BE557870A - - Google Patents

Description

       

   <Desc / Clms Page number 1>
 



   The present invention relates to novel therapeutic substances which have been identified as antibiotics of the demethyltetracycline group (including demethyltetracycline, chlorodemethyltetracycline and bromodemethyltetracycline) and their epimers.



   The new antibiotics according to the present invention are formed in a fermentation process in which mutant strains of Streptomyces aureofaciens are used. The new antibiotics appear to be closely related to members of the tetracycline family described previously, but are markedly different from them. They are characterized by marked stability in both alkaline and acidic environments, and are superior to tetracyclines in this regard. They have similar antibiotic activity and can serve the same uses, and in the same general way, as the tetracyclines currently known.



   According to the invention, there is provided a process for the preparation of antibiotics of the demethyltetracycline group (including demethyltetracycline, chlorodemethyltetracycline and bromodemethyltetracycline) and their epimers, which process comprises aerobic fermentation of an aqueous nutrient medium of 'a regulated content of ions' chloride and / or bromide, with a strain of S. aureofa- ciens producer of demethyltetracycline, to recover if desired the obtained demethyltetracycline antibiotics, from the fermentation medium, and, if desired , in adjusting the pH of a concentrated solution of this antibiotic to obtain the epimers.

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   Each of the previously known tetracycline antibiotics which are produced by fermentation with S. aureofaciens under specific conditions, namely, tetracycline, chlorotetracycline and bromotetracycline has its counterpart in the new series of antibiotics. . In other words, according to the process of the present invention, by using certain mutant strains of S. aureofaciens which will be more particularly described below, one obtains an antibiotic substance similar to tetracycline, another similar to chlorotetracycline and another similar to bromotetracycline. The particular new antibiotic which is formed as a result of the fermentation process depends on the nature of the nutrient medium and other conditions of the process.

   In a chloride-free medium, the predominant antibiotic is the unchlorinated analogue of tetracycline. In a medium rich in bromine, the brominated antibiotic is formed; and in a medium rich in chlorine, the predominant antibiotic will be a chlorinated antibiotic similar to chlorotetracycline. Usually, tetracycline, chlorotetracycline and / or bromotetracycline are also formed during the fermentation process, and their relative proportions depend on the chloride and / or bromide ion content of the medium, and on the strain of. microorganism used - By careful selection of the strain of S. aurecfaciens, it is possible to almost completely eliminate the formation of these known antibiotics of the tetracycline group.



   New antibiotics according to the present invention can be converted to epimers as described below, similar to the conversion of tetracyclins to quatrimycins.



   Chlorinated and brominated demethyltetracyclines according to the present invention can be dehalogenated by

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 hydrogenation, with a palladium-on-charcoal catalyst, as well as converting chlorote-gracycline or bromotetracycline into pure and simple tetracycline.



   Chemical analyzes, and examination of the chemical and physical properties of the new compounds, indicate that they differ essentially from the corresponding tetracyclines in that they contain one less methyl group. The methyl group in question is most likely that which occupies the 6-position on the naphtacene ring of tetracyclins. Accordingly, the tetracycline analog will be called 4-dimethylamino-1,4,4a, 5,5a, 6,11., 12a-octahydro- 3,6,10,12,12a-pentahydroxy-1,11 -dioxo-2-naphtacene-carboxamide. In the case of chlorine and bromine analogues, the halogen substitute should be in the 7-position, and will be named accordingly.

   Appropriate common names for these new antibiotics would be demethyltetracycline, chlorodemethyltetracycline and bromodemethyltetracycline, and this nomenclature will be used herein. But it is understood that the postulated structure has not been proven by unequivocal synthesis, and could be "later found to be erroneous. The present invention relates to novel compounds which possess certain properties, and is not limited by a particular system of nomenclature nor by a probable structure.



   The new antibiotics according to the invention are produced by certain mutant strains of S. aureofaciens.



  These strains are undoubtedly of the species Streptomyces aureofaciens, since they derive from the primitive strain - A-377 isolated by Dr. BM Duggar, and deposited at the Northern Regional Research Laborabories in Peoria (Illinois, United States), and indexed in this Laboratory under n NRRL 2209. Mutations involving treatment of the strain

 <Desc / Clms Page number 4>

   A-377 by mutagens, successively comprising ultraviolet irradiation, nicotine and 2,2-dichloro-N-methyldiethylamine hydrochloride, were used to create one of the strains. Various other strains have been obtained by spontaneous mutation and by treatment with mutagens.

   These various strains, which produce the new antibiotics in different proportions and with varying yields, exhibit the general characteristics of the .aureofaciens species. They differ a little from each other and also from the strains of S. aureofaciens previously described, mainly by their pigmentation and their property of producing new antibiotics. In most cases, a reddish-brown pigment is noted when the microorganisms are grown on ordinary culture media.



   The undertones of the pigment vary from a peach or coppery brown shade to a dark mahogany, depending on the particular strain and the nutrient medium used. Although the property of producing the novel antibiotics of the present invention is generally associated with strains which exhibit this type of pigmentation, this pigment formation and the production of the new antibiotics are not necessarily integral properties. All the strains studied produce chlorotetracycline, and generally tetracycline, although the relative ratios of these antibiotics vary considerably depending on the nature of the fermentation medium, the fermentation conditions and the particular strain. chosen.

   In some cases, the production of chlorotetracycline and tetracycline is very low, of the order of a few% of the total antibiotic produced by fermentation.



   The mutant strains which produce the novel antibiotics of the present invention have the same general characteristics as the strains which produce

 <Desc / Clms Page number 5>

 tetracyclines, and they differ from each other in the same general way that tetracyline-producing strains differ from each other, as has been explained in several published scientific articles. Although many strains of S. aureofaciens are available to the public in culture collections and have been described in the scientific literature, the following data will serve to illustrate the type of variation of the new strains, compared to the primitive strain A-377 which is under the designation NRL 2209.



     Comparison of S. aureofaciens strains
S-604 and A-377 on various environments.
Streptomyces aureofaciens strain S-604 which produces chlorodemethyltetracycline and demethyltetracycline was compared to Streptomyces aureofaciens strain A-377 (NRRL 2209) by observing the growth characteristics on various media incubated at 26-27 C.



   The observations made are as follows:
1.- Glycerol, asparagine, beef extract, agar-agar:
 EMI5.1
 
<tb> Glycerol <SEP> 1%
<tb>
<tb>
<tb>
<tb>
<tb> Asparagine-L <SEP> 0.05%
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Extract <SEP> of <SEP> beef <SEP> 0.2%
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> KH2PO4 <SEP> 0.05%
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Agar-agar <SEP> Bacto <SEP> 1.5%
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Distilled <SEP> water <SEP> q.s. <SEP> for <SEP> adjustment <SEP> of <SEP> pH <SEP> 100%
<tb>
<tb>
<tb>
<tb> by <SEP> KOH <SEP> at <SEP> 50%:

   <SEP> 7.0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> pH <SEP> after <SEP> sterilization <SEP> 7.1
<tb>
 

 <Desc / Clms Page number 6>

 Streptomyces aureofaciens
 EMI6.1
 
<tb> Strain <SEP> S-604 <SEP> Strain <SEP> A-377
<tb>
<tb>
<tb>
<tb>
<tb> Growth <SEP> Abundant, <SEP> color <SEP> Fairly <SEP> abundant
<tb>
<tb>
<tb>
<tb> "Venetian <SEP> Red;

   <SEP> ()
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Hyphae <SEP> aerial <SEP> Abundant, <SEP> white <SEP> to <SEP> White, <SEP> unifor-
<tb>
<tb>
<tb>
<tb> "Rose <SEP> gray" <SEP> () <SEP> mes
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Sporulation <SEP> Slight, <SEP> becoming <SEP> None
<tb>
<tb>
<tb> abundant
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Pigment <SEP> diffusi- <SEP> Reddish brown <SEP> Yellow
<tb>
<tb>
<tb>
<tb> ble
<tb>
 
