DE1041213B - Process for the production of antibiotics of the desmethyltetracycline series - Google Patents

Description

Process for the production of antibiotics of the desmethyltetracycline series The invention relates to a method for producing therapeutically active substances, which are known as desmethyltetracycline antibiotics, and desmethyltetracycline, Chlordesmethyltetracycline and Bromdesmethyltetracycline include, as well as epimers of these Links..

The new antibiotics are produced in a fermentation process, in which mutant strains of Streptomyces aureofa, ciens are used. the new antibiotics seem; already known members of the tetracycline series being very close, however, differ significantly from these. they draw excellent stability under both alkaline and acidic Conditions and are the tetracyclines. superior in this regard. They have similar antibiotic effectiveness and can apply for the same. Purposes and, in the can be used in the same way as the already known, tetracyclines. .

The invention consists in a process for the production of De @ smethyltetracyc: linantibiotika, to those desmethyltetra, cyclin, chlorodesmethyltetracycline and bromodesmethyltetracycline belong, as well as epimers of the same, with an aqueous nutrient medium with a controlled Chlorine and / or bromine ion content by means of a desmethyltetracycline. generating Strain of S. aureofaciens a.erob is fermented and the formed Desmethyltetracyclina, ntib, iotika optionally obtained from the fermentation medium, and optionally the p11 value a concentrated solution thereof is adjusted to form their epimers.

Any of the previously known, by fermentation using S. aureofaciens Tetracycline antibiotics produced under special conditions, namely Tetra.cyclin, Chlortetracycline and bromotetracycline, has a counterpart in the new range of Antibiotics. In the method according to the invention is namely using certain Mutant strains of S. aureofaciens, which are described in detail below, produces an antibiotic substance that corresponds to the tetracycline, another, which corresponds to the chlortetracycline, and finally another one that corresponds to the bromotetracycline is analog. Which new antibiotic each goes through fermentation depends on the nature of the culture medium and other process conditions. In one chloride-free medium becomes the predominant, antibiotic the chlorine-free analogue of the tetracycline. The production of nutrient medium with reduced halogen ion content is described in patent 959,584. In a bromine-rich medium, the bromine-containing Antibiotic is formed, and in a medium rich in chlorine ions it becomes the predominant one Antibiotic can be a chlorine-containing antibiotic that is analogous to chlortetracycline is. Usually some tetracycline is also added during the fermentation process, Chlortetracycline and / or Bromtetracycline formed, the proportions of these substances on the chlorine and / or bromine ion content of the medium and the one used Depend on the strain of the microorganism. Through careful selection of the tribe of S. aureofaciens it is possible for the formation of this well-known Te.tracyclinantib, iotika almost completely switched off.

The new antibiotics, as described below, can be used in are converted into their epimers in a similar way as the tetracyclines are converted into the Quatrimycins.

The antibiotics containing chlorine and bromine according to the present invention can also by hydrogenation using palladium on activated carbon as Catalyst can be dehalogenated in the same way as chlorotetracycline or bromotetracycline to tetracycline itself.

Chemical analysis and a consideration of the chemical and physical Properties of the new connections show that they differ from the corresponding tetracyclines, inasmuch as they are a methyl group exhibit less. The methyl group in question is most likely that which is in the 6-position on the naphthacene ring of the tetracycline. Accordingly the Tetracyclina, naloge of the present invention as 4-Dimethyla, mino-1,4,4a, 5,5a, 6,11,12a-octahydro-3,6,10,12,12a-pentaoxy-1,11-dioxo-naphthacene (2) -carboxamide are designated. In the case of chlorine and. Bromine analogs are likely to be the halogen substituent stand in 7-position and. the connection must be designated accordingly. As suitable Trivial names come for these new antibiotics desmethyltetracycline, chlordesmethyltetracycline and bromodesmethyltetra.cycline are considered, and this notation is used in the following applied, however, it should be noted that the derived structure is not has been proven by a clear synthesis and will therefore prove to be erroneous in the future can prove.

The new invention. Antibiotics are from certain mutant strains formed by S. aureofaciens. These strains undoubtedly belong to the species streptomyces aureofaciens because they were obtained from the original strain A-377, the by Dr. Duggar isolated and at the, North.ern Regional Research Laboratories in Peoria, Illinois, where it was assigned registration number NRRL 2209. Mutagenic treatment of A-377 with mutagenic agents, as repeated Ultraviolet radiation, nicotine, and nitrogen mustard, were used to develop one applied to the tribes; several other strains were created by spontaneous mutation. and obtained by treatment with mutagenic agents. All of these tribes. the the new antibiotics in different proportions. and different effectiveness produce have the general characteristics of the species S. auroofaciens. Both among themselves and from already described. Tribes of. S. aureofaciens they are primarily related to pigmentation and their ability to create new ones Producing antibiotics differently. In most cases it will be a reddish brown Pigment found when the microorganisms were grown on common culture media will. The pigment shades range from peach leather tones to copper brown to deep brown mahogany or burgundy, depending on the particular strain and nutrient medium, which are used. The property of the new antibiotics according to the invention It is true that it is generally associated with strains that produce this type of pigmentation show., yet represent this pigment production and the production of the new antibiotics not necessarily a common characteristic. All examined; Tribes produce some chlorotetracycline and usually some tetracycline, but they can Proportions of these antibiotics depending on the nature of the fermentation medium, the fermentation conditions and the selected strain vary considerably. In some cases. the formation of chlortetracycline and tetracycline is very low, on the order of a few percent of the total in fermentation educated antibiotic.

The mutant strains forming the new antibiotics of the invention have the same general characteristics as the tetracycline forming strains and differ from one another in the same general manner as the tetracycline strains do from one another, as shown in a number of scientific publications that have already appeared., was reported. Albeit many tribes of. S. aureofaciens open to the public through the Culture Callections. and many have been described in patents and the scientific literature, the following data should be presented to illustrate the nature of the deviation of the new strains from the original A-377 strain available as NRRL 2209. Comparison. the S. aureo_faai.ens strains S-604 and A-377 on various media. The Streptomyces aureofaziens strain S-604, which produces chlorodesmethyltetracycline and desmethyltetracycline, was with Streptomyces aureofaciens strain A-377 (NRRL 2209) Based on the observation of the growth characteristics, compared on different media which were incubated at 26 to 27 ° C.

