEP> 29.1 <SEP> 46
<tb>
<tb>
<tb>
<tb> second <SEP> washing <SEP> with <SEP> butanol <SEP> pH <SEP> 2 <SEP> 7.5 <SEP> 45
<tb>
<tb>
<tb>
<tb> aqueous <SEP> residue <SEP> 23.5 <SEP> 876
<tb>
<tb>
<tb>
<tb> first <SEP> extract <SEP> with <SEP> butanol <SEP> pH <SEP> 7 <SEP> 7,

   <SEP> 26 <SEP> 497
<tb>
<tb>
<tb>
<tb> second <SEP> extract <SEP> with <SEP> butanol <SEP> pH <SEP> 7 <SEP> 3, <SEP> 9 <SEP> 208
<tb>
<tb>
<tb>
<tb> Third <SEP> extract <SEP> with <SEP> butanol <SEP> pH <SEP> 7 <SEP> 1.43 <SEP> 80
<tb>
 
The first and second extracts were combined, the solvent removed, concentrated and lyophilized to give a material which had 60 micrograms of activity per mg.



     EXAMPLE VIII.-
New purification Adsorption column.



   An adsorption column is prepared as follows: 3000 g of "Celite 545" and 1000 g of "Cardo G-60" are thoroughly mixed in a kneader. 2000 cm3 of a 50% acetone and 50% water solution are added thereto, and the whole is mixed thoroughly. The final homogeneous mixture is compressed with compression in an inverted 20-liter carboy, the bottom of which has been cut to form a column 28 cm in diameter and 33-35 cm. from above.

 <Desc / Clms Page number 13>

 



   Samples prepared by the method of Examples V and VI were pooled to give about 125 µl of bruto antibiotic. This material contained a total of about 126 x 106 activity units, or 1000 units per mg, and dissolved in 1 liter of 50% acetone and 50% water solution, and the resulting solution is added to the top of the column. The column is developed with 46 liters of a 50% acetone and 50% water solution.

   The percolation product is fractionated as follows
 EMI13.1
 
<tb> Fraction <SEP> Volume <SEP> in <SEP> liter <SEP> Solids <SEP> totals <SEP> Activity <SEP> units
<tb> in <SEP> g <SEP> total
<tb>
<tb> A <SEP> 8.5 <SEP> 7.56
<tb>
 
 EMI13.2
 B 6, 9 in, 6 19, 4 x 106 c la, 2 7, 7 799.8 x 106 D 9, 4 3, 8 31.2 x 1Q6
 EMI13.3
 
<tb> 29.66 <SEP> 130 <SEP> x <SEP> 106
<tb>
 
The solvent is removed from the last three fractions under reduced pressure) and lyophilized with the following results:

   
 EMI13.4
 
<tb> Fraction <SEP> Weight <SEP> in <SEP> grams <SEP> Units <SEP> per <SEP> mg
<tb>
<tb> B <SEP> la, 3 <SEP> 1500
<tb>
<tb> C <SEP> 7.7 <SEP> 8640
<tb>
<tb> D <SEP> 3.6 <SEP> 8500
<tb>
<tb> 21.6
<tb>
 
The total recovery of activity units in the three dried samples is approximately 112 x 106 units (1 microgram crystalline antibiotic = 45 activity units).



   EXAMPLE IX.-
New purification - - Fractionated column.-
The n-butanol and water are stirred together in a separating funnel and separated to give two mutually saturated solutions. About 500 cm3 of n-butanol are added to about 15 liters of water-saturated butanol. so that the solvent phase used in this column is formed from butanol incompletely saturated with water.



   1 kg of "Celite 545" washed with ecid, is intimately mixed with 500 cm3 of aqueous phase :, and the mixture is packed to form a column 42 cm2 in cross section and about 83 cm high.



   Samples from Examples V and VI, weighing 3.6 g and containing about 8850 activity units per mg, or a total of about 14.9 g of partially purified antibiotic containing about 129 x 10, are put together. activity units, and we add it all to about 153 cm3

 <Desc / Clms Page number 14>

 of aqueous phase, then the mixture is stirred constantly at room temperature while bringing the pH to 2.0. After further stirring, the mixture is filtered to remove a small amount of insoluble material.