 EMI6.2
 Reverse ItBro \ "1l1 Mahogany" () Yellow to yellow
 EMI6.3
 
<tb> orange <SEP> light
<tb>
 () Designation of shades according to "Color
Harmony Manual "3rd edition, edited by
Container Corporation of America 2 .- 'Dextrin, Czapek-Dox, agar-agar .-
 EMI6.4
 
<tb> Dextrin <SEP> 1%
<tb>
<tb> NaN03 <SEP> 0.2%
<tb>
<tb> K2HP04 <SEP> 0.1%
<tb>
 
 EMI6.5
 ²S04.7H20 0,

  05%
 EMI6.6
 
<tb> KC1 <SEP> 0.05%
<tb>
 
 EMI6.7
 . FeSO.?H0 0.001%
 EMI6.8
 
<tb> Agar-agar <SEP> Bacto <SEP> 1.5%
<tb>
<tb> Distilled <SEP> water, <SEP> q, <SEP> s. <SEP> for <SEP> 100.0%
<tb>
<tb> pH <SEP> after <SEP> sterilization <SEP> 7.0
<tb>
 
 EMI6.9
 Streptomyces aureofaciens -
 EMI6.10
 
<tb> Strain <SEP> S-604 <SEP> Strain <SEP> A-377
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Growth <SEP> Rare, <SEP> hyaline <SEP> Profuse
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Hyphae <SEP> aerial <SEP> Absent <SEP> Abundant, <SEP> "
<tb>
<tb>
<tb>
<tb> lead <SEP> gray "<SEP> ()
<tb>
<tb>
<tb>
<tb> Superficial <SEP> blood cells
<tb>
<tb>
<tb> colorless
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Sporulation <SEP> None <SEP> Abundant
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Pigment <SEP> diffusible <SEP> None <SEP> Light:

   <SEP> pale yellow <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Reverse <SEP> no <SEP> pigmented <SEP> No <SEP> pigmented
<tb>
 () see previous note.

 <Desc / Clms Page number 7>

 
 EMI7.1
 3. -Maceration liquor of corn, èJ.1ar-agar:
 EMI7.2
 
<tb> Liquor <SEP> of <SEP> maceration <SEP> of <SEP> but <SEP> 0.4%
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Sucrose <SEP> 1.0%
<tb>
 
 EMI7.3
 jgSO, .7H 0 0.025% 1 \ H2P04 0.2% (NH4) zHP04 0.2% Agar-agar Bacto 2.0
 EMI7.4
 
<tb> Water <SEP> from <SEP> tap, <SEP> q. <SEP> s.

   <SEP> for <SEP> 100.0%
<tb>
<tb> pH <SEP> after <SEP> sterilization <SEP> 63,
<tb>
 
 EMI7.5
 Streptomyces aurenfaciens
 EMI7.6
 
<tb> Strain <SEP> S-604 <SEP> 'Strain <SEP> A-377
<tb>
<tb>
<tb> Growth <SEP> Profuse <SEP> Profuse
<tb>
<tb> Hyphae <SEP> aerial <SEP> Abundant <SEP> "pink <SEP> Abundant,
<tb> taupe "<SEP> (') dark <SEP>" Beaver "
<tb>
 
 EMI7.7
 Sporulation Very abundant, LÎr.èfūab.ondteJ,
 EMI7.8
 
<tb> uniform <SEP> uniform
<tb>
<tb> Pigment <SEP> diffusible <SEP> Very <SEP> concentrated, <SEP> Yellow <SEP> greenish
<tb> "deep <SEP> brown" <SEP> to <SEP> clear
<tb> "deep <SEP> brown <SEP> Mahogany"
<tb>
 
 EMI7.9
 Reverse "Brown 3Mariogany" "Covert Brown"
 EMI7.10
 
<tb> dark <SEP> () <SEP> (<SEP>) <SEP>
<tb>
 () see previous notes.

   

 <Desc / Clms Page number 8>

 
 EMI8.1
 4.- Other medium-x Streptomyces aureofaciens
 EMI8.2
 
<tb> Strain <SEP> S-604 <SEP> Strain <SEP> A-377
<tb>
<tb>
<tb>
<tb> Agar-agar <SEP> nutri- <SEP> Poor growth <SEP>, <SEP> Growth <SEP> fairly <SEP> a-
<tb>
<tb> tif <SEP> "Tauppe <SEP> brown" <SEP> to <SEP> bouncy. <SEP> No <SEP> of hy-
<tb>
<tb> "dark <SEP> brown" (). No aerial <SEP> <SEP> phes. <SEP> Reverse
<tb>
<tb>
<tb> of aerial <SEP> hyphae. <SEP>:

  Pale <SEP> yellow Pigment
<tb>
 
 EMI8.3
 Yellow to yellow soluble "Taupe brown" reverse
 EMI8.4
 
<tb> (). Pigment <SEP> soluble <SEP> not <SEP> brownish <SEP> clear.
<tb>
<tb> brown <SEP> reddish
<tb>
<tb> Glucose, aspara- <SEP> Abundant <SEP> growth. <SEP> Growth <SEP> fairly <SEP> a-
<tb>
<tb> gine, <SEP> extract <SEP> from <SEP> Large <SEP> aerial <SEP> hyphae <SEP> bounding.Hyphae <SEP> a-
<tb>
<tb> meat, <SEP> agar-agar <SEP> variegated: "pink <SEP> taupe" <SEP> nothing <SEP> white <SEP> deve-
<tb>
 
 EMI8.5
 or "fawn" or IIcamelll (O) more and more.
 EMI8.6
 
<tb>



  -Sporulation: abundant.
<tb> () .Pigment <SEP> soluble <SEP> lying. Reverse: yellow
<tb> reddish brown <SEP>. <SEP> clear Pigment <SEP> soluble <SEP> yellow <SEP> clear.
<tb>
<tb>



  Apple <SEP> of <SEP> earth <SEP> Profuse <SEP> growth <SEP> hu- <SEP> Profuse <SEP> growth
<tb> mide, smooth, nodulated: <SEP> smooth, wet, nodu-
<tb>
 
 EMI8.7
 "Dark broim j-iahogaily" lée: "Light Nie llon () with trace of yellow" () to "asti-" peàch tan "(). Hyphae that pink" () .Hyphae
 EMI8.8
 
<tb> air <SEP>: absent <SEP> to <SEP> subscriber- <SEP> air: trace.No
<tb>
<tb> dants, becoming <SEP> white <SEP> of <SEP> pigment <SEP> soluble.
<tb>
 
 EMI8.9
 at 11 C ame! (). Sporula-
 EMI8.10
 
<tb> <SEP> abundant <SEP> in
<tb>
<tb> the <SEP> zones <SEP> of <SEP> strong <SEP> for-
<tb>
<tb> <SEP> mation of aerial <SEP> hyphae.
<tb>
<tb>



  Pigment <SEP> soluble <SEP> "choco-
<tb>
 
 EMI8.11
 late "()" chocolate
 EMI8.12
 
<tb> broi, ni "<SEP> (<SEP> <SEP>) <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Milk <SEP> purple <SEP> Light <SEP> collar <SEP> of <SEP> believe- <SEP> Light <SEP> collar <SEP> of
<tb>
<tb>
<tb>
<tb> session <SEP> "Deep <SEP> red <SEP> Maho- <SEP> growth <SEP> white <SEP> to
<tb>
<tb>
<tb>
<tb> gany "(). <SEP> Little <SEP> of <SEP> varia- <SEP> pale yellow <SEP>.

   <SEP> Pe <SEP> u <SEP> from
<tb>
<tb>
<tb> significant <SEP> <SEP> of <SEP> pH, little <SEP> variation <SEP> notalde
<tb>
<tb>
<tb>
<tb> of <SEP> peptonization <SEP> appa- <SEP> of <SEP> pH, <SEP> little <SEP> of <SEP> pept
<tb>
<tb>
<tb> rent.Light <SEP> reaction <SEP> apparent <SEP> nization
<tb>
<tb>
<tb> colored <SEP> falsely <SEP> alca- <SEP> in <SEP> 15 <SEP> days
<tb>
<tb>
<tb>
<tb> line <SEP> due <SEP> to <SEP> the <SEP> broadcast
<tb>
<tb>
<tb>
<tb> of <SEP> soluble <SEP> pigment.
<tb>
 



  () see previous notes.