The observations were as follows: 1. Glycerine-asparagine-meat extract agar Glycerin ....................... 1.0 0/0 L-asparagine .................... 0.050 / 0 Meat extract .................. 0.2 0/0 K H2 P O4. . . . . . . . . . . . . . . . . . . . . . 0.05010 Altar .......................... 1.5% Distilled water qs ....... 100.0 0lo pA + KOH .. .. .. .. .. .. .. .. .. .. ... 7.0 pli after sterilization. . .. .. ... 7.1 + Setting with 50o / oiger. Streptomyces aureofaciens Strain S-604 strain A-377 Growth Abundant, Venetian red + _ (venetian.-red): Moderately ` Aerial Hyphae Abundant, White to Rose Gray + (white to rose gray) white, uniform Sporulation Weak, becomes abundant None Spreadable pigment reddish brown yellow Brown Mahogany underside (brown-mahogany-colored) yellow to light orange-yellow + Color Harmony Manual, 3rd Edition, Container Corporation of America. 2. Dextrin Czapek Dox Agar Dextrin. .............. ......... 1.0 0/0 NaN03 ....................... 0.2 0/0 K2 HP 04. . . . . . . . . . . . . . . . . . . . . . 0.1 0/0 MgS04 # 7H20. .. .. .. ... ...... 0.05% K C1 ........................... 0.05% Fe S 04 - 7H20 . . . . . . . . . . . . . . . . 0.0010 / 0 Agar .......................... 1.5 0/0 Distilled water qs. . . . . . . . 100.0 0 / a pH after sterilization. .. .. ... 7.0 Streptomyces aureofaciens Strain S-60.4 strain A-377 Growth sparse, transparent, abundant Aerial hyphae None Abundant, Lead Gray +, water-clear surface, globules Sporulation None Abundant Spreadable pigment None Weak: pale yellow Underside pigmentless pigmentless + Color Harmony Manual, 3rd Edition, Container Corporation of America. 3. Corn steep agar Corn steep water ................. 0,4 ° / o Sucrose ...................... 1.0% Mg SO - 7 H2 O. . . . . . . . . . . . . . . . . 0.0250 / 9 K H2 P 04. . . . . . . . . . . . . . . . . . . . . . . 0.20 / 0 (N H4) 2 HP 04 .... . . . . . . . . . . . . . . 0.2% Agar ........................... 2.0% Tap water qs ............. 100.0% pH after sterilization ... . . . . . . 6.3 Streptomyces aureofaciens Strain S-604 1 strain A-377 Growth over-rich over-rich Aerial hyphae Reichlich, Dk. Rose Taupe + (dark pink) Abundant, Beaver + (beaver) Sporulation Very abundant, uniform. Very abundant, uniform Dispersible pigment Very concentrated; Deep Brown + to Deep brown Mahogany + (deep brown to deep brown mahogany) light greenish yellow Underside Dk. Brown Mahogany + (dark brown- mahogany colored) Covert Brown + (covered brown) Color Harmony Manual, 3rd Edition, Container Corporation of America. 4. Other media i Streptomyces aureofaciens Strain S-604 @ Strain A-377 Nutrient Agar Poor growth. Taupe brown + Moderate growth, no aerial hyphae. to darkbrown + (top brown to dark- underside: pale yellow, yellow to light- Brown). No aerial hyphae, sub-brownish-yellow soluble pigment side: taupebrown +, (top brown) red- light brown soluble pigment Glucose-Asparagine- Abundant growth. Vigorous, airy growth, white aerial hyphae, Meat extract agar hyphae spotted: rose taupe + to the point of increasing spore formation Fawn + to camel + (rose-potted increasingly gray. to deer-colored to camel-colored) Spo underside: light yellow, light yellow soluble rulation: plenty. Underside: taupe pigment brown + (top brown), reddish brown soluble pigment continuation Streptomyces aureofaciens Strain S-604 j strain A-377 Potato Schnitzel Over-rich moist soft modu- Over-rich moist soft modu- lated growth: darkbrown grained growth: light melon yel- gany + (dark brown mahogany colored) low + (light melon yellow) to antique with a trace of peach-tan + (peach-rose + (old rose), aerial hyphae: trace, buff), aerial hyphae: no to no soluble pigment plentiful, white to camel + (camel colors) becoming. Copious sporu- lation in areas of strong air hyphae formation. Chocolate to choco- latebrown + (chocolate-colored to chocolate brown) soluble pigment Purple milk Slight deeped red mahogany + (weakly weak white to pale yellow wax (purple milk) dark deep red mahogany colored) tumskranz. Minor p $ change Growth wreath. Slight pH change, no noticeable peptic Change, no obvious nization in 15 days Peptonization. Weak false al- Potassium color reaction due to a Diffusion of the soluble pigment * Color Harmony Manual, 3rd Edition, Container Corporation of America. On all agar with the exception of dextrin-Czapek-Dox agar, but also when cultivated with potato pulp and potato slant, S. aureofaciens strain S-604 forms a characteristic deep pigmentation that usually occurs with growth. The characteristic soluble reddish-brown pigment is also easy to observe. 5. Microscopic observations Streptomyces aureofaciens Medium strain S-604 strain A-377 Mycelium spores mycelium 1 spores Glvcerin .Asparagin- Curved, conti- [Sphaeroid to a- Curved, conti- Sphaeroid to a- Meat extract agar natural, shaped, clear, shaped, clear branches, diameter 1.5 to branches, diameter 1.5 to knife 0.8 to 2.0 h knife 0.7 to 2.0 R > 1.0u. > 1.0 @. Maiquellwasser Agar Curved, conti- Sphaeroid to a- Curved, conti- Sphaeroid to a- nuierlich, ver,. shaped, clear, deformed, straight branches, diameter 1.5 to branches, diameter 1.5 to knife 0.8 to 2.0 u, knife 0.8 to 2.0 [. > 1.0 N, > 1.0 R The mycelial and spore morphology for strain S-604 is apparently similar to that observed for strain A-377. Both trunks show a continuous, curved, branched mycelium with an occasional tendency of the aerial hyphae to form spirals. Characteristic spores have an egg-shaped to spliaeroid shape.

