DE2327990A1 - BIOLOGICAL TRANSFER OF ANHYDROTETRACYCLINES INTO 6-DESOXYTETRACYCLINE - Google Patents

Description

Dr. Ing.E. Berk

Dipi.ing · H. Sertc

Patent attorneys

5 Cologne 41, P.O. Box 410149
Universitätsstrasse 31

V / f Oj 6 Ji

Biological conversion of anhydrotetracyclines into 6-De soxytetracyclines.

The invention relates to a biological process for the production of 6-deoxytetracyclines, in particular ot-6-deoxy-5-thiydroxytetracyclines, known as doxycyelin through a biological conversion of anhydro-5-hydroxytetracycline with a subsequent isolation of the doxyeyelin.

The chemical production of 6-deoxytetracyclines was published by Stephens et al. in the Journal of American Chemical Society, Volume 80, page 5324 (1958), McCormick et al., ibid., Volume 82, page 3381 (1960), as well as U.S. Patents 3,019,260, 3,069.4-67 and 3,200,149. Another chemical process is described in Portuguese patent ^{specification 1} 52.217 and German Auslegeschrift 2.131.944.

The chemical process for the production of 0 ^ -6-deoxytetracycline runs over three to four process steps, as well as a further process step for purification before the production of the pharmaceutically acceptable derivatives. The total yield of these process steps, starting from the oxytetracycline to the recovery of the 0 ^ -6-deoxytetracycline in the form of a base or hydrochloride, is between 5 and 16 % w / w.

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The chemical processes are more or less dangerous and also require the use of expensive reagents, such as the use of anhydrous hydrogen fluoride; they also require a catalytic one Hydrogenation or catalytic reduction in the presence of a noble metal as a catalyst. The present invention relates to a ' biological conversion through which "all of these disadvantages are overcome and a good yield is achieved.

The present invention is based on the discovery that certain mutant strains of Streptomyces alboflavus the anhydrotetracyclines convert into 6-deoxytetracyclines, namely by the fact that the double bond at C5 to C6 is saturated. The conversion is achieved by converting an anhyrotetracyelin to a aqueous nutrient solution, which is submerged under aerobic conditions with mitent strains of a species of the Streptomyces genus, such as Streptomyces alboflavus or Streptomyces lusitanus is fermented.

The anhydrotetracyclines can easily be derived from tetracyclines Dehydration can be produced by means of a strong mineral acid. The manufacture of Anhydrochlortetracycline is in U.S. Patent 2,744,931 and the manufacture of anhydrotetracycline is described in U.S. Patent 2,744,932, the preparation of the various anhydrotetracyclines is given in the Journal of American Chemical Society, Volume 74, page 4981 (1952) and in Volume 79, pages 2849 and 4653 (1957).

A typical mutant of Streptomyces alboflavus, strain M-434-001, was deposited with the National Collection of Industrial Bacteria, Aberdeen, Great Britain, in June 1971 under the number NCIB 10588. This mutant is derived directly from the Waksman's Streptomyces alboflavus strain obtained from the Centraalbureau Voor Sehimme! Cultures, Baarn, Holland. The non-spore-forming strain was first converted into one which forms spores, namely by alternating between two

232799Q

Culture media was inoculated, namely on oatmeal agar and Calium Citrate Glycerin Agar ,. until a colony with good sporulation was obtained. After four successive selections of a spore-forming strain, a mutant was found which produced about 6,500 mcg / ml oxytetracycline; these selections were performed under a sterile vaccination cabinet with two Unscreened Philips ultraviolet lamps with 30 W each was; each lamp was 60 cm above the work table. This strain was taxonomically identical to that in the US patent 3,637,463 (Villax) described trunk with the deviation, that it reduces nitrate to nitrite while Streptomyces alboflavus M-IO8-OX (ATCC 15388) does not. It then became a well sporulated slant culture on glycerol-calcium citrate-agar this oxytetracycline-producing microorganism was accidentally kept at 18 to 20 C for 28 months. The glycerin-calcium citrate agar medium had the following composition:

Agar 20th G Calcium citrate VJl G Dipotassium phosphate 0 , 5 g Ammonium chloride 0 , 5 g Glycerin 10 G water 1 1-

pH 6.9

After this period, 5 ml of sterile water and three sterile Glass spheres added; the tube was shaken well and then a plate culture was made. Developed after incubation at 28C found only five colonies, all of which had lost the ability to produce oxytetracycline.