 <Desc / Clms Page number 9>

 
 EMI9.1
 



  On all agar-agars, 'except dextrin' Czapek-Doc ,, but including the tilted c'u1.t \\ 1.r'e on potato, the .aurèofacien9 strain 'S-.604, produces a characteristically strong pigmentation, usually appearing with growth. Likewise, the characteristic reddish brown soluble pigment is easy to observe.
 EMI9.2
 5.- Croscopic observations.
 EMI9.3
 



  'Stre tom ces aureofacièzis
 EMI9.4
 
<tb> Medium <SEP> Strain <SEP> S-604 <SEP> Strain <SEP> A-377 <SEP>. <SEP>
<tb>
<tb>



  Mycelium <SEP> Spores <SEP> Mycelium <SEP> Spores
<tb>
<tb> Glycerol, <SEP> Sinuous, <SEP> Spheroidal <SEP> Sinuous, <SEP> Spheroidal
<tb>
 
 EMI9.5
 asparagine, continuous, to ovoid. areinu, -to ovolate. branched extract. Branched diameter. a ovo aux. beef, Diameter 1.5 - 2.0 Diameter diameter agar-agar o8 to microns 0.7 to 1.3 - 270, l, a micron 1.0 micron microns Liquor of Sinuous, Spherodidals Sinuous, Spheroidal maaeration continuous, to ovoid . continuous, to ovoid.
 EMI9.6
 
<tb> of <SEP> but <SEP> branched. <SEP> Branched <SEP> diameter !.

   <SEP> Diameter
<tb>
 
 EMI9.7
 agar-agar Diameter l5 - 2O a Diameter 1.5 - 2.0
 EMI9.8
 
<tb> 0.8 <SEP> to <SEP> microns <SEP> 0.8 <SEP> to <SEP> microns
<tb>
 
 EMI9.9
 ]., 0 micron] 1.0 micron
The morphology of the mycelium and the spores for strain S-604 is apparently similar to that observed for strain A-377. Both strains show a continuous, sinuous, branched mycelium with occasional tendency to
 EMI9.10
 aerial hyphae with spiralisatioii. The characteristic spores are ovoid to spheroidal.

 <Desc / Clms Page number 10>

 



   To illustrate the color variations among the different strains of s.aureofacieus which produce demethyltetracyclines, four selected strains were grown on agar-agar with corn maceration liquor, and the following observations were made:
Color observations (); Streptomyces aureofa- ciens:
Agar-Agar with corn maceration liquor (AP):: Incubation 4, days at 27 C.
 EMI10.1
 
<tb>



  Strain <SEP> Isolated <SEP> colonies <SEP> Growth <SEP> in <SEP> mass
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> S-604M1 <SEP> "Deep <SEP> red <SEP> mahogany" <SEP> "Deep <SEP> red <SEP> mahogany"
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> S1071 <SEP> "Copper <SEP> brown" <SEP> "Deep <SEP> brown <SEP> mahogany"
<tb>
<tb>
<tb> to <SEP> "Red <SEP> mahogany"
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> V-62 <SEP> Large <SEP> zone <SEP> marginal <SEP> "Light <SEP> copper <SEP> brown"
<tb>
<tb>
<tb> "peach <SEP> tan" <SEP> becoming
<tb>
<tb>
<tb> "light <SEP> copper <SEP> brown"
<tb>
<tb>
<tb> at <SEP> center <SEP> of <SEP> the <SEP> colo-
<tb>
<tb>
<tb> denies.
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>



  B-740 <SEP> "Dark <SEP> Wine" <SEP> "Burgundy"
<tb>
   () colors according to the work already cited /
A relatively simple method of selecting a strain of S. aureofaciens capable of producing the new antibiotics is to choose a strain of S. au- reofaciens which shows dark brown colonies when grown on agar-agar medium. and corn maceration liqueur. Inocula were prepared with these colonies and fermentation was carried out as explained in Examples 1 and.
2 below. At the end of the fermentation period, a certain amount of the fermentation liquor is acidified to a pH in the region of 1.5 to solubilize the antibiotic and filtered to obtain a clear aqueous solution.

   One or two drops of the filtrate are placed on a strip of paper and chromatographed, by ordinary methods of development.

 <Desc / Clms Page number 11>

 chromatographic method. As the solvent, butanol equilibrated with a 0.3 M aqueous phosphate buffer at pH 3 is used.



  The paper bands are developed by chromatography on the basis of the solvent phase of the equilibrated mixture. The location of the spots corresponding to the demethyltetracyclines is easily determined by inspection with ultraviolet rays. Another, more sensitive method consists of placing the strip on an agar-agar tray inoculated with microorganisms sensitive to antibiotics such as Bacillus cereus or Bacillus subtilis, - and incubating the plates. teaux. Zones of inhibition appear in the regions corresponding to the antibiotic spots.

   Comparison of the spots with control samples of pure antibiotic and the "Rf" indices given below, provide a basis for detecting the presence of the new antibiotics in the fermented liquor, and a semi-quantitative basis for determining. - determine the quantity present.



   This technique can be illustrated with the aid of Figure 1 of the drawing which shows how paper tape chromatography can separate and detect the various tetracyclines and products of the present invention. The drawing shows a strip of paper, 1, on which has been placed, at point 2, a small amount of an antibiotic solution containing tetracycline, chlorodemethyltetracycline, demethyltetracycline and chlorodemethyltetracycline. The total antibiotic activity of the solution placed at point 2 is 30 micrograms, expressed as tetracycline. The strips of paper are then developed as described above.

   After a certain time, the bands are examined with ultraviolet rays, with the results shown in the drawing. The solvent front has advanced to the point indicated by the solid line, 3. The tetracycline component of the mixed antibiotic solution has developed.

 <Desc / Clms Page number 12>

 advanced to point 5 and the chlorotetracycline is swallowed up to point 7. The new antibiotic, demethyltetracycline, has advanced to point 4, and the antibiotic chlorodemethyltetracycline is found at point 6. The clues Rf of these antibiotics are also indicated in the drawing.



  The Rf index is by definition the distance between the spot and the point of origin, 2, divided by the distance between the solvent front, 3, and the origin, 2, and of course it is always less than 1. A variety of solvent systems can be used to derive a series of Rf indices for a particular antibiotic, and these indices are considered to be very valuable in identifying antibiotics and distinguishing them from others.



   The Rf numbers of some tetracyclines, fourth-mycines and demethyltetracyclines in two precise solvent systems indicated in the following table.
 EMI12.1
 
<tb>



  Antibiotic <SEP> Ethyl acetate <SEP> / <SEP> 90/10 <SEP> chloroform /
<tb>
<tb>
<tb> pH <SEP> 4.7, <SEP> buffer <SEP> butanol,
<tb>
<tb>
<tb> citrate-phosphate <SEP> H3PO4 <SEP> aqueous <SEP> 0.3M
<tb>
<tb>
<tb>
<tb> (Mac <SEP> Elvain) '<SEP> 0.1% <SEP> acid <SEP> tri-
<tb>
<tb>
<tb> chloroacetic,
<tb>
<tb> pH <SEP> 1.9
<tb>
<tb>
<tb>
<tb> Chlorotetracycline <SEP> 0.76 <SEP> - <SEP> 0.80 <SEP> 0.61
<tb>
 
 EMI12.2
 Bromotetracyclititis 0.76 - 0.80 0.61 Tetracycline 0 .6 - 0 .7 0 z0 Quatrimycin 0.14 - 0.16 $ 0 12 Chloroquatrimycin OY27 - O28, 33 Bromoquatrimycin, 27 - 0) 28 o33 Oxyquatrimycin OYOO - 0, 06 0.11
 EMI12.3
 
<tb> Chlorodemethyltetracycline <SEP> 0.68 <SEP> - <SEP> 0.72 <SEP> 0.39
<tb>
<tb> Demethyltetracyclitle <SEP> 0.27 <SEP> 0.2q
<tb>
<tb> Epichlorodmethyltetra-
<tb>
 
 EMI12.4
 cyclin, 25 - 0.27 0,

  20
 EMI12.5
 
<tb> Epidemethyltetracycline <SEP> 0.09 <SEP> 0.10
<tb>
 

 <Desc / Clms Page number 13>

 
Several strains of S. aureofaciens which produce the new antibiotics have been deposited with the American Type Culture Collection in Washington; and are cataloged under ATCC Nos. 12551, 12552, 12553 and 12554. It is understood that mutants which also produce the new antibiotics can be derived from these strains by current methods, and indeed, it is to be expected that some of them have the property of producing higher yields of one or more of the new antibiotics, under favorable conditions.