To explain the color changes. in the case of the various strains of S. aureofa.ciens which produce the novel desmethyltetracyclines according to the invention, four selected strains were grown on corn steeple agar and. The following observations made: Color observations +: Streptomyces aureo @ fa, ciens corn steep water Aga.r (A P4) 4-day incubation at 27 ° C Strain I single colonies 1 mass growth S-604 lIL Deep red mahogany Deep red mahogany (deep red maha- (deep red maha- goni colored) goni colored) continuation Stem single colonies mass growth S-1071 Copper brown Deep brown maho- (copper brown) gany to red ma hogany (deep- brown mahogany colors to red mahogany colored) V-62 Further outskirts Light copper brown peachtan (peach- (light copper- leather-colored), brown) is used at colo- center point light copper brown (light- copper brown) B-740 Dark Wine (dark Burgundy wine-colored) (burgundy) + Color Harmony Manual, 3rd edition, Container Corpora- tion of America. A relatively straightforward procedure for selecting a strain of S. aureofaciens which produces the novel antibiotics of the present invention is to select one which exhibits dark maroon colonies when grown on corn coarse agar medium. Vaccines are prepared from these colonies and fermentation is carried out such as that described in Examples 1 and 2 given below. At the end of fermentation, a certain amount of the fermentation mash is added to one. Acidified to pH about 1.5 to solubilize the antibiotic and filtered to give a clear aqueous solution. One or two drops of the filtrate are placed on a strip of paper and chromatographed according to the usual chromatographic development methods. Butano1 equilibrated with 0.3 molar aqueous phosphate buffer of px 3 is used as the solvent. The paper strips are developed in descending chromatography with the solvent phase of the equilibrated mixture. The location of the stains corresponding to the antibiotics of the present invention can easily be determined by examination under ultraviolet light. Another and even more sensitive method is to apply the strips to an agar plate containing microorganisms sensitive to the antibiotic, e.g. B. Bacillus cereus or Bacillus subtilis, is inoculated, and these plates are to be incubated. In the areas corresponding to the antibiotic stains. zones of inhibition emerge. The comparison of the stains with comparison samples of the pure antibiotics and the Rf values given below provide. a basis for the detection of the new antibiotics in: the fermentation mash = and a semi-quantitative basis for determining the existing ones. Amounts.

An explanation of this mode of operation will be given with reference to FIG. 1 of the drawing. which shows how the various tetracyclines and the. Products of the method according to the invention can be separated from one another and determined. The drawing shows a paper strip 1 to which a small amount of an antibiotic solution containing tetracycline, chlortetracycline, desmethyltetracycline and chlorordesmethyltetracycline was applied at point 2. The total antibiotic activity of the solution applied at point 2 was 30 ", expressed as tetracycline. The paper strips were then developed as described above. After a period of time, the strips were examined by means of ultraviolet light and the results shown in the drawing were observed The solvent front had reached the position shown by the solid line 3. The tetracycline component of the antibiotic mixture solution had reached point 5 and the chlortetracycline had migrated to point 7. The new antibiotic De.smethyltetracycline had migrated to point 4, and the antibiotic Ch .lordesmethy ltetracy clin was found at point 6. The Rf values of these antibiotics are also shown in the drawing. The Rf value is defined as the quotient of the distance of the stain from the starting point 2 and the distance of the solvent front 3 from the starting point 2 and is of course always smaller as 1. Various solvent systems can be used to obtain a variety of Rf values for a particular antibiotic, and these values are believed to be of great value in identifying antibiotics and distinguishing them from other antibiotics. -The RF values of a number of tetracyclines, ouatrimycins and the new products according to the invention in two specific solvent systems are given in the following table: P ethyl acetate 4.7 90. 10-chloroform Butanol / aqueous 0.3 m Antibiotic citrate-phosphate buffer Ha P 04 -f- 0.1 ° / a Tri- (Mc E 1 vain) chloroacetic acid p. 1.9 _ Chlorte tracyclin Q, 76 to 0.80 _ - 0.61 , _omtetracycline "............. 0.76 to 0.80. 0.61 - - -Tetracycline -. .-. ::. . . :. . . :. . . -. 0.46 to 0.47 0.20 Ouatrimycin. . . . . . . . . . . . . ...:. . 0.1 = 1 to 0.16 0.12 Chlorquatrimycin. . . . . . . . . . . . . . . 0.27 to 0.28 .. 0.33 Bromquatriinycin. . . . . . . . . . . . . . 0.27 to 0.28 0.33 Oxyquatrimycin. . . . . . . . ... . . . . . 0.00 to 0.06 0.11 Chlordesmethyltetracycline ....... 0.68 to 0.72 0.39 Desmethyltetracycline ........... 0.27 0.22 Epichlorodesmethyltetracycline .... 0.25 to 0.27 0.20 Epidesmethyltetracycline ........ 0.09 0.10 Several strains of S. aureofaciens that make up the new antibiotics have been deposited with the American Type Culture Collection, Washington, DC. They are registered with the Collection as ATCC 12551, 12552, 12 553, and 12 554. It goes without saying that mutants, which also produce the new antibiotics, can be derived from them by conventional methods. Tribes can be obtained. Indeed, it can be expected that some of these will, be able =, higher yields of one or the other of the new. Form antibiotics under favorable conditions. These mutants may show variations in general morphological characteristics to a certain extent, as is the case with the various strains of the species S. aureofaciens. It is also to be expected that further desmethyltetracycline-producing strains of S. a, ureofaaiens will be found in nature and can be developed from currently isolated strains of S. aureofaciems which do not produce these new antibiotics.

One of the main advantages of desmethyltetracyclines over the previously known tetracyclines is their increased level. Stability against acids and alkalis. The instability of te'racycline to acids and the instability of chlortetracycline to alkali are well known. In an aqueous solution with a sodium carbonate buffer, chlorotetracycline has a half-life of 29.2 minutes at p $ 9.85 at 23 ° C. In contrast, chlorodesmethyltetracycline does not lose more than 6% of its effectiveness within 2-1 hours under the same conditions. Tetraevcline will take less than 5 minutes. completely destroyed in 1N sulfuric acid at 100 ° C. In contrast, chlorodesmethyltetracycline loses only about 2% of its effectiveness at 100 ° C. after 15 minutes in 1 ml-schiveic acid. In a corresponding manner, the new antibiotic desmethyltetracycline in 1N hydrochloric acid at 100 ° C. loses only about 33% in 6 minutes. its activity. In 0.1N: sodium hydroxide solution at 50 ° C, desinethyltetracycline has a half-life of. about 20 hours versus 3.3 hours for tetracycline. These surprising properties. are very valuable insofar as the instability of tetracycline to acids and the instability of chlorotetracycline to alkali limit or completely exclude the use of these valuable substances in many cases. Because of the much larger. With the stability of the new antibiotics, the manufacture of many pharmaceutical products is possible which cannot be prepared in a satisfactory manner with the tetracycline. The increased stability also enables the recovery and purification processes to be improved, since more drastic pA and temperature conditions can be used and the effectiveness of various stages in the process can be increased as a result.