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The colonies were then subjected to successive conventional selections as above, and the various well sporulating colonies were first checked to see whether they could form oxytetracycline or not. The strains which have no inhibitions ^ one against Staphylococcus aureus were further selected and tested for anhydro-5-hydroxytetracycline in Convert 6-deoxy-5-hydroxytetracycline.

The experiment was carried out by forming streak cultures on agar plates in Petri dishes containing either calcium citrate-glycerol agar or oatmeal agar and a clear crystal of anhydro-5-hydroxytetracycline about 1 cm from the surface -Culture lays away. The plates wurden-, ^incubated at 28 ° C and observed daily under an ultraviolet lamp was provided with a ^ cobalt glass filter. The colonies were selected which lead to a yellow fluorescence of the agar between the non-fluorescent anhydro-oxytetracycline crystal. 'and the emergence culture. Plate cultures that were bluish or lacking fluorescence were discarded.

The strains that caused yellow fluorescence to appear were then fermented in a standard shake flask culture at 28 ° C. After an incubation of 48 hours, 500 meg / ml anhydrooxytetracycline was added and aliquots were subjected to thin layer chromatography; the chromatographic plate was coated with a kieselguhr suspension in water containing 0.5% ethylenediaminetetraacetate and 5 % glycerol; stationary phase: Mcllvain buffer pH 3.7 with 5 % glycerol - mobile phase: ethyl acetate, saturated with Mcllvain buffer pH 3.7 and containing 5 % glycerol; the aliquots were compared to simultaneous runs of doxycycline, oxytetracycline and anhydro-oxytetracycline. After seven successive selections, the strains M-434-OO1 and M-Hl were chosen, the more

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than 50 % of the added anhydrooxytetracycline were able to convert into o (, - 6-deoxy-5-hydroxytetracycline. These two strains have been deposited with the National Collection of Industrial Bacteria under the names NCIB IO588 and IO589. These strains are equally capable of the anhydroderivatives the tetracyclines apart from oxytetracycline to be converted into the corresponding 6-deoxytetracyclines.

In a parallel experiment, Streptomyces lusitanus (NCIB 9585 - ATCC 15842) became another mutant strain in the same way which did not produce chlortetracycline but did produce the anhydrotetracyclines converted to 6-deoxytetracyclines. This mutant strain is designated M-15842-NHIO. . ■ "

Different from the many cultures studied Streptomyces species were only the two aforementioned cultures Anhydrotetracycline in 6-deoxytetracyclines convert _s and these were from randomly dried cultures.

An examination of Streptomyces alboflavus mutant NCIB IO588, by an electron microscope that the resulting _s spores have a smooth surface, are ovate 1.0 to 1.6 μ size of 0.7 to 1.0 μ χ; the spore carriers belong to the group "Retinaculum Apertum" according to Pridham "

A mature culture of Streptomyces alboflavus M-43 * 1-001 (NCIB

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- -> &, "■

10588) after eighteen days when incubated at 26 ° C following properties:

Gelatin medium :

Meat extract 1.5 g

Peptone 2.5 g

Gelatin ■ 80 g

Distilled water 500 ml

pH 6.2 * before sterilization

No sporulation, weak growth, no pigment

Tyrosine agar (Ettlinger's melanin-forming medium): yeast extract 1 g

L-tyrosine 1g-

NaCl 8.5 g '

Agar 16 g

Tap water 1,000 ml

Good sporulation, pale pink pigment after two days Incubation that did not get stronger after four days, color, the spores "en masse", pearl or clamshell tint (2ba)

Potato slopes:

A potato slope (diameter 1.5 cm, height 3 to cm) was _{washed with a 10 percent Na 9} CO, solution and sterilized in a test tube at the same time with 1.5 cm of distilled water.