  On 3 cc of the filtrate, titration and determination of the total solids are carried out and the remaining 150 cc is mixed with 300 g of acid washed "Celite 545". The resulting mixture is packed at the top of the column, a further 25 cm is added thereto, to give a total height of 108 cm.

   The column is then developed with the solvent phase, and the percolation product is fractionated as follows
 EMI14.1
 
<tb> Fraction <SEP> Volume <SEP> in <SEP> cm3 <SEP> Accumulated volume <SEP> <SEP> Total solids <SEP> <SEP> in
<tb>
<tb>
<tb> mg / cm3
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F1 <SEP> 1.120 <SEP> 4.3
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F2 <SEP> 1.070 <SEP> 2.190 <SEP> 0.52
<tb>
<tb>
<tb>
<tb>
<tb> F3 <SEP> 675 <SEP> 2.865 <SEP> 0.48
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F4 <SEP> 800 <SEP> 3.665 <SEP> 0.38
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F5 <SEP> 900 <SEP> 4. <SEP> 565 <SEP> 0.40
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F6 <SEP> 1.070 <SEP> 5. <SEP> 635 <SEP> 0.22
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F7 <SEP> 1.020 <SEP> 6. <SEP> 655 <SEP> 0.23
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F8 <SEP> 1.040 <SEP> 7.

   <SEP> 695 <SEP> 0.21
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F9 <SEP> 1.200 <SEP> 8. <SEP> 895 <SEP> 0.14
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F10 <SEP> 1.060 <SEP> 9.955 <SEP> 0.45
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Thread <SEP> 640 <SEP> the <SEP> * 595 <SEP> 1, <SEP> 21 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F12 <SEP> 4.000 <SEP> 14.595 <SEP> 0.56
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> F13 <SEP> 0.94
<tb>
 
Fractions F10, Fil and F12 were combined, concentrated under reduced pressure to remove the solvent, and lyophilized to give 3.34 g of product.



   EXAMPLE X.



   Crystallization of the new antibiotic.
3.34 g of partially purified antibiotic are dissolved in 80 cm3 of water at 65 C, and the pH of the solution is adjusted to 1.5 with acid. Dilute hydrochloric acid The mixture is filtered to remove some insoluble gum. The pH is adjusted to 4.0 with dilute aqueous sodium hydroxide, a seed crystal is added, and the solution is left to stand overnight at 4-5 C. The next morning, the crystals are filtered off, washed with water. cold water, and dried in a vacuum desiccator over "Drie- rite" at a pressure of less than 1 mm, for about 24 hours; yield 1.19 g.

 <Desc / Clms Page number 15>

 



   EXAMPLE XI. -
Crystallization of the new antibiotic after extraction of impurities with butanol.



   Several samples purified by the first adsorption, for example the samples of Example VI, are combined and subjected to a second purification in an adsorption column, as described in Example VIII. The antibiotic-rich portion of the infusion product is concentrated under reduced pressure to a small aqueous volume and lyophilized as in Example VIII. A sample weighing 1.67 g and containing a total of about 12.8 x 106 units of activity is dissolved in 80 cm 3 of water, and hydrochloric acid is added until the solution is obtained. decinormal with respect to this acid. This solution is extracted twice, each time with 80 cc of n-butanol, thus removing about 1.07 g of total solids but only about 9% of the activity.

   The pH of the residue is adjusted to 6.5 and the solvent removed from the resulting solution under reduced pressure, then concentrated to 5-10 cm3 and kept under refrigeration. The crystalline product is filtered, taken up in about 10 cm3 of water at pH 3-4 while heating slightly, and filtered. The filtrate is cooled, the crystals obtained are filtered off, washed with cold water and dried; yield, 145 mg of white crystals.