   These mutants may vary somewhat in their general morphological characteristics, as may the various strains of the species S. aureofaciens.



  It is also expected that other strains of S. aureofaciens producing demethyltetracycline could be found in nature and could be created from the strains. currently isolated from S. aureofaciens which do not produce these new antibiotics.



   One of the most important advantages of demethyltetracycline over the tetracyclines described previously is its increased stability in acidic and alkaline media.



  The instability of tetracycline in an acidic medium and the instability of chlorotetracycline in an alkaline medium are well known. Chlorotetracycline in aqueous solution with sodium carbonate buffer at pH 9.85 has a half-life of 29.2 minutes at 23 C. On the other hand, chlorodemethyltetracycline does not lose more than 6% of its activity in 24 hours in same conditions. Tetracycline is completely destroyed within 5 minutes in 1N sulfuric acid at 100 C.



  On the other hand, chlorodemethyltetracyline loses only about 2% of its activity in 1N sulfuric acid at 100 ° C. after 15 minutes. Likewise, the new antibiotic demethyl-

 <Desc / Clms Page number 14>

 tetracycline loses only about 33% of its activity in 1N hydrochloric acid at 100 C in 6 minutes. In 0.1N sodium hydroxide at 50 C, demethyltetracycline has a half-life of approximately
20 hours, against 3.3 hours for tetracycline. These unexpected properties are very valuable, since the instability of tetracycline in an acidic medium and the instability of chlorotetracycline in an alkaline medium limit or completely prevent the use of these valuable substances in many applications. .

   Due to the much better stability of the new antibiotics, it is transferable to prepare many pharmaceuticals which could not / could not be prepared satisfactorily with the tetracyclines. The increased stability also improves the recovery and refining processes, since these more stringent conditions of pH and temperature can be used, and thus the efficiency of various stages can be improved. process weight.



   Chemical analyzes of highly purified samples of chlorodemethyltetracycline agree with the calculated empirical formula C21H21C108 'the error being within the experimental range. The Kuhn-Roth method for the determination of methyl groups gives an extremely low value, indicating that there is no methyl group at position 6 of the naphthocene ring. Accordingly, the name chlorodemethyltetracycline appears appropriate. This is further confirmed by the comparison of the ultraviolet and infra-red absorption spectra of the products, with two of the different tees = tracylclines.



   The melting point of chlorodemethyltetracycline as free base, measured on a hot platform, is 170-175 C with decomposition. The melting point of chloro tetracylline is 168-169 C. Demethyltetracycline free base melts at 170-177 C, with decomposed

 <Desc / Clms Page number 15>

 tion, while tetracycline melts on the hot platform at 160-168 C.



   Here are other comparisons between chlorotetracycline and chlorodemethyltetracycline. Chlorodemethyltetracycline has a pHa value of 4.45 in a 50/50 mixture of dimethylformamids and water. Optical rotation of chlorodemethyltetracycline in hydrochloric acid
0.03N at a concentration of 0.5% is [o (725D = 261; for chlorotetracycline, -243 The demethyltetracyclines appear to be more soluble in water than the corresponding tetracyclines. For example, chlorodemethyltetracycline hydrochloride is soluble in water at a rate of approximately 45 mg / cm, compared with approximately 14 mg / cm for chlorotetracycline.



   The antibacterial activity of tetracyclines and 'demethyltetracyclines is generally similar, but with marked differences as will be seen from the following table.



   This table compared the concentrations of chlorotetracycline and chlorodemetyyltetracycline which are necessary to achieve half-maximum inhibition of growth, expressed in micrograms of antibiotic per cm3 of solution, over time periods varying from 4 to 48. hours with various microorganisms. Low numbers indicate more effective inhibition of growth.

 <Desc / Clms Page number 16>

 



  TABLE I. -
Antibiotic
 EMI16.1
 Microorganism chlorotetracycline ch10rodeTIethyltestracycline Times in hours Time in hours ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯4 8 4 1 $ - 8 24 48
 EMI16.2
 
<tb> Staphylococcus
<tb> aureus <SEP> ATCC <SEP> 6538 <SEP> P <SEP> 0.041 <SEP> 0.072 <SEP> 0.57 <SEP> 4.1 <SEP> 0.065 <SEP> 0.094 <SEP> 0.41 <SEP> 0.68
<tb>
<tb> Staphylococcus
<tb> albus <SEP> 7.5 <SEP> 20.5 <SEP>> 50.0 <SEP>> 50.0 <SEP> 16.5 <SEP> 50.0 <SEP>> 50.0 < SEP>> <SEP> 50.0
<tb>
 
 EMI16.3
 Escherichia coli 0.12 0.27 1.5 10.2 0.16 0.3 0.66 ±. 1.56 0.8 Salmonella galli- 0 0.74 465 37.0 0.1 0.56 1.06 1.1 narum Bacillus cereus (0.02;

  5 0.035 0.15 1.1 0.015 0.039 0.077 0.19 Pseudomouas
 EMI16.4
 
<tb> aeruginosa <SEP> 0.31 <SEP> 0.56 <SEP> 6.3 <SEP> 35.0 <SEP> 0.94 <SEP> 1.3 <SEP> 4.3 <SEP> 6 , 7
<tb> Streptococcus
<tb>
 
 EMI16.5
 pyrogenes beta 11.5 1r3.0> 50.0> 50.0 14, A 41.0> 50.0 50.0
 EMI16.6
 
<tb> hemolytic
<tb>
 
 EMI16.7
 Proteus vulgsris 0> 28 0.56 9.2 50.0 0.62 1.12 JY38 4.4
 EMI16.8
 
<tb> Escherichia <SEP> coli
<tb>
<tb> ATCC <SEP> 9637 <SEP> 0.54 <SEP> 1.18 <SEP> 6, <SEP> 6 <SEP> 34.0 <SEP> 0.43 <SEP> 0.67 <SEP> 0.97 <SEP> 1.8
<tb>
 As will be seen by examining the figures of the
 EMI16.9
 table above, chlorotetr: cyclizze, in general; is slightly more active within 4 hours.



  However, it will be noted that after a certain standing time of the solutions, bacterial growth resumes in the case of chlorotetracycline, but that it is still generally inhibited in solutions which contain chlorodeme thyltetracycline. This is indeed the better stability of the latter antibiotic. Similar results are observed when comparing tetracyline to demethyltetracycline and bromotetracycline to bromodemethyltetracycline.



  It has also been found that emethyltetracyclines are considered
 EMI16.10
 significantly superior to ltoxytetracycline in the same type

 <Desc / Clms Page number 17>

 
The demethyltetracyclines were also compared with the tetracyclines in in vivo tests, and it was found that there is no appreciable difference in the antibacterial activity of the demethyltetracyclines compared to their tetracyclines analogues. It is therefore concluded that these new antibiotics can be used for the treatment of the same diseases in the same general way as the tetracyclines currently used.



   The ultraviolet absorption spectra of tetracyclines and demethyltetracyclines are very similar.



  Figure 2 of the drawings shows the comparison between the ultraviolet absorption spectrum of chlortetracycline and chlorodemethyltetracycline at the same concentration.



  As will be appreciated, the shape of the curves is essentially the same, but there is, however, a slight fluctuation in the absorption spectrum of chlorodemethyltetracycline near 260 millimicrons.