Chemical analysis of highly purified samples of chlorodesmethyltetracycline agreed with the calculated empirical formula C21H21Cl08 within the experimental Error limit match. The Kuhn-Roth method for the determination of methyl groups gave an extremely low value, which indicated that in the 7-position of the Naphthacen.rings no methyl group is present. The name Chlordesmethyltetracycline therefore seems justified.

The comparison of the ultraviolet and. Infrared absorption spectra of the Products and, the various tetracyclines provides further confirmation for this fact.

The melting point of chlordesmeth.yltetracycline in the form of the free base was 170 to 175 ° C. when measured on a hot stage with decomposition. The melting point of chlorotetracycline is 168 to 169 ° C. Desmethyltetracycline as the free base melts at 170 to 177 ° C with decomposition, whereas tetracycline melts at 160 to 168 ° C on the hot stage.

Further comparisons between chlortetracycline and chlordesmethyltetracycline led to the following. Results: Chlordesmethyltetracycline has a pKä value of 4.45 in dimethylformamide-water (50:50). Chlortetracycline has in the same System has a value of 4.50. The optical rotation of: Chlordesmethyltetracycline in 0.03 normal hydrochloric acid at a concentration of 0.5% was [a] ö = -2610, of chlortetracycline against it. -243o. The desmethyltetracyclines are apparently stronger water soluble than the corresponding tetracyclines. So dissolve. for example about 45 mg of chlorodesmethyltetrarycline hydrochloride in 1 ml of water versus about 14 mg chlortetracycline / ml water.

The antibacterial activities: the tetraryclines and desmethyltetracyclines are broadly comparable but have certain differences as can be seen from the following table.

This table compares the concentrations of chlortetracycline and chlordesmethyltetracycline required to produce half the maximum g growth inhibition in periods of 4 to 48 hours in different microorganisms, expressed in micrograms of antibiotic / ml of solution. Lower words correspond to more effective truth inhibition. Table I. antibiotic Microorganism Chlortetracycline Chlordesmethyltetracycline Time in hours 4 8 1 24 48 4 8 '24; 48 I. Staphylococcus I i aureus ATCC 6538P. .... 0.041 0.072 0 0.065 0.094 5 7 4.1; 0.41 0.68 Staphylococcus. albus. . . . . . . . % .5 20.5:>50.0> 50.0 16.5 50.0! >50.0> 50.0 Escherichia coli 0.12 0.27 1.5 10.2 0.16 0.3 0.66 I <1.56 > 0.8 Salmonella I f gallinarum ... 0.4 0.7-1, 4.65 j 37.0 0.4 i 0.56 1.06 1.1 Bacillus cereus <0.025 0.035 0.15 1.1 <0.015 0.039 0.077 0.19 Pseudomonas I aeruginosa .. 0.31 0.56 6.3 i 35.0 0.94 1.3 4.3 6.7 Streptococcus i pyrogenic ß-hemolytic 11.5 43.0>50.0> 50.0 14.4 - '41.0 i> 50: 0> 50.0 Proteus vulgaris 0.28 0.56, 9.2 50.0 0.62 I 1.12 1.38 4.4 Escherichia coli j A I'CC 9637 .. 0.5-1 1.18 6.6 _ 34.0 0.43 0.67 0.97, 1.8 The values given in the table above show. that chlortetracycline is generally somewhat more active after 4 hours. It should be noted, however, that if the solutions are left to stand for a while, bacterial growth occurs again in the case of chlortetracycline, whereas in those solutions containing chlordesmethylitetracycline, it is still generally hibernated. This fact is due to the greater stability of the latter antibiotic. Corresponding results are observed when comparing tetracycline with methyl tetracycline and bromotetracycline with bromethyl tetracycline. It has also been found that the desmethyltetracyclines are considerably superior to the oxytetracycline under the same experimental conditions.

The desmethyltetracyclines were also used in invivo experiments compared to tetracyclines and found no noticeable differences in the antibacterial activity of the methyl tetracyclines and the corresponding Tetracycline analogs. These new antibiotics can therefore be used for treatment serve the same sicknesses and in the same way as the tetracyclines.

The ultravialet absorption spectra of the tetracyclines and methyltetracyclines are. very similar to each other. The Fig.2 of the drawing shows. a comparison between the ultraviolet absorption spectrum of chlortetracycline and chlordesmethyltetracycline with the same concentration. It can be seen from this that the shape of the curves. practically is the same, but with a slight deviation in the chlorodesmethyltetra, cyclin absorption spectrum can be observed in the area of 260 m # t.

A further comparison of the ultraviolet absorption spectra of the tetracyclines is given in Table II in which the coefficients E; ä, (spectrophotometric absorption of a 10% solution, measured in a 1 cm cell) of the maxima and minima of several antibiotics are listed. The values are calculated from the ultraviolet absorption curves by returning the concentration at which the samples were measured to 1 0/0. All samples were measured in 0.1N sulfuric acid solution. Table II Antibiotic maxima minima maxima minima maxima E t% m, u, u Ei m y E t tec a ! m ( , Ettx em 1 ml, E 1 tx, m t nm nn Chlortetracycline hydrochloride ..... 209 368 135 302 356 265 288 242 343 228 Epichloretetral cyclin-N H4 ...... 170 368 100 302 332 254 290 242 342 228 Tetracycline hydrochloride ..... 305 355 182! 299 393 268 191 232 289 217 Epitetracycline-NH4 290 355 140 299 326 254 200 I 232 275 I 216 Desmethy ltetra- cyclin, neutral .... 292 353 169 298 395 268 202 232 299 217 Epidesmethyltetra- j cyclin hydrochloride 310 355 162 299 367 255 228 232 306 217 Chlorodesmethyltetra- cyelin hydrochloride 249 368 160 299 362 268 270 238 357 227 Epichlorodesmethyl tetracycline hydro chloride .......... 242 368 130 300 348 254 289 238 346 227 3 through 6 of the drawings show the infrared absorption spectra of various of the novel compounds of the present invention. Fig.3 represents the infrared absorption spectrum of chlorode desmethyltetracycline hydrochloride, and. 4 shows a comparison of the infrared absorption spectra of tetracycline as the free base and desmethyltetracycline as the free base. In addition to the similarities, a number of clear differences can be seen from this. Fig. 5 shows the infrared absorption spectrum of epichlorodesmethyltetracycline hydrochloride and Fig. 6 shows the absorption spectrum of epidesmethyltetracycline hydrochloride. These curves were obtained with a Perkin-Elmer model 21 infrared self-recording spectrophotometer on the solid phase antibiotics which had been compressed into a tablet with potassium bromide. Additional experimental data are as follows: Prism: Na.Cl; Resolution: 2; Sensitivity: 1-1; Gain: 5: Speed: 1 / E minute / .; Suppression: 2: and scale: 5.08 cm (2 ") / g. Various working methods have already been developed for testing the content of tetracyclines. Some of these can be used to determine desmethyltetracyclines after suitable modification Increase in fluorescence due to the formation of an isochlorotetracycline under alkaline conditions. The sample to be determined is dissolved in an alkaline phosphate buffer, and the fluorescence of the solution becomes isomerization at time 0 (immediately after preparation of the solution) and again after standing The increase in fluorescence corresponds to the amount of chlortetracycline present. However, the presence of chlordesmethyltetracycline in the solution can interfere with the chlorotetracycline detection and accordingly lead to a surprisingly low chlorotetracycline value. A more reliable method of determination 4 "0n chlortetracycline in mixtures with chlordesmethyltetracycline is based on the fact that the alkaline old building product of chlortetracycline shows no absorption at 380 mg. The sample is prepared using carbonate buffer at pH 10, and. the change in the absorption at 380 m [u] after standing for 30 minutes (about three times the half-life of chlorotetracvcline) is measured. Before the alkaline degradation, both chlortetracycline and chlordesmethyltetracycline show an absorption which, however, is only shown by chlordesmethyltetracycline after the degradation treatment. The reduced absorption is a measure of the chlortetracycline originally present in the sample.

