Antibiotic a-4696

POLAND PATENT DESCRIPTION OF THE REPUBLIC

PEOPLE __

Additional patent to patent no. MKP Cl2d 9/14

<img file="PL88977B1_D0001.tif" />

Reported: February 23, 72 (P. 153634) Cl.² C12D 9/14

OFFICE

PATENT

PRL

Priority: 25/02/71 United

States

America

The application was announced: 30.05.73

Patent description published: 30.09.1977

READING ROOM

Patent Office I IzBczyospoimj LiUnoj

Inventor: Patent holder: Eli Lilly Company, Indianapolis (USA)

Production method of the new antibiotic A-4696

The subject of the invention is a method of producing a new antibiotic, designated as A-4696, and pharmaceutically acceptable addition salts of this antibiotic.

Teeth deterioration and gum disease are serious health problems. Observations show that dental calculus is the cause of tooth decay and periodontal defects, or both at the same time. There is a need for prophylactic prevention of this type of scaling or for keeping deposits below the level at which toxic reactions occur. Antibiotics that inhibit the growth of sediment-forming microorganisms have been improved, but there is still a need for more effective agents to prevent and treat tooth and gum disease.

The main task of breeding is more efficient production of animal protein for food purposes. A number of feed additives, including numerous antibiotics, effectively affect the weight gain of chickens and pigs. There is a constant demand for improved, safe and economical feed additives that increase animal weight gain and feed efficiency. The discovery of new factors that meet this goal represents real progress in the field of breeding slaughter animals.

The method of producing the new antibiotic A-4696 and its pharmaceutically acceptable addition salts is by cultivating the microorganism Actinoplanes sp. strain ATCC 23 342 in liquid medium, containing sources of available carbon, nitrogen and inorganic salts under conditions of aerobic fermentation of the subsurface, until a significant part of the antibiotic activity transferred from the cultured microorganism to the medium, isolation of the obtained antibiotic and possible transformation into a pharmaceutically acceptable and salt.

Antibiotic A-4696 is isolated from the medium by filtration and separation by adsorption on the activated adsorbent and elution with acidic solvent, followed by¹⁵ is formed into a pharmaceutically acceptable salt. It is particularly advantageous to convert the obtained antibiotic A-4696 into the hydrochloride or sulfate solution in aqueous methanol and precipitate the addition of crystalline salt acetone, which²⁰ the hand is filtered off.

Antibiotic A-4696 exhibits antibacterial and growth promoting activity.

The new antibiotic obtained by the method of the invention is a basic compound that forms salts with the corresponding acids. The data presented below refers to the antibiotic A-4696 in the form of the hydrochloride. Isolation and characterization of the antibiotic A-4696 as hydrochloride is convenient, although in a known manner can

977

977 also make other pharmaceutically acceptable salts of this antibiotic.

• Antibiotic hydrochloride A-4696 is a white crystalline body with a melting point above 220 ° C. It is soluble in water and insoluble in solvents such as methanol, acetone, ether, chloroform, pyridine, benzene, aliphatic hydrocarbons and the like. It is very stable in solutions with a pH value in the range 1.0-10.0, at a temperature up to 27 ° C.

Potentiometric titration curve of the antibiotic hydrochloride A-4696 in water or a mixture of dimethylformamide with water in a ratio of 2: 4 has in the pH range^e6.9-13.0 shape similar to a straight line with an inclination of about 0.14. The elemental composition of the A-4696 hydrochloride, calculated as the average of Miku microamalizations, is as follows: C — 51, ^ 3 °%, H — 5.79%, N — 5.46%, Ό — 30, ^ 96%, Cl— 6.72%. The apparent molecular weight, determined by the method of measuring osmotic pressure, is 1153. Specific rotation of the antibiotic hydrochloride A-4696 [a]_D²⁵= —42.3 / c = 1, H ^ O /. The absorption spectrum of antibiotic A-4696 hydrochloride in ultraviolet shows in acid and neutral solutions a single maximum at 276ηημ and the extinction coefficient

1%

E, _ = 45. In alkaline solutions, the maximum cm of absorption shifts to 3'00 πιμ, and the extinction coefficient is E