   Another comparison of the ultraviolet absorption spectra of tetracyclines is seen in Table II, which shows the extinction coefficients E 1cm (spectrophotometric absorption of a 1% solution measured in a cell of 1 cm) measured at the maximum and minimum of the minimum of the different antibiotics. The values were calculated from the UV absorption curves, correcting to 1% the concentrations at which the tests were made.



  All samples were measured in 0.1N H2SO4 solution.

 <Desc / Clms Page number 18>

 



    TABLE II
 EMI18.1
 
<tb> Max. <SEP> Mih. <SEP> Max. <SEP> Min. <SEP> Max.
<tb>



  Antibiotic <SEP> e <SEP> 1% <SEP> milli- <SEP> E1% <SEP> milli- <SEP> E1% <SEP> milli- <SEP> E1% <SEP> milli- <SEP> E1% <SEP> milliE1cm <SEP> micron <SEP> E1cm <SEP> micron <SEP> E1cm <SEP> micron <SEP> E1cm <SEP> micron <SEP> e1cm <SEP> micron
<tb> Chlorotetracycline <SEP> hydrochloride <SEP> 209 <SEP> 368 <SEP> 135 <SEP> 302 <SEP> 356 <SEP> 265 <SEP> 288 <SEP> 242 <SEP> 343 <SEP > 228
<tb> Epichlorotetracycline <SEP> -NH4 <SEP> 170 <SEP> 368 <SEP> 100 <SEP> 302 <SEP> 332 <SEP> 254 <SEP> 290 <SEP> 242 <SEP> 342 <SEP> 228
<tb> <SEP> Tetracycline Hydrochloride <SEP> 305 <SEP> 355 <SEP> 182 <SEP> 299 <SEP> 393 <SEP> 268 <SEP> 191 <SEP> 232 <SEP> 289 <SEP > 217
<tb> Epitetracycline-NH4 <SEP> 290 <SEP> 355 <SEP> 140 <SEP> 299 <SEP> 326 <SEP> 254 <SEP> 200 <SEP> 232 <SEP> 275 <SEP> 216
<tb> Demethyltetracyline <SEP> neutral <SEP> 292 <SEP> 353 <SEP> 169 <SEP> 298

  <SEP> 395 <SEP> 268 <SEP> 202 <SEP> 232 <SEP> 299 <SEP> 217
<tb> Epidemethyltetracycline hydrochloride <SEP> 310 <SEP> 355 <SEP> 162 <SEP> 299 <SEP> 367 <SEP> 255 <SEP> 228 <SEP> 232 <SEP> 306 <SEP>, <SEP> 217
<tb> Chlorodemethyltetracycline <SEP> hydrochloride <SEP> 249 <SEP> 368 <SEP> 160 <SEP> 299 <SEP> 362 <SEP> 268 <SEP> 270 <SEP> 238 <SEP> 357 <SEP > 227
<tb> Epichlorodemethyltetracycline hydrochloride <SEP> <SEP> 242 <SEP> 368 <SEP> 130 <SEP> 300 <SEP> 348 <SEP> 254 <SEP> 289 <SEP> 238 <SEP> 346 <SEP> 227
<tb>
 

 <Desc / Clms Page number 19>

 
The infrared absorption spectra of several of the new products are shown in the other figures of the drawing.

   Figure 3 is a representation of the infra-red absorption spectrum of chlorodemethylteracycline hydrochloride. FIG. 4 represents the comparison of the infrared absorption spectra of free base tetracycline and free base demethyltetracycline. As we can see, there are several clear differences, and also similarities.



  Figure 5 shows the infrared absorption spectrum of epichlorodemethyltetracycline hydrochloride, and Figure 6 is the absorption spectrum of epidemethyltetracycline hydrochloride. These curves were obtained on a Perkin-Elmer model 21 automatic infrared recording spectrophotometer, with the solid phase antibiotics, compressed into a disc with KBr. Here are other data: prism - NaC1; resolution - 2; answer - 1 - 1; gain - 5; , speed 1/2 min / micron; deletion - 2; and scale: 50.8 min for one micron.



   Various methods have been developed to titrate tetracyclines, and some of them can be used to titrate demethyltetracyclines, of course with appropriate modifications. The fluorometric titration of the. Chlorotetracycline is based on an increase in fluorescence due to the formation of an isochlorotetracycline in an alkaline medium. The sample to be titrated is dissolved in an alkaline phosphate buffer, and the fluorescence of the solution is determined in a fluorophotometer at time zero (immediately after preparation of the solution), and again after standing to allow isomerization. to occur.



  The increase in fluorescence corresponds to the amount of chlorotetracycline present. but the presence of chlorodemethyltetracycline in the solution may interfere with the reaction of chlorotetracycline, tending to give the illusion

 <Desc / Clms Page number 20>

 with a lower content of chloroterracycline. A more reliable method for determining chlorotetracycline in mixtures with chlorodemethyltetracycline is based on the fact that the alkaline degradation product of chlorotetracycline does not absorb at 380 m millimicron. The sample is prepared at pH 10 with a carbonate buffer, and the absorption change is measured at 380 m millimicron after standing for 30 minutes (about three half-lives of chlorotetracycline).

   Chlorotetracycline and chlorodemethyltetracycline both absorb before alkali degradation, but only chlorodemethyltetracycline absorb after degradation treatment; and the decrease in absorption is the measure of the chlorotetracycline originally present in the sample.



   A specific titration of chlorodemethyltetracycline in the presence of other antibiotics of the tetracycline group is based on the Hiscox method. A moderate acid treatment for 15 minutes at 100 ° C. with 1N HCl destroys all the tetracyclines except chlorodemethyltetracycline. Then a portion of the sample thus treated is subjected to a vigorous acid treatment for 15 minutes at 100 ° C. with 6N HCl, in order to destroy the chlorodemethyltetracycline. The chlorodemethyltetracycline content is calculated by measuring the spectrophotometric absorption loss at 369 m millimicron and the increase at 430 m millimicron, which is achieved by the vigorous acid treatment, and comparing with a control.



   A mixture containing only two constituents, for example a mixture containing the two members of an epimeric couple,: can be titrated by spectrophotometry. We determine the spectrophotoinometric absorptions for two wavelengths, and we interpose linearly the ratio of these two absorptions between the known values in the consti-

 <Desc / Clms Page number 21>

 pure killers. It is thus possible to determine the percentage composition of the mixture.



   The easiest way to titrate bromodemethyltetracycline is to test for. bromine content by common methods of microanalysis.



   As indicated above, bromodemethyltetracycline can be obtained as the main fermentation antibiotic by controlling the halogen content of the fermentation medium. Generally, the aqueous nutrient medium should contain at least 50 parts per million bromide ions and the chloride ion content should be kept as low as possible, less than about 50 parts per million chloride ions. The bromide ion can be provided by any water soluble bromide, for example potassium bromide, which releases bromide ions to serve in the biological synthesis of the antibiotic.



   Apart from controlling the halogen ion content of the fermentation, the process for preparing bromode-methyltetracycline is similar to that for preparing demethyltetracycline and chlorodemethyltetracycline, described above. Strains of S. aureofaciens which produce demethyltetracycline can also be used to produce the brominated analog.



   Bromodemethyltetracycline has approximately the same antibacterial activity as chlorodemethyltetracycline, and it can be used for the same uses and in the same way.



   It is not necessary to separate broinodenethyltetracycline from other antibiotics which may concurrently form during fermentation because a mixture. of the various demethyltertracyclines is useful for many uses; for example, the fermented nutrient medium can be concentrated and used directly for its antibiotic content.

 <Desc / Clms Page number 22>

 ticks. Another way to use it is to acidify the fermented liquor, filter to remove mycelium and insolubles; to neutralize and concentrate to obtain a dry powder which can be mixed with animal feed. Mixed antibiotics are useful in stimulating the growth of many animals.



   A crude form of bromodemethyltetracycline, and mixtures of this with other antibiotics, can be used to preserve meats, poultry and fish in the same way that tetracyclines are currently used.