Slightly yellowish-brown diffusible pigment, good « Sporulation of pearl-colored or clamshell-colored tint (3ba)

Bennett Medium;

Yeast extract 0.5g

Meat extract 0.5 g

Hydrogenated Casein 1 g

Glucose ■ 5 g

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Agar-agar 10 g

Distilled water . 500 ml

Good sporulation, color of the spores "en masse" ivory- ' colored tint (2cb), brownish diffusible pigment

Glycerine asparagine medium;

Glycerine. '5g

Asparagine 0.25g

Meat extract 1 g

K ₂ HPO ₄ 0.25g

Agar-agar 7.5 g

Distilled water 500 ml

pH 6.9 after sterilization

Very good growth, good sporulation of pearl-colored / or.

Shell-colored tint (2ba), pale yellow, diffusible pigment

Czapek Dox medium;

Glucose 20 g

NaNO ₃ . ■ 1g

K ₂ HPO ₄ 0.5 g

MgSO ₄ .7H ₂ O 0.25g

KCl 0.25g FeSO ₄

Distilled water 500 ml

Agar-agar 7.5 g

pH 7.1 after sterilization. Weak growth, no sporulation

Emerson medium;

Yeast extract 2 g

Soluble starch 7.5 g

K ₂ HPO ₄ 0.5 g

MgSO ₄ .7H ₂ O 0.25g

Agar-agar 10 'g

Distilled water 500 ml

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■ «

pH 7 after sterilization

Sparse growth with weak speculation (2db), pale brown pigment

Czapek Dox Starch Agar:

Soluble starch, 5 g

NaNO ₃ '1g

K ₂ HPO ₄ 0.5 g

MgSO ₄ .7H ₂ O 0.25g

KCl "0.25g FeSO ₄

Agar-agar 7.5 g

Distilled water 500 ml

pH 7 after sterilization

Good growth, good sporulation (2db), pale yellow pigment

Oatmeal agar; .

60 g of oatmeal were heated in 800 ml of water on a water bath for fifteen minutes; Then it was filtered through gases and 20 .g agar like water was added up to one liter. The pH was adjusted to 6.3 with 15% sodium hydroxide.

Very good growth and very good sporulation of natural (or fibrous) color (2dc), dark brown diffusible pigment

Peptone yeast extract iron agar ;

Bacto-pepton 15 g.

Proteose peptone, Difco 5 g

Ferric ammonium citrate 0.5 g

Dipotassium phosphate 1 g

Sodium thio sulfate 0.08g

Bacto agar 15 g

Yeast extract 1 g distilled water 1,000 ml

V 10/30 9851/1168.

Good sporulation of oyster-light color (b), weak smoky gray pigment that slowly becomes less pronounced after ten days

The strain NCIB 10589 shares the same taxonomic characteristics.

All color names and the related code numbers are taken from the Color Harmony Manual (Container Corp. of America), 3rd edition. -

The cooling tower for fermentation contains an assimilable Nitrogen and carbon source and mineral salts used for • are conducive to the growth of the microorganism. The fermentation takes place in the usual manner by the aerobic submerged process at a temperature in the range from 24 to 38, preferably from 26 to 28 C. The anhydrotetracyclines can be used either as a fine sterile - & ä aqueous suspension or as a solution in Dimethylformamide at the beginning of the fermentation, but preferably 36 to 60 hours after the start of the fermentation, "can be added.

In order to be consistent and satisfactory in large-scale operations To achieve results, the microorganism - as in the Fermentation industry of antibiotics is common - are subjected to continuous selections, and the composition of the Culture medium must be adapted to each new strain in order to achieve the highest possible Achieve yields.