1% cm

65. The infrared spectrum of antibiotic A-4696 hydrochloride in mineral oil shown in Fig. 1 shows in the range of 2 to 15 maxima at 3.0, 5.8, 5.9, 6.03, 6.15, 4.2 ", 6 , 63, 6.85, 7.27, 7.75, 8.1, 8.25, 8.9, 9.4, 9 # and 18, Ιμ, Rf values ​​of the antibiotic hydrochloride A-4696 in Whatman paper chromatography No. 1 is summarized in Table 1. Oiromatograms were biologically induced using BaciRus subtHis as the test strain.

Chemical composition of chromatographic systems:

1. Water saturated with butanol + 1% p-toluenesulfonic acid

2. Water saturated with metytolebutyl 5 ketone + 1% p-toluenesulfonic acid

3. Water saturated with methyl isobutyl ketone + 1% p-toluenesulfonic acid + 1% piperidine

4. Water: methanol: acetone / 12: 3 * 1 /, Doio solution is carried out with NH4OH to a pH value of 10.5, and then to a pH value of 7.5.

5. Methanol: 0.1 n HC1 / 3: 1 /

6. Methylisobutyl ketone solution (1%) and NH4OH / 0.5% / in water

7. Sodium chloride solution (7%) and 2.5% methylisobutyl ketone solution in water.

8. 10% solution of propanol in water

9. Butanol: ethanol: water / 150: 15 * 13.5 /

10. Propanol: pyridine: acetic acid: water / 15: 10: 3: 12 /

11. Water: ethanol: acetic acid / 70: 24: 6 /

Pharmaceutical addition salts of the A-4696 antibiotic are acid salts ²⁵ inorganic such as hydrochloric, hydrobromic, sulfuric, phosphoric etc. and with organic acids such as citric, tartaric, maleic, p-toluenesulfonic, salicylic, fumaric, acetic, propionic etc. Antibiotic salts with ³⁰ acids can be produced, for example, by acidifying the antibiotic with the appropriate acid in the form of the free base and by treating the solution with ten times the volume of acetone. In some cases, salts may be prepared by exchange³5 ions on a column with suitable resin or other methods used to make salts of antibiotics.

The new antibiotic is produced according to the method of the invention by the newly discovered Actinoplanes strain, grown in an appropriate medium under aerobic conditions. An antibiotic can be isolated and purified by various methods known and used. Degree of purification₄5 the antibiotic may be lower when used as a feed additive than when used for medicinal purposes.

Table 1

Paper chromatography of the antibiotic hydrochloride A-4696

<td>System</td><td></td><td>Microorganism, used in the process according to</td>

<td>chromatography</td><td>Rf values ​​* /</td><td>of the invention for the production of antibiotic A-4696, &lt;</td>

<td></td><td></td><td>50 identified as a strain of the Actinoplanes species</td>

<td> 1</td><td> 0,88</td><td>the Actinoplanaceae family. Actinoplanaceae are new</td>

<td> 2</td><td> 9,72</td><td>a family of microorganisms belonging to the order Actl-</td>

<td> 3</td><td> 0,89</td><td>nomycetales, which was first described by Dr. John '&lt;</td>

<td> 4</td><td> 0,59</td><td>N. Oouch, Jour. Elisha Mitchell Sci. Coc., 85,</td>

<td> 5</td><td> 9,35</td><td>55 315-318 / 1949 / and 88, 87-92 / 1-959 /, Trans. New Yo'Tk</td>

<td> 6</td><td> 0,77 ;</td><td>Aead. Sci., 16, 315-318 / 1954 /, Jour, Elisha Mitchell '</td>

<td> 7</td><td> 0,89</td><td>Sci. Soc., 71, 148-155 and 460/1955 /, Bergeyls Manual</td>

<td> 8 ;</td><td> 9,74 .</td><td>of Determinative Bacterioiogy, 7th Edition, 825-829 ¹</td>

<td> 9</td><td> 0,87</td><td>/ 1957 / and Jour. Elisha Mitchell Sci, Soc., 79,</td>

<td> 19</td><td> 9,59</td><td> 60 53—79 /1963/.</td>

<td> 11</td><td> 9,77</td><td>The Actinoplanes sp. Strain, used to produce</td>

0 / The ratio of the antibiotic path, calculated from the point Mmiesiem to the distance from the front of the system · from this point.

can You ^ Cidture Ofllcction in Maiytmd and registered as ATCC 33 342, and is also achieved

0gramc2eń. The ton strain was extracted 'from the soil

977 in the Cascade mountains in Washington State. In the collection of Dr. John N. Couch from the University of North Carolina, it is marked with number 581.