  In these compositions, little need be paid to the proportions of the various tetracyclines which they may contain.



   In the event that it is desired to separate the bromodemethyltetracycline from the other antibiotics contained in the fermented liquor, this can be done by several means which will be apparent to those skilled in the art. For example, treatment with acid destroys tetracycline, and treatment with alkali destroys chlorotetracycline and chlorotetracycline in the fermented liquor. Bromodemethyltetracycline can be recovered from the resulting solutions; through fractional chromatography using diatomaceous earth in the column. A mixture of chloroform and butanol of about pH 2.0 elutes antibiotics from the diatomaceous earth.

   The bromodemethyltetracycline exits the column ahead of the demethyltetracycline and can be recovered from the eluent. Bromodemethyltetracycline can also be separated into a single antibiotic component, using Craig's countercurrent delivery technique.



   The epidemethyltetracyclines of the present invention are considered to be isomers of demethyltetracy-

 <Desc / Clms Page number 23>

 clines. The structural difference appears to be based on a rearrangement of the dimethylamine group on the carbon atom in position 4. Apparently, under certain conditions which will be described in more detail below, the inversion occurs and a mixed mixture is obtained. the equilibrium of deinethyltetracycline and its epimer. The two can be separated to obtain practically pure products.

   For each of the bodies, demethyltetracycline, chlorodemethyltetracycline and bromodemethyltetracycline, there is a corresponding isomer which will be called epidemethyltetracycline, epichlorodemethyltetracycline, and epibromodemethyltetracycline. Demethyltetracyclines can be converted to their isomeric forms by simply adjusting the pH of a concentrated solution of the antibiotic to between 3.0 and 5.0 and allowing the solution to stand until the isomerization has reached equilibrium. The most important conditions that need to be set in order to convert the demethyltetracyclines to their isomers are concentration, pH, time and temperature.



   The most convenient is to carry out the isomerization at room temperature, but at higher temperatures a higher conversion rate is obtained. The pH should be between about 3.0 and 5.0, preferably between 3.5 and 4.5. Some epimerization occurs at pHs outside this range, and even in distilled water; but the speed is very low. The concentration of the antibiotic in the aqueous solution should be as high as possible if faster epimerization rates are to be obtained. Complete equilibration may take about 24 hours at 25 ° C., but satisfactory equilibration can be obtained in a much shorter time under specified conditions.

   Usually, however, best results are obtained by allowing the solution to stand for

 <Desc / Clms Page number 24>

 durations of a week or more. It seems that the equilibrium is reached, in most cases, at about 50%; that is, at equilibrium about half of the demethyltetracycline is converted to the epimer.



   Since concentration is an important factor in obtaining high yields in short periods of time, a solvent system should be chosen which gives the highest concentration of demethyltetracycline. These solvent systems must be buffered to obtain a pH within the range of dawi (whatever the preferred range. The various solvents include methanol, ethanol, butanol, acetone, 2-ethoxyethanol, 2-methoxypropanol, tetrahydrofuran, dimethylformamide and mixtures of these solvents. Still other solvents can be used. A preferred buffer is monosodium phosphate, but other buffers and pairs of buffers can be used which maintain the pH in the desired range. .



     The epimers of the demethyltetracyclines can be recovered from aqueous solutions, in the same general way as the tetracyclines are recovered. Although they have mild antibacterial activity in vitro and in vivo than the demethyltetracyclines, they are still very useful in the treatment of diseases caused by bacteria, due to their appreciable antibacterial activity.



   As might be expected, the demethyltetracyclines form salts and complexes of the same type, and in the same general way, as the tetracyclines. Treatment with acids at a pH below about 4 results in the formation of acid salts. The free base can be obtained at a pH of about 4 to 6; and at pH greater than 8, we obtain

 <Desc / Clms Page number 25>

 holds salts with bases, for example calcium salt.



  Likewise, epimers are formed from salts.



   As indicated above, the process for preparing the new antibiotics according to the present invention will generally be the same as the processes for producing chlorotetracycline, tetracycline and bromoteter cycline, the main differences residing in the process. made .
 EMI25.1
 that we choose a mutant strain of S. aureofaciens which produces the desired demethyltetracycline instead of one of the tetracyclines.

   The composition 'of the fermentation medium, the degree of aeration, the time, the temperature', the control of hydrogen ions, the mode of inoculation and the like, applied in the production of chlorotetracycline, bromotetracycline and tetracyclines are suitable for the production of the new demethyltetracyclines.



   In order that the fermentation process according to the invention may be more fully understood, it will now be described with reference to the specific examples which follow.



   EXAMPLE 1. -
A suitable medium for the preparation of inocula intended for fermentation can be prepared with the following substances:
 EMI25.2
 
<tb> Sucrose <SEP> 30 <SEP> g / 1
<tb>
 
 EMI25.3
 (NH) 50, -S'J 2 g / 1
 EMI25.4
 
<tb> CaC03 <SEP> 7 <SEP> g / 1
<tb>
 
Corn maceration liquor 16.5 cm3 / 1
The pH of the medium thus obtained is approximately 6.8.



  We measure an 8 cm3 portion which is introduced into a 20 cm Brewer tube, and sterilized at 120 ° C. for 20 minutes. The sterilized medium is then inoculated with 0.5 cm3 of an aqueous suspension of spores from a strain of S. aureofa.
 EMI25.5
 ciens capable of producing chiarodemethyltetracycline,

 <Desc / Clms Page number 26>

 for example strain S-604, the suspension containing about 40-60 million spores per cm3. The inoculated medium is incubated for 24 hours at 28 ° C., on a reciprocating shaker operating at 110 cycles per minute.



   A suitable fermentation medium contains water, and a typical medium which is suitable for producing chlorodemethyltetracycline is as follows:
 EMI26.1
 
<tb> starch <SEP> from <SEP> but <SEP> 55 <SEP> g / 1
<tb>
<tb> CaCO3 <SEP> 57 <SEP> g / 1
<tb>
<tb> (NH4) 2SO4 <SEP> 2g / 1
<tb>
 
 EMI26.2
 FeS04-7H20 l0 z: ng / 1 MnS04-4H20 50 rr / 1
 EMI26.3
 
<tb> ZnSO4.7H2O <SEP> 100 <SEP> mg / 1
<tb>
 
 EMI26.4
 CoCl 2 ¯6H 2 0 5 mg / 1
 EMI26.5
 
<tb> liqueur <SEP> of <SEP> maceration <SEP> of <SEP> but <SEP> 30 <SEP> g / 1
<tb>
<tb> flour <SEP> of <SEP> seed <SEP> of <SEP> cotton <SEP> 2 <SEP> g / 1
<tb>
<tb> Oil <SEP> from <SEP> lard <SEP> 2.0% <SEP> by <SEP> volume
<tb>
 
When the chloride ion content of this medium is restricted, it tends to increase the amount of demethyltetracycline at the expense of the production of chlorodemethyltetracycline.



   EAMPLE 2. -
Do we carry out fermentation in shaker flasks? which results in the formation of a certain quantity of demethyltetracycline and chlorodemethyltetracycline, with a medium prepared as follows:
 EMI26.6
 
<tb> liqueur <SEP> of <SEP> maceration <SEP> of <SEP> but <SEP> 30 <SEP> g / 1
<tb>
<tb> <SEP> lard oil <SEP> <SEP> 2% <SEP> by <SEP> volume
<tb>
<tb> starch <SEP> from <SEP> but <SEP> 55 <SEP> g / 1
<tb>
<tb> flour <SEP> of <SEP> seed <SEP> of <SEP> cotton <SEP> 2 <SEP> g / 1
<tb>
<tb> CaC03 <SEP> 7 <SEP> g / 1
<tb>
<tb> (NH4) 2SO4 <SEP> 5 <SEP> g / 1
<tb>
 

 <Desc / Clms Page number 27>

 
 EMI27.1
 
<tb> NH4C1 <SEP> 1,5g / 1
<tb>
 
 EMI27.2
 ZnS04..7H20 100 n / 1 MhSO, .4HO 50 m /.