A specific content test for Chlordesiiie, tliyltetracycline in the presence of other Te: tracyclinantibiotika is based on the so-called Hiscoxllethode: By a mild acid treatment for 15 minutes at 100 ° C with 1N hydrochloric acid all tetracyclines except - Chlordesmethyltetra: cyclin are destroyed. A portion of the sample treated in this way is then subjected to a strong acid treatment for 1.5 minutes at 100 ° C. with 6N hydrochloric acid in order to destroy the chlorodesmethyl tetracycline. The Clilordesmethvltetracyclineha.lt is calculated by measuring the decrease in the spectrophotometric absorption at 368 mu or the increase at 430 m # t caused by the vigorous acid treatment and comparison with a standard.

One only two components, for example the two members of an epimeric The pair containing mixture can be determined spectrophotometrically. The spectrophotometric Absorptions are determined at two wavelengths, and the ratio of these absorptions is linearly interpolated between the known values for the pure connections. In this way the percentage composition of the mixture can be determined.

Bromdesniethvltetracycline can easily be determined by bromine analysis customary oil analysis methods can be determined.

As mentioned above, bromodesmethyltetracycline can be the main one Antibiotic obtained during fermentation by adjusting the halogen content of the fermentation will. The aqueous nutrient medium should usually be at least 50 parts per million bromine ions contain. and the chlorine ion content should be kept as low as possible, at less than about 50 parts / million. Preferably the medium should be about 100 to = 1500 parts / million bromine ions and less than about 10 parts / million chlorine ions contain. The bromine ion content can be determined using any water-soluble bromine salt, z. B. Potassium chromide * can be provided, which contributes to the bromine ions for consumption the biological synthesis of the antibiotic supplies.

Apart from controlling the halogen ion content during fermentation corresponds to the process for the production of BromdesmethylteträcycIin essentially that for the production of desmethyltetracycline .. and Chloydesinethylxetracycline, such as it is described here .-- Desmeth "ylfetracycline-producing strains of S. aureofaciens -can also be used to generate the bromine alc; en.

Brydesmethyltetracycline has practically the same antibacterial properties Activity like chlordesmethyltetracycline and k; lnn for the same purposes and in in the same way as the latter.

It is not necessary to remove Broind.esmethyltetracycline from the other, to separate simultaneously formed antibiotics, as a mixture of different Desmethyltetracvclinen is useful for many purposes. For example, this can be fermented nutrient medium and concentrated directly due to its antibiotic content be used. Another use is to acidify the fermentation mash, to remove mvcel and insoluble. To filter and neutralize components and concentrate, - # by means of which a dry powder is obtained, which can be used as animal feed can mix. The mixed antibiotics are suitable for stimulating growth many animals.

Bromodesmethyltetracycline in raw form and its mixtures with others Antibiotics: can be used to preserve meat, poultry and fish in the same To be used in the same way as the tetracvcline is currently used. In such compositions one needs the proportions of the various tetracyclines present in it little to consider.

If you get the bromodesmethyltetracycline from the others in the fermentation mash If you want to separate contained antibiotics, you can do this on several to the specialist well-known @@ Tegen. For example, treatment with acid destroys it the tetracycline and treatment with an alkali chloruni brometracycline in the fermentation mash. The bromodesmethyltetracvcline can be obtained from the solutions obtained in this way by partition chromatography using a column of diatomaceous earth be won. Using a mixture of choroform and butanol at a pH value of about 2 antibiotics can be extracted from diatomaceous earth. Bromodesrcethyltetracycline escapes from the column before desmethyltetracycline and can be obtained from the eluate will. Also through. Using the Craig countercurrent distribution technique, one can use bromodesmethyltetracycline separate as an individual antibiotic component.

The epidesmethyltetracyclines according to the invention can be used as isomers the desmethyltetracyclines. The structural differences are based apparently due to a rearrangement of the dimethylamino group on the C4 carbon atom: m. Under certain conditions, which are to be described in detail below, apparently there is an inversion, with an equilibrium mixture. from Desmetllvltetracvclin and its epimer is formed. The two components can be used to achieve practically pure products are separated. each of the desmethyltetracyclines, viz Desmethyl tetracycline, chlordesinethyl tetracycline and bromodesmethyl tetracycline a corresponding. 'Opposite isomer, which is called Epi- "desmethyltetracyclin ,, EpichlordesYnethyltetracyclin and Epibroiridesrnethyltetracyclin: bezeichri: et should be.

The Desmethyltetracycline can by simply adjusting the hydrogen ion concentration a concentrated solution of the antibiotic to a range from pH 3.0 to 5.0 and let the solution stand until the isomerization reaches equilibrium has to be converted into their isomeric forms: en ,. The main conditions. That of both Conversion of the desmethyltetracyclines into their isomers must be complied with, are concentration, hydrogen ion concentration, time and temperature.