The physical and breeding properties are given below. Actinoplanes sp. Strain ATCC 23 542: Color names are given according to the Ridgeway Color Standards and Nomenclature / 1012 / system.

Microscopic morphology and general culture characteristics of Actinoplanes sp. ATCC 23 342.

Nfflnroscopic morphology. Skimpy mycelium on pollen of Styrofoam / Liąuidamher / in water. Hyphae rDogdfpEżoie with septum diameter 02-1.5 μ. They fill pollen grains, spreading in the water at a distance approximately equal to the diameter of the grain. Sporangophores spread above the water surface. Sporangial stems usually single, sometimes branched; on one sporangiophore two, less often a larger number of sporangia. Shafts 1.0-1.5μ thick with partitions. Sporangia small diameter 4-11 μ almost spherical, sometimes spherical, usually with an irregular wall. Mature spores arranged on a sporangium in one or. more blurred scrolls. The sporangium rupture is caused by the swelling of the inter-spore substance, which causes the sporangia to expand and assume an almost smooth, spherical shape. Mobile spores with a diameter of 1.0-1-1 &gt;&gt; μ, spherical or almost spherical.

Characteristics of breeding on agar Czapeka / SA Waksman, The Actinomycetes, 1950 /. Good growth, approximately 8 cm in diameter within 8 weeks around the point culture. Central bulge, flattened smear - with slight unevenness Zinc orange mycelium, pale to slightly yellow agar. Sporangia rarely formed.

Culture characteristics on Czepek '* agar with peptone / 5 g peptone in place of 2 g NaNO * /. Growth similar to that observed on Czapek agar »» with more pronounced furrows and ridges. Sporangia do not form.

As previously mentioned, the antibiotic A-4626 is obtained; - from Actinoplanes ep. Culture, strain ATCC 23 342. Various media are used for culturing, however, due to the cost, efficiency and ease of isolation - the use of certain media is particularly advantageous for the antibiotic. .. So, for example, starch is one of the beneficial sources of carbohydrates, and soy flour is the preferred source of nitrogen. Molasses, glucose, dextrin, glycerin, etc. are also suitable sources of carbohydrates. Suitable nitrogen sources include mixtures of amino acids, peptones, etc.

As nutrient inorganic salts, you can use commercial salts, which include sodium, potassium, ammonium, calcium, phosphate, chloride, sulfate, acetate, carbonate ions, etc. A positive effect on the production of A-4W antibiotic - it has an addition to the medium of sources of growth factors such , such as soluble distilleries and yeast extracts. Necessary! the ingredients are trace elements. They are usually contained in other nutrient components.

The strain that produces antibiotic A-4696 develops over a relatively wide range of pH values, but it is preferable to bring the pre-culture medium to a pH value of 6.5-7.2. As with other Actinomycetes, the pH of the medium is gradually changed, increasing. at the end - fermentation period up to 7.0-7.8.

The conditions favorable for the production of the antibiotic A-4696 are the conditions of subsurface oxygen culture. Small amounts of the antibiotic can be made in a shaker flask; in order to produce large amounts, it is preferable to carry out sub-dry - culture in sterile tanks. The medium in sterile tanks can be inoculated with a spore suspension of mycelium, but due to the longer development time of the culture - in the case of spore suspension, it is beneficial because it enables inoculation - a vegetative form of culture. .

It is preferable to obtain vegetative culture by inoculating a small amount of the medium with spores or mycelium of the microorganism and then transferring the young, active culture to a large tank. The same media can be used for plating preparation as for large-scale fermentation. It is also possible to use other media.