   
 EMI27.3
 
<tb> CoC12.6H2O <SEP> 5 <SEP> mg / 1
<tb>
 We measure a portion of 25 cm3 that we introduce
 EMI27.4
 in a 250 cc Erleiimeyer flask, sterilize during. 20 minutes at 120 C and inoculated with 1 cm3 of mycelion culture prepared as in Example 1. After incubation for 120 hours at 26 C on a ratative shaker at 186 t / mm, the liquor is titrated and it is found that '' it does not contain more than approximately 70 micrograms of chlorotetracycline per cm3,
 EMI27.5
 about 450 micrograms of demethyltetracycline per cm3, and about 900 micrograms of chlorodemethyltestracycline per cm3.



   EXAMPLE 3. -
Fermentation in vats.
A fermentation of 40 liters is carried out in. a pilot tank, essentially according to the method described in example 2. After having prepared the medium, it is sterilized for 25 minutes at 125 C and inoculated with an inoculum of strain S-604 prepared as indicated by Duggar, U.S. Patent No. 2,482,055 of September 13, 1949.



   The fermentation is carried out with continuous agitation at a temperature of 28 ° C. for the first 24 hours and 25 ° C., until complete, in 135 hours. Sterile air is introduced at a rate of 0.3 1 / mm during the first sixteen hours, then at a rate of 0.5 1/1 / mm until the. harvest.



   The following examples illustrate the recovery, refining and separation of antibiotics.

 <Desc / Clms Page number 28>

 



   EXAMPLE A. Refining of chlorodemethyltetracycline and of demethyltetracycline.
25.8 liters of liquor are prepared as in Example 3, treated with 200 cc of concentrated hydrochloric acid to adjust the pH to 1.5. The treated liquor is filtered, after having mixed it with 2.8 kg of a filter aid, to obtain 16.3 liters of filtrate. The filter cake is taken up in 25 liters of water at a temperature of about 50 ° C. and adjusted to pH 1.5. After stirring for 30 minutes, the diluted cake is filtered and the filtrate is combined with the previous filtrate, to form a mass of 42.3 liters.



  The combined filtrate is treated with 6.76 kg of sodium chloride and extracted four times with butanol at 15% of the volume of the filtrate. The extracts are combined, clarified by filtration and concentrated in vacuo at 25-30 ° C to a final volume of 0 liters.



   EXAMPLE B. -
 EMI28.1
 Chromatographic separation of chlorodemethyltetracycline and demethyltetracycline.
The butanol concentrate of Example A - is divided into two equal portions, one is concentrated to 900 cm3 and the other is concentrated to 610 cm3 under vacuum. The concentrates are filtered off and adjusted to pH 2 with 50% sodium hydroxide.



   To prepare the solvent system used for the chromatographic separation, a mixture of 80% butanol and 20% chloroform is equilibrated with water adjusted to pH 2 by means of hydrochloric acid. The columns are 15 cm diameter glass columns packed with 2.8 kg of acid washed diatomaceous earth. The columns are equilibrated with the aqueous phase of the solvent system before use.

 <Desc / Clms Page number 29>

 



   The two concentrates are treated with butanol, on. separate columns. The concentrates are slowly poured into the top of the columns. The columns are then eluted with the solvent phase which is passed through at a rate of about 10 liters per hour. In each case, 40 slices of 500 cm3 are taken and the antibiotic content of each is determined by chromatography on a strip of paper. In the 900cc portion, slices 6 through 17 contain chlorodemethyltetracycline and chlorotetracycline, and slices 24 through 40 contain demethyltetracycline and tetracycline.



  In the 610 cc pot, slices 7 to 18 contain chlorodemethyltetracycline and chlorotetracycline, and slices 19 to 31 contain demethyltetracycline and 1a. tetracycline.



   EXAMPLE G. - Recovery of chlorodemethyltertrcycline from column fractions.
The fractions containing chlorodeme-thyltetracycline from Example B are combined to give a volume of about 12 liters. An equal amount of water is added, and the mixture is concentrated in vacuo to 1810 cm3 of aqueous solution. The pH of the concentrate is adjusted to 2.3-1.9 by adding hydrochloric acid. The acidified concentrate is washed twice with 180 cm3 of chloroform, filtered and concentrated in vacuo to a volume of 360 cm3. The pH of the concentrate is adjusted to between 1.2 and 2.8 with 50% sodium hydroxide.

   The solution was dried by freezing to give 18.56 g of amorphous chlorodemethyltetracycline hydrochloride titrating 550 micrograms per mg.



   EXAMPLE D. Recovery of demethyltetracycline from column fractions.
The slices of example B are combined which contain

 <Desc / Clms Page number 30>

   holds demethyltetracycline to obtain a volume of 14 liters. An equal volume of water is added and the mixture is concentrated in vacuo at 24 ° C. to a volume of 565 cc of aqueous solution. The pH of the concentrate is adjusted to 1.65 by adding concentrated hydrochloric acid, and the solution is washed twice with 55 cm 3 of chloroform. The washed solution is filtered and adjusted to pH 5.8 with 505 sodium hydroxide solution.



  After aging for 3 hours at room temperature and 15 hours at 4 ° C., the crystalline slurry obtained is filtered and the product is washed with water and dried under vacuum at 40 ° C.



  A yield of 4.7 g of crude neutral demethyltetracycline is obtained, assaying 880 micrograms per mg expressed as hydrochloride. tetracycline drate which represents an overall yield of 73% from the tart concentrate.



   EXAMPLE E. Crystallization of chlorodemethyltetracycline hydrochloride with an acetone-ether mixture.
One takes the, 56 g of freeze-dried chlorodemethyltetracycline as in Example C, stirred in a mixture of 55 cn3 of acetone, 7.4 cm3 of water and 3.7 cm3 of concentrated hydrochloric acid. The solution was filtered and the solids rinsed twice with a total of 55 cm3 of acetone contained 1.85 cm3 of water. The filtrates are combined, the pH being 0.8, and treated with 55 cm3 of ether. The mixture is seeded, left to age for 18 hours at room temperature with constant agitation, and the crystals which form are removed by filtration.

   The product is washed with a mixture of acetone, water and ether, then with acetone and finally with ether, and dried under vacuum at 40 C. A yield of 4 is obtained. , 74 g of hydrochloride

 <Desc / Clms Page number 31>

 
 EMI31.1
 chlorodemetlylt ± tracycli: the lens titrating 1090 micrograms per mag, which -represents HIM lende; uelU 'd! op (: ratloll of 50.0;,;. The product contains 11 de chlor8t'3tracrcliile.



  (... "1C '.. J..-JJF, ¯ ¯ L, ristallisatii; du clil, riiv -dr de c] ilorodeinéthjrl.t4tracyclî, ie,' from a talazye butatzol-eel7¯orolve.- A 50.4 g portion of freeze-dried chlorodemethyltetracycline is taken, prepared as in
 EMI31.2
 Example G, and assaying 7fla3 micrograms per tztJ, oz1 dilutes it in 22.5 cm3 of cellosolve, and to the resulting solution is added a total of 205 cm3 of butanol. The pH is adjusted to 0.8 by adding 11 cm3 of concentrated hydrochloric acid and the mixture is allowed to age for 51 hours with stirring. The crystals obtained are removed by filtration, washed with a 90/10 mixture of butanol and cellosolve, then with chloroform and dried in vacuo.

   We obtain a yield of
 EMI31.3
 3036 g of chlorodemethyltetracycline hydrochloride, assaying 1053. micrograms by a yield of 4; j *.



  EXTMPLE G. - 4ecrystallization of cIl0r¯od6methyltetracyclil! .E so as to destroy chloro6etracirclin ;. -
1g of the chlorodemethyltetracycline prepared in Example E is taken, diluted in 3 cm3 of a mixture of 90% butanol and 10% cellosolve, and 3 cm3 of triethylamine is added thereto to adjust the pH to 10.4. The incomplete solution is allowed to stand at room temperature for 24 hours to destroy any chlorotetracycline which may be present, and at the end of this time, concentrated hydrochloric acid is added to lower the pH to 0.5, and the pH is reduced to 0.5. leash
 EMI31.4
 age the m'lau; for 4 hours with agitation.