The isomerization is most conveniently carried out 1.-i room temperature, although at higher temperatures a higher rate of conversion is achieved will. The p] I value should be in the range from about 3.0 to 5.0, preferably between 3.5 and 4.5, lie. Even outside of these areas and even in distilled There is a certain epimerization of water, but the speed is then very high low. The. Concentration of the antibiotic in the aqueous solution should be so high as possible to achieve greater epimerization rates. A complete Equilibrium can take about 24 hours at 25 ° C, however requires a satisfactory equilibrium under special conditions. a much shorter time. The best results are usually obtained from standing of solutions achieved during a week or more. The equilibrium arises in most cases apparently at about 50 per cent. i.e., about half of the desmethyltetracyc.line is converted into its epiineres in equilibrium.

As the concentration to achieve high yields in short periods of time. is an essential factor in choosing a solvent system with which the highest concentrations of desinethyltetracycline are obtained. Such Solvent systems should be used to set a pil value within the preferred, Range to be buffered. Solvents that can be used include: Methanol. Ethanol, butanol, acetone, 2-ethoxy-ethanol, 2-methoxy-propanol, tetra; -hydrofuran, Dimethylformamide. and mixtures of these solvents. A preferred buffering agent is Na, triumdihydrogenphosph, a, t, but can. also other buffers and buffer pairs, which keep the hydrogen ion concentration within the desired range, be used.

The epimers of the invention can from the aqueous solution in the obtained in the same way as the tetracycline. If they are also in vitro and in vivo a somewhat lower antibacterial activity than the Desmethyltetra.cycline they are useful in the treatment of bacteria induced Diseases, still highly useful.

It was also found that the Desmethyltetra.cycline salts and complexes of the same, kind and in the same. Make way like the tetracyclines. When treated with acids finitely a pH value of less than about 4 - the acid salts formed. The free base can have a pH in the range of about 4 to 6 can be obtained, and at p11 values of over 6 salts with bases, e.g. B. the calcium salt formed. The salts of the epimers are formed in a corresponding manner Way.

As already mentioned above, is used to produce the new invention Antibiotics practically the same process as for producing chlortet.racycliil, Tetracycline and bromotetracycline applied, the main difference being in the selection of a mutant strain of S. aureofaciens, the, the desired desmethyltetracyclines able to form in place of the tetracyclines. Composition of the fermentation medium, Ventilation speed, time, temperature, hydrogen ion setting, inoculation method and the like, as used in the production of chlortetracycline, bromotetracycline and tetracycline are used, are also suitable for the production of the new desmethyl, tetracygline.

The following examples explain this in the context of the present invention applied fermentation processes.

Example 1 A suitable medium for the production of vaccines, for such fermentations can be prepared with the following substances: Sucrose .. ................. 30 - / I (NH4) 2 SO ' . . . . . . . . . . . . . . , .... 2 g / 1 Ca C 03. . . . . . . . .-. . . . . . . . ,. . . . 7 g / 1 Corn Steep Liquor ... . . . . . . . . . . 16.5 m1 / 1 The pH value of the meditation prepared in this way is around 6.8. A portion of. 8 ml is placed in a Brewer tube about 20 cm (8 inch) and sterilized at 120 ° C for 20 minutes. The sterilized medium is then inoculated with 0.5 nil of an aqueous spore suspension of a strain of S. a.ureofaciens capable of producing chlorodesmethyltetracycline, for example S-604, the suspension containing about 40 to 60 million spores / ml. The inoculated medium is incubated at 28 ° C for 24 hours on a shaker with reciprocating motion at 110 oscillations / minute.

A typical medium suitable for the production of chlorodesmethyltetracycline is composed as follows: Cornstarch. ... . ...... ..: .. 55 g / 1 Ca C O3. . . . . . . . . -. . . . . . . . . . 7 g / 1 (N H4) 1 S 04. . . . . . . . . . . . . . 5 g11 NH4Cl. . . . . . . . . . . . . . 1.5 g / 1 Fe S 04.7 H2 O. . . . . . . . . . : 40 mg / 1 Mn S 04 - 4H2 0. . ... . . . . . . 50 mg / 1 Zn S 04 * 7H2 0. . . :. . . . . . . . 100 mg / 1 C0C12-6H20 ............ 5 mg / 1 Corn steep liquor. ° .-. . . . . . . 30 g / 1 Cottonseed flour ....... 2 g / 1 Lard oil. . . . . . . ... . . . . . . 2.0% v / v Limiting the chlorine ion content of this medium results in an increase in the amount of desmethyltetracycline at the expense of chlorodesmethyltetracycline production. EXAMPLE 2 A shake flask ferration in which both desmethyltetracycline and chlorodesmethyltetracycline were produced - would be carried out with a medium prepared as follows: Corn steep liquor. .. .. ... 30 g / 1 Lard oil. . . . . . . . _. . . . . . . . . 2 1 / o V / V Cornstarch. . . . . . .- -_. . :. . . . . . 55 g / 1 Cottonseed meal `,. .... ... 2 g / 1 CaCO3 ....: ...: -__-..-- ..: .. 7 g / l (N H4) 2S04. . . . . . -. . . . . . . 5 g / 1 l \ TH4C1. ... ..: ..:. . . .. .. ... 1.5 g / 1 FeS 04 - 7I12.0 -. ... . . . . . . 40 mg / 1 Zn S 04 - 7H2 0. . : r: ... . . . . . . 100 mg / l Mn S 04 "4H2 O .... 50 mg / l Co-C12 "6 H2 O.. -..;......... 5 ing / I A portion of 25 ml was placed in a 250 ml Erlenmeyer flask, sterilized for 20 minutes at 120 ° C. and inoculated with 1 ml of the mycelial growth produced as described in Example 1. After incubating for 120 hours at 26 ° C on a rotary shaker (186 rev / min :), the mash was checked for its content. It contained no more than about 70 y / ml chlorotetragycline and about 450 l / ml desmethyltetracycline and 900 y / ml chlorodesmethyltetrac_vcl i n.

Example 3; Tank fermentation A 40-1 fermentation was carried out in an experimental tank essentially according to that described in Example 2 Way of working carried out. After preparation, the medium was sterilized at 125 ° C. for 25 minutes and inoculated with a vaccine of strain S-604 which, as described in U.S. Pat 2,482,055. The fermentation was taking place continuously Stir at a temperature of 28 ° C for the first 24 hours and 25 ° C led to completion after 135 hours. Sterile air was used during the first 16 hours at a rate of 0.3 1/1 and minute and 0.5 1/1 and Minute to harvest.