Antibiotic A-4696 strain Aetinopanes ATCC 23342 develops at 20-4g ° C. Naóótten &amp; Rivnejisse production of anti-alcohol is observed at a temperature of about 36 ° C.

Under aerobic culture conditions, sterile air is blown through the medium, in an amount of 0.1 to 0 volumes per volume unit of medium per minute. The highest performance of an antibiotic is achieved by blowing 0.5 volumes of air per unit volume of medium per minute.

The build up of A-4696 antibiotic concentration in the medium can be monitored by determining the antibiotic activity of the samples taken relative to the test organisms. A suitable test organism is Baeillus subtil », Assays (b-ol ^ generic are carried out using the standard method - bidimetric method, cylinder-plate method or method-discs - paper on agar plates.

Usually, the maximum concentration of the antibiotic, both in shaker flasks and in tanks, occurs after 4-6 days of breeding,

Antibiotic A-4696 can be isolated from the culture by adsorption or extraction. Adsorption methods are more beneficial because they avoid handling large amounts of solvents.

After fermentation, both wort and mycelium exhibit antibiotic activity. The mycelium is filtered off, the filter cake is washed with water and then broken in water adjusted to pH 105 with NaOH, stirred vigorously for 30 minutes and filtered again. The combined filtrates and rinsing water are filtered through an adsorbent bed such as activated carbon, acid activated alumina, polyamide resin, cellulose, silica gel, etc. luh adds one of the above adsorbents to roz88887

8 of the mixture, thoroughly mixed for 20 —Θ minutes and then drained. The crude antibiotic A-4696 is recovered from the adsorbent by elution.

The crude antibiotic A-4696 is purified by converting it into the addition salt with picric acid, which in turn into the hydrochloride, lulb also by adsorption and elution on a suitable adsorbent or ion exchange resin. ZM polyamide resin is a suitable adsorbent for this purpose. Woelm, 'Eschwege, West Germany, acidic alumina, neutral alumina, basic alumina, silica gel, Amberlite XAD-2 / Rohm - and HAAS, Philadelphia /, Amberlite XAD-4 / Rohm and Haas, Philadelphia /, Dowex 50 / H + / / Dow Chemical, Midland, -Michigan.

The new antibiotic obtained by the method of the invention inhibits the growth of many pathogenic microorganisms in relation to humans, animals and plants. Table 2 summarizes the minimum concentrations of A-4696 antibiotic hydrochloride, which inhibit the growth of examples of microorganisms. Minimal inhibitory concentrations / MIC / were determined by the dilution method on agar / adix shortcut / or in broth / abbreviation bd / and expressed in micrograms per milliliter Ąg / ml /.

In the agar dilution method, the test organism is seeded in 'agar' plates containing antibiotic A-4696 hydrochloride at various concentrations. Plates are incubated at 37 ° C for 48 hours. The lowest concentration of antibiotic is used, which inhibits the growth of the test strain.

In the broth dilution method, a series of tubes with the broth containing the hydrochloride, antibiotic A-4696 in various concentrations are inoculated, and incubation is carried out at 37 ° C for 24 hours. The MIC is the lowest concentration of the antibiotic at which no growth of the organism is observed in the tube.