The anhydrotetracyclines can be added in amounts of 1 to 8 g / l and the total fermentation time is 90 to 150 hours. After fermentation, the mash becomes acidified with hydrochloric acid, filtered and the mycelium is with 30-bis 70 percent aqueous dimethylformamide containing concentrated hydrochloric acid extracted. The 6-deoxytetracyclines are then from the filtrate by precipitation as water-insoluble acid addition salts, Metal lates or molecular complexes obtained. The 6 ~ besoxytetracyclines are then obtained from the precipitate obtained in this way released in a known way »The preferred way of

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Precipitation takes place in the form of 3-hydroxy-2-naphthylates, methylene-5,5-disalicylates, 5-sulf © salicylates or as complexes of N, W ¹ -dibenzylethylenediamine or N, N'-dibenzylethylenediamine. The precipitation can also take place by means of sodium hexametaphosphate.

The θί-6-deoxytetracyclines can be obtained from the crude product obtained in this way can be isolated in the pure state in a known manner. The preferred form of such purification is in German Ausl-egeschrift 2.131.9 ^ 5 described.

The invention is illustrated in the following examples; for all Tap water was used in the media described in the examples.

1. It became a sterilized medium of the following composition used:

Cornruellwasser sugar

10 IV) 0.25 G 15th 1 ß 2 G he
O G 1

KH ₂ PO ₄ . ; MgSO ₄ .7H ₂ O water

The nutrient medium was adjusted to pH 6.6 and 30 ml this nutrient medium with 0.5 ml of a sterile aqueous spore suspension of a freshly selected strain of Streptomyces alboflavus M-434-001 (NCIB 10588) and inoculated in an Erlenmeyer flask kept in motion on a reciprocating shaker at 28 ° C for about two and a half hours.

A series of 300 "ml Erlenmeyer flasks, each containing 30 ml of a sterile culture medium of the following composition:

Corn steep water 30 g

Sugar 12.5 g

10/3 & §SS1 / f

CaCl ₂ 9.5 g

(NH ^) ₂ HPO ₄ 2.6 g

Water 1 1

pH adjusted to 6.3

inoculated with one milliliter of the aforementioned preculture and true

incubated.

and for fifty-four hours with stirring at 28 ° C

Then 0.6 ml of a dimethylformamide solution of anhydrooxytetracycline hydrochloride (concentration 250 mg / ml) was added under sterile conditions and the incubation of the shaking cultures was continued for forty-eight hours. In each case, an aliquot removed under sterile conditions _..Bedin-% from the bottles, the pH adjusted to 1.5 with hydrochloric acid and einpr.ozentiger a thin layer chromatogram using a sample of o ^ -6-deoxy-5-hydroxytetracycline (doxycycline ) as a comparison. All aliquots showed a specific spot like doxycycline at the same R ~.

After an additional forty-eight hours of incubation, the contents became of the bottles combined, acidified to a pH of about 0.1 with concentrated hydrochloric acid and washed. Of the the mycelium-containing solid was with a mixture of one part dimethylformamide, one part water and one part concentrated hydrochloric acid and filtered, and the wash waters were added together to the first filtrate.

Then 4 g / l of 5-sulfosalicylic acid crystals were added to the clear filtrate, the precipitated crude sulfosalicylate was filtered, washed with water, aqueous methanol (60%) , acetone and ether. The dried product consisted essentially of ct-6-deoxy-5-hydroxytetracycline, E ^ ' 185 at 349 mu (in Me-

I Olli *

ethanol, which contained 1 % concentrated hydrochloric acid) corresponding to a purity of 83 %, i.e. 58 % of the total

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put amount of anhydro-5-hydroxytetracycline was inc? (, - 6-deoxy-5-hydroxytetracycline converted.