The following examples illustrate the recovery, purification and separation the antibiotics. Example A Purification of Chlordesmethyltet.racycline and Desmethyltetracycline 25.8 l of the mash prepared as described in Example 3 were mixed with 200 ml of concentrated hydrochloric acid are added to adjust the pH to 1.5. the Mash treated in this way was filtered and mixed with 2.8 kg of filter aid gave 16.31 filtrate. The filter cake was again with 25 l of water at about 50 ° C slurried and adjusted to pH 1.5. After stirring for 30 minutes the reslurried cake filtered off and the filtrate with the original The resulting filtrate was combined to give a total of 42.31. That united The filtrate was admixed with 6.76 kg of sodium chloride and four times with 15% of its volume extracted on butanol. The combined extracts were clarified by filtration and concentrated in vacuo at 25 to 30 ° C to a final volume of 61. Example B Chromatographic Separation of Chlordesmethyltetracycline and Desmethyltetracycli.n Das according to the example A butanol concentrate obtained was divided into two equal parts for further processing divided. One of these was concentrated to 900 ml and the other to 610 ml in vacuo. The concentrates were filtered off and adjusted to a p11 value with 50% sodium hydroxide solution set by 2.

The solvent system used for the chromatographic separation was made by equilibrating a mixture of 800% butanol and 20% chloroform with Water adjusted to pH 2 with hydrochloric acid was prepared. There were glass pillars about 15 cm in diameter, which was mixed with 2.8 kg of acid-washed diatomaceous earth were filled. The columns were flushed with the aqueous phase of the solvent system before use equilibrated.

The two butanol concentrates were processed on separate columns and carefully poured onto the upper ends of the pillars. The pillars were then eluted with the solvent phase at a rate of about 101 / hour was enforced. In both cases 500 ml fractions were collected, whose antibiotic content was determined by paper strip chromatography.

In the 900 ml batch, fractions 6 to 17 contained the chlorodesmethyltetracycline and chlortetracycline and fractions 24 to 40 desmethyltetracycline and tetracycline. In the 610 ml batch, fractions 7 to 18 contained the chlorodesmethane tetracycline and chlortetracycline and fractions 19 to 31 the desmethyltetracycline and tetracycline. Example C Obtaining Ch.lordesmethyltetracycline from the column fractions Die fractions containing chlordesmethy 1-tetraeyclin obtained according to Example B were combined to give a volume of about 121. After adding a equal volume of water, the mixture in vacuo to 1810 ml of aqueous solution constricted. The pH of the concentrate was adjusted to 2.3 by adding hydrochloric acid set to 1.9. The acidified concentrate was washed twice with 180 ml of chloroform washed, filtered and concentrated in vacuo to a volume of 360 ml. The p11 value the concentrate was brought from 1.2 to 2.8 with 50% sodium hydroxide solution. The solution was freeze-dried to give 18.56 g of amorphous chlorodesmethyltetracycline hydrochloride with an active ingredient content of 550 y / mg. Example D Obtaining Desmethyltetracycline from the column fractions containing the Desmethyltetracycli.n obtained according to Example B Fractions were combined to give a volume of 14 liters. After adding one same. By volume of water, the mixture was reduced to volume in vacuo at 24 ° C concentrated from 565 ml of aqueous solution. The pH of the concentrate was adjusted by adding of concentrated hydrochloric acid adjusted to 1.65 and the solution twice with 55 ml of chloroform. The washed solution was filtered and the p.1 value brought to 5.8 with 50% sodium hydroxide solution. After three hours of aging at room temperature and aging at 4 ° C for 15 hours, the resulting crystal slurry was filtered and the product washed with water and dried in vacuo at 40 ° C. One received a yield of 4.7 g of crude neutral desmethyltetracyciins with an active ingredient content of 880 y / mg, calculated as tetracycline hydrochloride, giving a total yield of 73% from the aqueous concentrate. Example E Crystallization from chlorodesmethyl tebracycline hydrochloride from acetone-ether 18.56 g of the freeze-dried chlorodesmethyltetracycline obtained according to Example C were concentrated in a mixture of 55 ml of acetone, 7.4 ml of water and 3.7 ml Hydrochloric acid introduced. The solution was filtered and the solids were washed twice with a total of 55 ml of acetone containing 1.85 ml of water. the combined filtrates, their. pH was 0.8, were treated with 55 ml of ether. The mixture was seeded and kept at room temperature for 18 hours Aged with stirring and the crystals which formed were filtered off. The product was with a mixture of acetone, water and ether, then with acetone and finally washed with ether and dried at 40 ° C in vacuo. A yield was obtained of 4.74 g of crystalline chlorodesmethyltetracycline hydrochloride with an active ingredient content of 1090 "/ mg, which corresponded to a yield of 50.6% in this stage. The product contained 110% chlorotetracycline. Example F Crystallization of Chlordesmethyltetracyclin-hydrochlori, d from butanol-ethoxyethanol 50.4 g of according to Example C prepared freeze-dried chlorodesmetyltetracyclnhs with a Active ingredient content of 700 y / mg were suspended in 22.5 ml of ethoxyethanol, and the mixture thus obtained was admixed with a total of 205 ml of butanol. The pH was adjusted to 0.8 by adding 11 ml of concentrated hydrochloric acid and that Mixture aged for 51 hours with stirring. The crystals formed were filtered off, with a mixture of 90% butanol and 10% ethoxyethanol and. then with chloroform washed and dried in vacuo. A yield of 30.6 g of chlorodesmethyltetracycline hydrochloride was obtained with an active ingredient content of 1053 y / mg, corresponding to a yield of 840/0.

Example G Recrystallization of chlorodesmethyl tetracycline for destruction des Chlortetraeyclins 1 g of the chlorodesmethyltetracycline prepared as described in Example E with an active ingredient content of 1090 y / mg was in 3 ml of a mixture of 90% butanol and 10% ethoxyethanol slurried and to adjust the pH to 10.4 mixed with 3 ml of triethylamine. The not completely dissolved mixture was 24 hours left at room temperature to remove any chlorotetracycline present destroy, after which concentrated hydrochloric acid was added to lower the pH to 0.8 and the mixture was aged with stirring for 24 hours. The crystalline product was filtered off with a mixture of 90% butanol and 10% ethoxyethanol, then washed with chloroform and dried at 40 ° C in vacuo. A yield was obtained of 0.36 g of chlorodesmethyltetracycline hydrochloride with an active ingredient content of 1228 y / mg, which contained about 2.5% chlorotetracycline, which corresponded to a yield of 40%.