Table 2

<td>Test organism</td><td>Minimal inhibitory concentration / pg / ml /</td>

<td>Staphylococcus aureus 3055</td><td>125 ad</td>

<td>Bacillus subtilis</td><td>6.25 na 0.78 ad</td>

<td>Mycobacterium- avium</td><td>0.4 ad</td>

<td>Streptococcus faecalis</td><td>3.12 ad</td>

<td>Trichophyton mentagrophytes</td><td>0.2 ad</td>

<td>Vibrio cola</td><td>12.5 na</td>

<td>Mycoplasma gallisepticum</td><td>50.0 na</td>

<td>Staphylococcus auTeus</td><td>10.0 na</td>

<td>/ resistant to penicillin / Staphylococcus aureus</td><td>5.0 bd</td>

<td>/ resistant to methicillin / Diplococcus pneumoniae</td><td>3.12 na</td>

<td>Clostridium perfringens</td><td>1.25 na</td>

<td>Clostridium tetani</td><td>2.5 na</td>

<td>Corynebacterium gravis</td><td>1.25 na</td>

<td>Lactobacillus casei</td><td>&gt; 100 ad</td>

<td>Leuconostoc citrovorum</td><td>&gt; 100 ad</td>

<td>Escherichia coli</td><td>&gt; 100 ad</td>

dc table 2

<td>Proteus sp.</td><td>. -&gt; 100 ad</td>

<td>Pseudomonas sp.</td><td>&gt; 100 ad</td>

<td>Salmonella sp.</td><td>&gt; 1 TO AD</td>

<td>Vibrio metschnikovii</td><td>&gt; 100 ad</td>

<td>Saccharomyces pastorianus</td><td>&gt; 100 ad</td>

<td>Candida albicans</td><td>&gt; 100 - ad</td>

Antibiotic hydrochloride A-4696, introduced subcutaneously in mice, has activity in vivo against a number of pathogenic organisms. For example ED₅o / dose giving 50® · o15 protect test animals / with two injections is ·: in relation to Staphykcoccuis - aureus 9.2 mg / kg, in relation to Streptococcus pyógeńes 0.68 mg / kg and in relation to Diplococcus pneumonies 0.76 mg / kg.

²⁰ The new antibiotic obtained by the method of the invention also effectively inhibits the growth of microorganisms contributing to the development of periodontal disease and tooth decay. For example - a solution - antibiotic hydrochloride A-4696

2⁵ exhibits activity against stone-forming organisms in a test carried out as follows: tubes with nutrient broth containing 5® · sucrose are inoculated with caries-causing microorganisms. For probation³⁰ The rods are introduced into glass rods and incubated at 37 ° C overnight. A precipitate forms on the surface of the rods, consisting of bacterial cells and dextran. The sticks are transferred to solutions - A-4696 α-4696 hydrochloride at various concentrations - and left in them within 5, 19 and 15 minutes. After the assumed time has elapsed, the sticks are washed with sterile, inverted water and immersed in uninoculated sucrose-containing medium. Incubation is carried out at 37 ° C overnight, then bromothymol blue is added to each medium. Growth - of the micro-organism is observed as a change in color from blue to yellow, caused by acidification of the medium by the developing micro-organism.

A 0.046® antibiotic hydrochloride solution A-4696 turned out to be active in relation to the unidentified caries-forming Streptococcus sp. Strain 5 przy, when contacted with a sedimented rod within 10 minutes. In the event of contact lasting 5 minutes, the growth of the microorganism was inhibited by a 0.1® · solution. In addition, the A-4696 hydrochloride solution inhibits the growth of the stone-forming microorganism - Odontomyces viscosus, at a concentration of 0.25 g / ml as determined by the dilution method in the broth.

The admixture of antibiotic A-4696 or its addition salt to a suitable toothpaste, powder, gel, etc., to a suitable rinse aid or other oral disinfectant can be an effective way to inhibit tooth decay and periodontal disease. Alternatively, no, a solution of the antibiotic A-4696 or its ad salt can be applied to the surface of the gums and teeth using a suitable tampon.

Antibiotic A-4696 is also an effective means of stimulating animal growth. Antibiotic hydrochloride A-4696, added to the basic food ration in the amount of 45.4 g / t, causes increased weight gain of chickens and increased nutrition efficiency; Table 3 presents data obtained in four separate experiments carried out to determine the effect of the antibiotic hydrochloride A-4696 on the weight of chickens. The tests were carried out in a culture carried out at a temperature of about 25 ° C. Antibiotic dosing was started in individual trials from day one to day five of animal life. The dosing time was 10-17 days. The tests were carried out with 24/7 lighting and unlimited nutrition.