Each part of the process product thus obtained was mixed with two and a half parts of 70 percent aqueous dimethylformamide and two parts of concentrated aqueous hydrochloric acid mixed. The acid mixture was heated to 68 ° C and thirty minutes kept at a temperature of 68 to 7 ° C. It was cooled quickly and one-half part sulfosalicylic acid was added. After stirring for two hours at 15 ° C - the crystalline precipitate was taken up by filtration and with Washed 60 percent aqueous methanol. The process product was dried and each part of the product was then Suspended in a mixture of two parts of ethanol and half a part of water and with stirring became a stoehiometric Amount (JL mol / 1 mol) of triethylamine mixed with an equal amount of ethanol was added. After the start of crystallization A further four parts of ethanol were added to the free base. The product was filtered and washed with ethanol, acetone and Ether washed. The free o (.- 6-deoxy-5-hydroxy-

tetracycline base (melting point 175 ° to 177 ° C, E ^ % 420 at

ι cm

268 np and 331 at 3 ^ 9 τημ) was then dissolved by mixing one part each with one part ethanol, half a part water and one fifth part concentrated aqueous hydrochloric acid. When this solution was mixed with two parts of ethanol containing 17% hydrochloric acid and half a part of concentrated aqueous hydrochloric acid, pharmaceutical grade doxycycline hydrochloride hemihydrate hemiethanolate was

o 1 96 loot won by 41.4 % . Melting point 210 C, Ej ⁷ Z _1n 373 at 268 mji and 292 at 349 mju, / <X / _D -105 (c = 1 in methanol containing 1 % concentrated hydrochloric acid). The. Infrared curve was identical to that of an authentic sample.

2. Example 1 was carried out with the microorganism Streptomyces Iusitanus, Repeat strain M-15842-NBK10. The overall yield of pure doxycycline was 38.9%.

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3. Example 1 was repeated, but instead of 5-sulfosalicylic acid, 3-hydroxy-2-naphthalic acid was added to the acidified filtrate and the pH was slowly increased with 10 percent NaOH until the precipitation was complete. The overall yield, expressed as 100 % doxycycline base, was 40.7 %.

4. Example 1 was repeated after the free base had been obtained, but using anhydrotetracycline instead of anhydro-oxytetracycline. A pure o (-5-deoxytetracycline base was isolated with a yield of 33.1 % ; E ^ 333 at 350 mp.

Claims

30th

Claims (3)

PATENT CLAIMS 1. A method for the biological conversion .von Anhydrotetracyclxnen into the corresponding 6-deoxycycline, characterized in that 'that an anhydrotetracycline of an aqueous nutrient solution at the beginning or during an aerobic fermentation with Streptomyces alboflavus M-434-OO1, NCIB IO588 or Streptomyces alboflavus M- Hl ₉ NCIB IO589 is added and the fermentation is continued until the anhydrotetracycline is practically converted into the corresponding 6-deoxytetracycline and the 6-deoxytetracycline is then isolated from the fermented broth. 2. The method according to claim 1, characterized in that the anhydrotetracycline 7-chloro-anhydrotetracycline, 5-hydroxy-anhydrotetracycline, ö-Desmethyl-T-ehlor-anhydrotetracyclin or 6-desmethyl-anhydrotetracycline as amphoteric free base, acid addition salt, Addition salt with an organic amine, metal chelate or used as a molecular complex. 3. The method according to claims 1 and 2, characterized in that that the 6-deoxytetracycline from the fermentation medium by precipitation as an acid addition salt, metal chelate or Molecular complexes isolated and then the 6-deoxytetracycline in the pure state from these derivatives in a per se known Way wins. 309851/1168 Process according to claims 1-3 _3, characterized in that 6-deoxytetracycline is used as 3-hydroxy-2-naphthelate, methylene-5,5'-disalycilate, molecular complex with Ν, Ν'-dibenzylethylene diamine, N, N ' Dibenzylethylenediimine or sodium hexametaphosphate isolated. 309851/1168