    EXAMPLE XII.- Recrystallization of the novel antibiotic - Preparation of samples ana- ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯lytics.-¯¯¯¯¯¯¯¯¯¯¯ ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ On'd dissolve, in 200 om3 of water at 60 C, 4522 g of various combined batches of crystalline antibiotic (prepared as described in example X), and the pH of the solution is adjusted to 2.5. The solution is filtered and the filtrate is adjusted to pH 4, and the mixture is left to stand overnight at 4-5 C. The crystals are filtered off and the same recrystallization operation is repeated four times. The final recrystallization comprises a decoloration step. with a small amount of charcoal o The final product is 1.89 g of analytical sample (after drying under a pressure of 2 mm).

   From this sample, 1.25 g are taken, which are further dried for 2 hours with an Abderhalden gun at the boiling point of chloroform. The product obtained is constituted by tangles of filiform crystals. The principal directions of vibration and the corresponding principal refractive indices are difficult to determine, and are not given, due to the possibility of error.

 <Desc / Clms Page number 16>

 



  EXAMPLE XIII.-
 EMI16.1
 
<tb> Dextrin <SEP> 1%
<tb>
<tb> N-Z <SEP> Amine <SEP>. <SEP> A <SEP> 1%
<tb>
<tb> NaCl <SEP> 0.2%
<tb>
 
 EMI16.2
 (NH 4) 2EPO4 0,
 EMI16.3
 
<tb> KH2PO4 <SEP> 0.15%
<tb>
<tb> K2HPO4 <SEP> 0.05%
<tb>
<tb> MgSO4.7H2O <SEP> 0.025%
<tb> Solution <SEP> Wiso.A-Z <SEP> 0.1% <SEP> (in <SEP> volume)
<tb>
<tb> Water <SEP> from <SEP> tap <SEP>: <SEP> q.s. <SEP> for <SEP> 100%
<tb>
 
100 cm3 of the above medium are taken, sterilized for 20 minutes in a 500 cm3 Erlenmeyer flask, under a vapor pressure of 1.05 kg / cm2, and inoculated with 3 cm3 of inoculum prepared in advance. The flask is incubated on an alternating shaker at 28 ° C for 72 hours. The fermented liquor is found to be active against Trypanosoma equiperdum.



   EXAMPLE XIV. -
Equal volumes of n-butanol and the above fermented liquor are stirred together at the pH indicated, then separated. The table below shows the activities in the butanol extracts and the aqueous residues, as well as the extraction coefficients.
 EMI16.4
 
<tb>



  Butanol <SEP> Water <SEP> K
<tb>
<tb>
<tb>
<tb> pH <SEP> 2 <SEP> 8 <SEP> 135 <SEP> 0.06
<tb>
<tb>
<tb> pH <SEP> 5 <SEP> 51 <SEP> 100 <SEP> 0.5
<tb>
<tb>
<tb> pH <SEP> 7 <SEP> 49 <SEP> 88 <SEP> 0.6
<tb>
<tb>
<tb> pH <SEP> 9 <SEP> 23 <SEP> 123 <SEP> 0.2
<tb>
 
EXAMPLE XV.-
A quantity of the above fermented liquor is saturated with ammonium sulfate. Five liters of this solution are extracted twice, each time with 2.5 liters of n-butanol. Another five-pound batch is taken and extracted twice, each time with 2.5 liters of acetone.



   The two extracts are combined with butanol, and they are found to contain almost all of the activity of the fermented liquor. The two extracts were combined with acetone and were also found to contain all the activity of the fermented liquor.

 <Desc / Clms Page number 17>

 



     EXAMPLE XVI.-
9 liters of a similar fermented liquor are stirred at harvest pH 11 with 55 g of "Darco G-60" for 1/2 hour. The suspension is filtered with a small amount of "Celite" and washed on a Buchner funnel with 500 cc of water.



   The washed cake is then stirred for about 20 minutes, with
300 cm3 of acetone, and filtered. The residual cake is again eluted twice, each time with 300 cm3 of 95% acetone. The three liquors eluting with acetone are combined and titrated; the results show a recovery of 35% of the activity of the fermented liquor. The overall solution is concentrated in vacuo until an aqueous concentrate is obtained which is freeze-dried, obtaining 3.64 g of solids, the titration of which determines that they represent a material eleven times purer than the antibiotic of fermented liquor.