The effect of the addition of antibiotic A-4696 hydrochloride to weaned piglets was also investigated. Before the start of the experiment, 4 to 5 week old piglets were kept on a diet without the addition of antibiotics for about a week. Piglets were weighed and divided into experimental groups depending on weight and litter. They were kept in closed rooms, properly heated and ventilated, providing them with draft¹⁰ the whole experience of food and water in unlimited quantities. In each trial, piglets were divided into two groups, one of which received the 'basic ration without the addition of antibiotics, and the other received the same basic ration with the addition of¹⁵ antibiotic hydrochloride A-4696. Table 4 presents the average weight gain and feeding efficiency of piglets fed with feed with the addition of antibiotic A-4696 hydrochloride and comparative animals.

Table 3

Weight gain and nutrition efficiency when feeding young chickens with feed with the addition of antibiotic hydrochloride A-4696

<td>Food ration</td><td>Antibiotic hydrochloride A-4696 g / t feed</td><td>Number of feeding days</td><td>number fed animals</td><td>Average increase weight g / animal</td><td>Efficiency of feed conversion weight feed increase weight animal</td>

<td>Basic reason</td><td> 0</td><td> 10</td><td> 40</td><td> 147</td><td> 1,53</td>

<td>Basic ration + antibiotic dihydrochloride A-4696</td><td> 45,4</td><td> 10</td><td> 40</td><td> 146</td><td> 1,47 *'</td>

<td>, Basic reason</td><td> 0</td><td> 10</td><td> 20</td><td> 146</td><td> 1,50</td>

<td>Basic ration + antibiotic hydrochloride A-4696</td><td> 45,4</td><td> 10</td><td> 20</td><td> 153</td><td> 1,45</td>

<td>Basic reason</td><td> ' 0</td><td> 17</td><td> 80</td><td> 295</td><td> 1,56</td>

<td>Basic ration + antibiotic hydrochloride A-4696</td><td> 45,4</td><td> 17</td><td> 80</td><td> 320</td><td> 1,46</td>

<td>Basic reason</td><td> 0</td><td> 17</td><td> 80</td><td> 300</td><td> 1,51</td>

<td>Basic ration + antibiotic hydrochloride A-4696</td><td> 45,4</td><td> 17</td><td> 80</td><td> 318</td><td> 1,43</td>

<td>Basic reason</td><td> 0</td><td> 17</td><td> 80</td><td> 287</td><td> 1,58</td>

<td>Basic ration + antibiotic hydrochloride A-4696</td><td> 45,4</td><td> 17 1</td><td> 80</td><td> 307</td><td> 1,47 1</td>

Table 4

Weight gain and nutrition efficiency when feeding weaned piglets with feed with the addition of antibiotic hydrochloride A-4696

<td>Food ration</td><td>Antibiotic hydrochloride A-4696 g / vol</td><td>Number of piglets in the experiment combined</td><td>Number of days of experience</td><td>Average daily increase weight / kg</td><td>Efficiency of feed conversion feed weight / weight gain animal</td>

<td>Basic reason</td><td> 0</td><td> 11</td><td> 28</td><td> 0,31</td><td> 1,91</td>

<td>Basic ration + antibiotic hydrochloride A-4696</td><td> 20</td><td> 11</td><td> 28</td><td> 0,43</td><td> 1,77</td>

<td>Basic reason</td><td> 0</td><td> 11</td><td> 34</td><td> 0,50</td><td> 2,41</td>

<td>Basic ration + antibiotic hydrochloride A-4696</td><td> 50</td><td> 11</td><td> 34</td><td> 0,55</td><td> 2,17</td>

Loam

Toxicity of the A-4696 antibiotic hydrochloride, determined 'on mice on intraperitoneal injection, expressed as 1&gt;₅0, is 2460 mg / kg.

The method according to the invention is illustrated by the following examples:

Example I. Grzybnia Actinopkanes sp. ATOG 22342, an agar bevel is grafted with the composition:

'977 cooked flour 160 g yeast 2&gt; 5 g κ * ηρο₄ 1.0 g dried soluble distilled wastes 5.0 g mineral salt solution according to

Cap * / 5, Q mi agar 25 g

Mineral salt set according to · Czapek: KCI - 190 g

MgSO₄.7H / O 100 g

FeSO₄.7H / O / dissolved in 2 ml concentrated HCl / 2 g demineralized water 1 liter. Incubation. carried out at 30^QC, within 0 days. In the medium of the above composition, the culture usually does not sporulate; in order to increase the number of potential growth centers, it is therefore necessary to macerate the mycelium with a flattened and pointed seed needle. The macerated mycelium is poured with distilled water and · with a sterile Spo Rządza stick · suspension.