The following examples illustrate the conversion of the antibiotics into different shapes.

Example H Chlordesmethyltetracycline Hydrobromide 1 g of ammonium chlorodesmethyltetracycline was dissolved in 20 ml of water and the solution with 0.6 ml of 48% hydrobromic acid to adjust the pH to 1.6. The mixture was stirred at Aged room temperature for 18 hours, after which long, needle-shaped crystals of chlorodesmethyltetracycline hydrobromide occurred. The product was filtered off with 6 ml of water triturated with hydrobromic acid had been adjusted to pH 2.2, washed and dried for 5 hours at 44 ° C in vacuo. A yield of 750 mg of chlorodesmethyltetracycline hydrobromide was obtained.

Analysis: C2, H ', N2 Cl Br 0, Calculated: C 46.2. H 4.1 N 5.1 Cl 6.5, Br 14.6, O 23.4; Found: C 45.94, H 4.44, N 4.71, Cl 6.78, 6.47; Br 13.74, O (difference) 24.39, 24.70. Example I Preparation of Chlordesmethyltetrac_vclinammoniumsalz 100 mg of the chlorodesmethyltetracycline prepared according to Example E were dissolved in 0.5 ml of a mixture of 90% butanol and 10% ethoxyethanol, and gaseous ammonia was introduced into the solution until a thick solid substance appeared. The slurry was diluted with a small amount of concentrated aqueous ammonia, allowed to stand at room temperature for 5 hours and filtered off. The product was washed with a mixture of 90% butanol and 10% ethoxyethanol and then with acetone and dried in vacuo. A yield of 41.4 mg of chlorodesmethyltetracycline ammonium salt with an active ingredient content of 1148 g / mg as chlorotetracycline hydrochloride (44%) was obtained. Example J Production of neutral chlorodesmethyltetracycline 50 mg of the chlorodesmethyltetracycline obtained according to Example E with an active ingredient content of 1090 μg / mg were dissolved in 1.3 ml of water and the solution was mixed with dry sodium carbonate up to a pH of 7 to 8. The amorphous precipitate obtained was centrifuged off and the clear, supernatant solution was adjusted to pg 5.4 after decanting and then aged for 16 hours at room temperature. The product was filtered off, washed with water and dried at 35 ° C. in vacuo. 21.9 mg of crystalline, neutral chlorodesmethyltetracycline with an active ingredient content of 1250 μg / mg (500% yield) were obtained.

Example K Reduction of chlorodesmethyl tetracycline to desmethyl tetracycline 12.5 mg of crystalline chlorodesmethylteteracycline hydrochloride was dissolved in water and mixed with a drop of triethylamine. The solution was 10% strength with 10 mg Palladium-carbon mixed and shaken in a hydrogen atmosphere for 20 minutes. The resulting solution was filtered to remove the catalyst. Both the Ultraviolet spectra as well as paper chromatography indicated that one Dechlorination to desmethyltetracycline had taken place.

As mentioned above, treatment with acids and bases can produce salts the desmethyltetracyclines can be obtained. Hydrochloric acid is a preferred acid, since the salts from this acid and the desmethyltetracyclines are very useful products represent. By using other acids, e.g. B. sulfuric acid, phosphoric acid, Acetic acid, boric acid, and other organic and inorganic acids can be certain Properties can be achieved. Salts with bases can be prepared by means of alkali hydroxides, e.g. B. sodium hydroxide, and the alkaline earths prepared in the manner described above will. The calcium salt is particularly suitable and can be easily adjusted an aqueous solution of any of the desmethyltetracyclines to pH between 6 and 10 in the presence of at least 1 molar equivalent of calcium in the solution getting produced. The barium, magnesium, strontium and other salts can also can be obtained in a corresponding manner. As in the case of the tetracyclines, make up too the desmethyl tetracycline complexes with boron, zirconium and other polyvalent metal ions.

If the fermentation medium contains neither chlorine nor bromine, then of course neither clordesinethyltetracycline nor bromodesmethyltetracycline are formed. In In this case the main antibiotic is desmethyltetracvcline. By doing Dimensions, how the chlorine ion content is increased, especially at over 50 parts / million, there will be substantial amounts of chlorodesmethyltetracycline formed because 1 part of chlorine ions form about 14 parts of chlorodesmethyltetracycline can lead. The amount of the latter antibiotic can be controlled. In the manufacture of this antibiotic, the fermentation medium preferably contains more a1> 50 parts / million chlorine ions, and this amount can be used for maximum generation of chlorodesmethyltetracycline can be increased to 1000 parts / million or above.

A low concentration of bromine ions in the fermentation medium of about 10 parts / million up to a few percent leads to the suppression of the formation of chlorodesmethyltetracycline and to a general increase in the production of Desmethyltetracvcline. If the fermentation medium is less than about 50 parts / million Chlorine ions and more than about 50 parts / million bromine ions will be generated favored by bromodesmethyltetracycline, and when using high yields on antibiotic-producing strains of S. aureofaciens the fermentation mash obtained Contain brointetracvcline as the primary antibiotic. y

Claims (6)

PATENT CLAIMS: 1. Process for the production of antibiotics of desmethyltetracy clin series and their epimers, characterized in that an aqueous nutrient medium is used with a controlled chlorine and / or bromine ion content by means of a Desmethyltetracvclin by spontaneous mutation and / or by mutagenic agents from Streptomyces aureofaciens obtained strain aerobically fermented, the antibiotics formed from the fermentation mash wins and, if necessary, by setting the antibiotic solution to a pH of 3.0 to 5.0 forms their epimers. 2. The method according to claim 1, characterized in that a practically chlorine ion-free medium is used, whereby mainly desmethyltetracycline is formed. 3. The method according to claim 1, characterized in that a medium with a content of at least 50 Parts / million chlorine ions used, creating predominantly chlorodesmethyltetracycline is formed. 4. The method according to claim 1, characterized in that one is a Medium containing at least 50 parts / million brorons and less than 50 parts / million chlorine ions. whereby predominantly Bromdesniethvltetracyclin is formed. 5. The method according to any one of claims 1 to -1, characterized in, that one uses: a medium with a content or an addition of alkaline earth ions and the pH of the medium after fermentation to a value between 6 to 10 sets. 6. Process for the production of desmethyltetracyclines, characterized in that that one can use Chlordesmethyltetracycline or Bromdesinethyltetracycline by catalytic Hydrogenation, preferably using palladium on activated carbon as a catalyst, dehalogenated.