   Butanol or acetone can be replaced in the above extraction with beta-methoxyethanol or beta-ethoxyethanol.



     EXAMPLE XVII.-
50 mg of the antibiotic, free base, of Example XII are dissolved in approximately 5 cm3 of water, adding 0.1N hydrochloric acid, then
5 cm3 of a saturated aqueous solution of picric acid. The resulting crystalline precipitate is centrifuged and washed with about 5 cc of cold water.



   The product is recrystallized three times from water, followed by hot filtration (60-75 C) in the final step, and dried for 24 hours under 1 mm, and finally dried for 4 hours over P2O5. , at 110 C and under 1 mmo Yield: 35 mg of bright yellow picrate crystals.



    .Analysis:
Calculated for C17H18O15H9S: C 32.9; H 2.91; N 20.3; S 5.17; 0 (by difference) 38.1;
Effective: C 33.19; H 3.13; N 20.19; s 5.70; 0 (by difference) 37.79.



   Melting point 143-144 C (uncorrected) with slight decomposition.



   Ultraviolet # max (millimicrons) 253.356 (20 micrograms cm3 in ethanol).



     EXAMPLE XVIII.-
150 mg of the antibiotic, free base, are dissolved in 5 cm3 of glacial acetic acid and treated with 5 cm3 of glacial acetic acid saturated with hydrochloric acid.



   The white hydrochloride precipitate which formed was removed by filtration, washed with 10 cm3 of glacial acetic acid and 10 cm3 of anhydrous ethyl ether, and dried for 24 hours over "Drierite" at 1 mm. and at room temperature.

 <Desc / Clms Page number 18>

 



   EXAMPLE XIX. -
A solution of 150 mg of the free base is taken in about 10 cm3 of absolute ethanol, the mixture is treated with a mixture of 5 cm3 of absolute ethanol and about 4 drops of concentrated sulfuric acid. The sulfate precipitate obtained is filtered off, washed with absolute ethyl ether, and dried under 1 mm for 24 hours. The product is amorphous.



   EXAMPLE XX.-
A concentrated aqueous solution containing about 150 mg of the free base is taken, and treated with a saturated aqueous solution of methyl orange to obtain a colored crystalline precipitate of hemianthate.



   CLAIMS.



   A process for the preparation of an antibiotic, in which an aqueous nutrient medium is fermented with a microorganism of the species Streptomyces T-3018.


    

Claims (1)

2. The method of claim 1, wherein the 'fermentation' is carried out between 17 and 42 ° C. 3. The method of claim 1, wherein the fermentation is carried out for a period of at least 48 hours. 4. The method of claim 1, wherein the -fermentation is carried out at a pH of 6 to 8. 5. A method according to any of claims 1 to 4,. wherein the water-soluble constituents of the fermentation liquor are contacted with an adsorbent, the adsorbent is separated from the liquid, and the antibiotic is recovered from the adsorbent by elution using 'a polar solvent. 6. - The method of claim 5, wherein the solvent is a mixture of acetone and water. 7. A process according to either of claims 5 and 6, wherein the adsorbent is columnar charcoal, and the eluent is passed through said column. 8. A process according to either of claims 5 and 6, wherein the elution product is passed through a column of activated carbon, the latter is developed by continuously passing a sol- flow through until the weakly adsorbed impurities are removed, and the elution is continued with a more vigorous eluent until the active principles have been removed, then the solvent phase containing the active principles is concentrated. 'separation from the antibiotic. 9. A process for producing an antibiotic, in substance, as described above. 10. Antibiotic, when produced by the process according to any one of the preceding claims. .il.- Antibiotic comprising, as elements, carbon, hydrogen, nitrogen, sulfur and oxygen, having an infrared absorption spectrum, in substance , as shown in figure 1 and a <Desc / Clms Page number 19> adsorption spectrum in the ultraviolet at pH 7 substantially as shown in the figure. 2, as well as the salts of this antibiotic.