The suspension obtained in the manner described inoculates a thousand 'ml sterile vegetative medium composed of:

<td>glucose</td><td>5.0 g</td>

<td>dextrin</td><td>20.0 g</td>

<td>soy flour</td><td>15.0 g</td>

<td>yeast extract</td><td>2.5 g</td>

<td>calcium carbonate</td><td>1.0 g</td>

<td>tap water</td><td>1 liter</td>

<td>The inoculated medium is incubated in</td><td>distemper-</td>

30 ° C, for 48 hours on a rotary shaker rotated at 250 revolutions per mdhuta.

; Ten mRiHtrami of the incubated vegetative medium are inoculated with 100 ml sterile medium with the same composition. The inoculated medium is incubated at 30 ° C for 24 hours on a rotary shaker rotated at 250 rpm. 0.4 ml of this medium is inoculated with 100 ml of production medium in a 500 ml Erlenmayer flask, sterilized at 120 ° C for 30 minutes.

The composition of the production medium

<td>dextrose</td><td> 1,0·/·</td>

<td>dextrin</td><td> 3,0·/·</td>

<td>peptone</td><td>lM / ·</td>

<td>soy flour</td><td> 0,5·/·</td>

<td>MgSO4.7H₂ABOUT</td><td> 1,5’/·</td>

<td>sugar beet molasses</td><td> 1,5’/·</td>

<td>corn soak extract</td><td> 0,5°/·</td>

<td>betaine</td><td> 0,1/·</td>

<td>KHPO4</td><td> 0,05’/·</td>

<td>demineralized water</td><td>25 ml</td>

Before sterilization, the pH of the medium is adjusted to 5 with 5N NaOH. - After sterilization, the pH value is about 5.2.

Production fermentation is carried out on rotary scrapers rotated at a rate of 250 revolutions per minute. At the end of the fermentation cycle, the pH of the medium is about 7.2. 25 liters · of production medium with the above composition, with the addition of Oow Corning antifoaming agent in the amount of 0.02%, is sterilized in an autoclave at 120 ° C for 30 minutes and introduced into a fermentation tank with a capacity of 45 liters. Fermentation is carried out at 39 ° C for 4 days. During fermentation, sterile air is passed through the medium in an amount of about half the volume of medium per minute. The mixing of air with the nutrient medium is achieved by means of a rotor mixer of the right size, rotated at the appropriate speed. During fermentation, the pH of the medium increases from an initial value of about 6.9 to about 7.2.

All the fermentation broth A-4696 described above is filtered with the addition of commercial, filter aid. The mycelial cake is washed with 32 liters of water, and on²⁵ it then suspends 32 liters of water in the next portion and the pH of the suspension is raised to 10.5 with 5N NaOH. The suspension is stirred for minutes and filtered. The filtrate and washers are combined and adjusted to pH = 4 with HgSO4, then passed through a column filled with 1 kg of activated carbon ZPitaburg, 12X40 /. The column is washed with water until a colorless leakage occurs. The antibiotic A-4696 adsorbed on the column is eluted with two H &lt;tra³⁵ m and 1% H2SO4 solution in a 1: 1 mixture of acetone and water. The sulfate ions are removed by quenching the eluate with a saturated Ba / OH / solution₂ and filtering off the precipitated BaSO4. · The filtrate containing the antibiotic A-4696 is evaporated under reduced pressure⁴⁰ to give approximately 89 g dry residue. About 89 g of the raw antibiotic · A-4696 dissolves in 5 liters of water and adds 599 g of activated carbon ZOarco G-69 Atlas Chemical,₄₅ Wilmington, OeLZ The suspension is stirred for an hour and filtered. The filtrate is discarded, and the carbon filter cake with absorbed antibiotic A-4696 is washed successively with 1 liter of water and 1 liter of 6.0Śn aqueous HCl, discarding the filtrate.