   Either filter the crystalline product, or wash it with a mixture of 90% butanol and 10% cellosolve followed by chloroform and

 <Desc / Clms Page number 32>

 che under vacuum at 40 C. A yield of 0.36 g of.
 EMI32.1
 c ". chlorodemethyltetracycline trihydrate, assaying 1228 micrograms per mg and containing approximately 2.5% of chlorotetracycline, which represents a yield of 40%
The following examples illustrate the conversion of antibiotics into various forms:
EXAMPLE H. - Chlorodemethyltetracycline broitihydrate.
1.0 g of chlorodemethyltetracycline-ammonium is dissolved in 20 cm3 of water and the solution is treated with 0.6 cm3 of 48% hydrobromic acid to adjust the pH to 1.6.

   The mixture was allowed to age for 18 hours with stirring at room temperature and at the end of this time elongated needle crystals of chlorodemethyltetracycline hydrobromide were found. The product is filtered, washed with 6 cm3 of water adjusted to pH 2.2 with hydrobromic acid, and dried under vacuum for 5 hours at 44 ° C. A yield of 750 mg of hydrobromide is obtained. of chlorodemethyltetracycline.
 EMI32.2
 



  Analysis calculated for ClI12ZNC1Br0 $: 0 = 46.2; H = 1,, 1; N = 5.1; Ci = 6.5; Br = 14.6; 0 = 23.4.



   Effective Analysis: 0 = 45.94; H = 4.44; N = 4.71; C1 = 6.78, 6.47; Br = 13.74; 0 (difference: 24.39; 24.70).



   EXAMPLE I. Preparation of the ammonium salt of chlorodemethyltetracycline.
A 100 mg portion of the chlorodemethyltetracycline prepared in Example E is taken, dissolved in 0.5 cm3 of a mixture of 90; 3 of butanol and 10% cellosolve and ammonia gas is bubbled through the solution. until a thick solid appears. The slurry is diluted with a small amount of concentrated aqueous ammonia,
 EMI32.3
 leash; re'fiY0 \ -ge #! ' at room temperature for 5 hours and filtered. The product is washed with a 90% mixture of

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 butanol and 10% cellosolve, then with acetone, and dried under vacuum.

   A yield of 41.4 mg of chlorodemethyltetracycline ammonium salt is obtained, assaying 1148 micrograms per mg of chlorotetracycline hydrochloride (44% yield).



   EXAMPLE J. -
Preparation of neutral chlorodemethyltetracycline. ':::
A 50 mg portion of the chlorodemethy1tetracycine obtained in Example E, titrating 1090 micrograms per mg, is taken, it is dissolved in 1.3 cm3 of dieàù and the solution is treated with dry sodium carbonate until pH 7-8 The amorphous precipitate obtained is removed by centrifugation and the clear settling liquid is adjusted to pH 5.4 and allowed to age 16 hours at ambient temperature.

   The product is filtered, washed with water and dried under vacuum at 35 ° C. to obtain 21.9 mg of chlorodemethl. crystalline neutral tetracycline assaying 1250 micrograms per mg (yield 50%)
EXAMPLE K. Reduction of Chlorodemethyltetracycline to Demethyltetracycline. -
A 12.5 mg sample of crystalline chlorodemethyltetracycline hydrochloride is dissolved in water and a drop of triethylamine is added thereto.

   The solution is mixed with 10 mg of 10% palladium on charcoal, and stirred under a hydrogen atmosphere for 20 minutes. The resulting solution is filtered to remove the catalyst. Ultraviolet spectra as well as paper chromatography indicate that dechlorination occurs to give demethyltetracycline.



   As indicated above, salts of demethyltetracyclines can be formed by treatment with

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 acids and bases. Hydrochloric acid is a preferred acid because the salts of this acid and of the demethyltetracyclines are very useful products. Special properties can be imparted by using other acids, such as sulfuric acid, phosphoric acid, acetic acid, boric acid, and other organic and inorganic acids. Salts can be prepared with bases, using alkali hydroxides such as sodium hydroxide and alkaline earth hydroxides, as indicated above.

   The calcium salt is particularly useful, and can be prepared by simply adjusting to pH 6-10 an aqueous solution of one of the demethyltetracyclines, with at least one molar equivalent of calcium in the solution. The salts of barium, magnesium, strontium [, etc ... can be prepared in a similar manner. Complexes of demethyltetracyclines are also formed with boron, zirconium and other polyvalent metal ions, as in the case of tetracyclines.



   Obviously, if the fermentation medium does not contain chlorine and bromine, neither chlorodemethyltetracycline nor bromodemethyltetracycline can be formed. In such a case, the main antibiotic will be demethyltetracyclin. When the content of chloride ions is raised, particularly above 50 parts per million, significant amounts of chlorodeinethyltetracycline are formed, since 1 part of chloride ion can cause the formation of about 14 parts of chlorodemethyltetracycline. The amount of the latter antibiotic can be adjusted.

   When preparing this antibiotic, it is preferable that the fermenta-. tion contains more than 50 parts per million of chloride ion, and this quantity can be increased up to 1000 parts per million or even more to have a maximum production

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 of chlorodemethyltetracycline.



   A small concentration of bromide ion in the fermentation medium, from about 10 parts per million to several%, tends to decrease the formation of chlorodemethyltetracycline, and the formation of demethyltetracycline is generally increased. When the fermentation medium contains less than about 50 parts per million of chloride ions, and more than about 50 parts per million of bromide ions, the
 EMI35.1
 formation of bromoderaethyltetracycline is favored; and with strains of S. aureofaciens which are good antibiotic processors, the obtained fermented liquor may contain bromotetracycline as the predominant antibiotic.



   CLAIMS.-
1.- Process for the preparation of antibiotics of the demethyltetracycline group (comprising demethyltetra-
 EMI35.2
 cycline, chlorodemethyltetra'elyelin and "Efohiodemethylteracycline), and their epimers, in which an aqueous nutrient medium is fermented under aerobic conditions with a controlled content of chloride and / or bromide ions. , with a strain of s.aureofaciens producing demethylteracycline, the antibiotics are recovered if desired from the fermentation medium and, if desired, the pH of a concentrated solution is adjusted to obtain the epimers.


    

Claims (1)

2. A process according to claim 1, wherein a medium substantially free of chloride ions is used and demethyltetracycline is obtained predominantly. 3. - Process according to claim 1, in which a medium containing at least 50 parts per million ion :: chloride is used and the predominantly obtained. EMI35.3 chloroddidbliylt6tracycliiie. 4. A process according to claim 1, wherein a medium containing at least 50 parts per thousand is used. <Desc / Clms Page number 36> bromide ions and less than 50 parts per million chloride ions, and the predominantly bromodemetethyltetracycline is obtained. 5. A process according to any of claims 1 to 4, in which the medium contains, or has been added thereto, ions of alkaline earth metals, and after fermentation, the mixture is adjusted. pH of the medium between 6 and 10, so as to precipitate the alkaline earth salts of the antibiotics. 6. - Process for the preparation of antibiotics of the demethyltetracycline group (comprising demethyltetracycline, chlorodemethyltetracycline and bromodemethyltetracycline), and their epimers, in substance, as described above, in particular in Examples 1 to 3. 7. - Antibiotics from the demethyltetracycline group (comprising demethyltetracycline, chlorodemethyltetracycline and bromodemethyltetracycline) and their epimers, when they are obtained by the process according to one or the other of the preceding claims. 8. A process for preparing demethyltetracycline which consists in dehalogenating chlorodemethyltetracycline or bromodemethyltetracycline by catalytic hydrogenation - preferably with a palladium catalyst on charcoal. 9. Demethyltetracycline prepared by the process of claim 8. 10.- As new substances for therapeutic use, antibiotics of the demethyltetracycline group (including demethyltetracycline, chlorodemethyltetracycline and bromodemethyltetracycline) and their epimers.