5₀ The washed cake is eluted by mixing it for 39 minutes with 590 ml of an aqueous acetone HCl solution / 9.95n HO: acetone solution = 7: 4 /. The mixture is filtered while maintaining the filtrate. Identical. the method is repeated four times more,_in keeping the filtrates. Five portions of the filtrate containing the antibiotic A-4696 are combined with each other and then evaporated to a volume of about 100 ml under reduced pressure and treated successively with 200 ml methanol and 2 liters of acetone. It turns off₆₀ crude hydrochloride of the antibiotic A-4696, which after draining and drying weighs 60.9 g.

g crude antibiotic hydrochloride A-4696 is dissolved in 20 ml of water. The solution is passed through a vein previously washed with water₆₅ polyamide wica / M. Woelm, Eschwege, NR / - in a glass column about 7X60 cm. The column is washed with water at a rate of about -10 ml per minute, the fractions showing the usual antibiotic activity are combined and evaporated under reduced pressure. The residue is dissolved in a mixture of 25 ml water and 50 ml methanol. The aqueous methanolic solution of the antibiotic A-4696 hydrochloride is acidified with 5n HO to pH = 2.0, then add about 1.5 liters of acetone to it. The precipitated hydrochloride antibiotic A-4696 is filtered off.

The residue on the filter dissolves in the least amount of water. Ethanol is added to the solution in a volume twice the volume of water and the whole is heated to a temperature of about 60 ° C, and then cooled, causing crystallization of the hydrochloride of antibiotic A-4696. The crystals are filtered off and dried, obtaining about 9 g of product.

{Example. 2.3 g raw antibiotic

A-4696, prepared as described in Example 1, is dissolved in 20 ml of a 50% aqueous methanol solution and passed through a 2X4 ohm column filled with rinsed alumina (Alcoa) acid, after which the column is washed immediately with methanol. The antibiotic is eluted from the column with 50% methanol, collecting fractions showing biological activity. The combined fractions are concentrated to a volume of about 100 ml and the addition of acetone precipitates the antibiotic A-4696. The precipitate is filtered off and dried in vacuo to give. about 930 g of pure, crystalline antibiotic A-4696.

The acid-washed alumina is prepared as follows: activated Alcoa F-20 alumina is stirred for 6 hours in water, acidified with HJSO4, down to pH = 3, maintaining the pH at 'this level. The alumina is then filtered off, washed with 50% aqueous methanol and dried at 100 ° C.

Example III. 500 mg of the raw antibiotic A-4696, prepared as described in Example 1, are dissolved in 25 ml of water. 25 ml of saturated aqueous picric acid solution are added to the solution with stirring. The whole is kept overnight at 5 ° C, then the precipitate is centrifuged and dried. Yes . the resulting precipitate is dissolved in 25 ml of me88 · toluene, the pH of the solution is adjusted to 1.5 with HCl and 500 ml of diethyl ether is added, precipitating the hydrochloride of antibiotic A-4696. The precipitated hydrochloride is centrifuged. washed with diethyl ether and dried under reduced pressure to give about 306 mg of a white crystalline product.

Example IV 2 g of crystalline antibiotic A-4696 hydrochloride, prepared as described in Example 1, are dissolved in 300 ml of water, the pH of the solution is adjusted to 7.5 with NaOH and 30 g of Darco carbon are added. G-60. The mixture is stirred for 30 minutes, filtered with the addition of commercial filter aid, and the filter cake is washed successively with 300 ml water and 306 ml 0.06 n

The antibiotic is rinsed out of the filter cake, adding 500 ml of a mixture of 70 *% 0.05 n HJSO4 and 30% acetone, stirring - all within

thirty minutes and draining the coal. The filtrate is evaporated to a volume of about 10 ml, about 20 ml of methanol is added and the whole is poured into 600 ml of acetone, which causes precipitation of the antibiotic sulfate A-4696. The precipitate is filtered off, washed with acetone. and dried under reduced pressure to obtain 662- mg of a white crystalline product.