Tuamarina c and method thereof

 

(57) Abstract:

Derived thimerisol, which shows antibacterial and antimycoplasmal properties derived from microorganisms of the genus Alteromonas and is called "Tamarina C". 1 C.p. f-crystals, 3 tables.

The invention relates to certain new derivative thimerisol, provides a way of obtaining them and relates to methods and compositions for use as antibacterial agents.

Tuamarina described, for example, in European patent application N 512824, which was published prior to the filing of this application, but later than the priority date. It can be represented by the following formula:

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It is produced by fermentation of microorganisms of the species Alteromonas, in particular Alteromonas rava strain SANK 73390. Found two derivatives thimerisol, which show a similar type of antibacterial activity, the original tuamarina.

Organisms of the genus Alteromonas can be extracted from sea water and, as has been shown, some of them are capable of producing compounds that are potentially suitable for use in therapeutic purposes. For example, a compound known under the name biakaveron was derived from a single species of the genus Alte CLASS="ptx2">

With regard to the structure thimerisol, there are several antibiotics that have a similar structure, which can be divided into four groups.

The first group includes pseudomonaceae acid, first isolated from microorganisms Pseudomonas spp. They include Pseudomonas acid A [produced by species of Pseudomonas fluorescens, is described in J. Chem. Soc. Perkin Trans, 1, 294 (1997)] Pseudomonas acid B [ibid, 318 (1977)] Pseudomonas acid C [ibid, 2827 (1982)] and Pseudomonas acid D [ibid, 2655 (1983)] Pseudomona acid A is available under the name "baktroban" (trademark firm Beecham) in the form of 2% skin ointment for antibacterial use. However, all of these are known from the technical field of the compounds have weaker antibacterial activity than derivatives thimerisol of the present invention.

The second group of substances that have similarities in structure to the compounds of the present invention, are antibiotics such as colomycin (Helv. Chim, Acta, vol. 42, 563 (1959)] pyrrhotite [J. Am. Shem. Soc. vol. 77, 2861 (1955)] thiolutin [Angew. Chem, Bd. 66, 745 (1954)] laureation [J. Am. Chem. Soc. vol. 74, 6304 (1952)], and others. These antibiotics are usually produced actinomycetoma and have in its composition structurai the chromophore. Xen is 75).

There have been numerous studies of derivatives of these two groups of compounds, but we don't have any information on compounds having a molecular structure similar to the structure of timorinab, or with similar properties.

The third group of compounds described in publications such as patent application Japan Kokai NN 52-102279, 54-12375, 54-90179, 54-103871 and 54-125672. It consists of derivatives of Pseudomonas acid in which the terminal carboxyl group is replaced by an amide group. These compounds do not exhibit comparable antibacterial activity and does not possess a wide spectrum of antibacterial activity. In fact, these compounds tend to have lower antibacterial activity than the original pseudomona acid.

The fourth group includes a connection similar to tuomarila that it is a physiological product of vital activity of marine bacteria [Abstracts 200 Annual conference of the American chemical society (August 26-31, 1990), part 2, N 139] However, the heterocyclic group attached to the terminal carboxyl group of this compound is 2-oxo-3-piperideine group. As was recently shown, it is about the essential connection is pseudomona acid A, and all tomaranai, i.e. himself tuamarina, tuamarina B and Tamarina C, are significantly more potent antibacterial activity than pseudomona acid A.

The aim of the present invention is the provision of some new derivatives thimerisol.

The next and more specific objective of the present invention is the provision of compounds which have excellent antibacterial and antimycoplasmal activity.

Other objectives and advantages will become clear upon consideration of the description.

In a General sense, the present invention provides two new derivatives thimerisol, one of which is S,S-dioxo derived and denoted as "Tamarina B", and is the subject of consideration in the application N 93053043/13 from which highlighted this, and the second is diethoxypropane and hereinafter referred to as "Tamarina C".

More specifically, the present invention provides a connection tuamarina C, which can be represented by the following formula:

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The invention also provides a method of obtaining thimerisol C, which involves the cultivation of producing tuamarina microart tuamarina C contains the number of asymmetric carbon atoms and a few multiple bonds. In particular, the possible isomerization ,b-unsaturated carbonyl fragment thimerisol. Thus, tuamarina can form various stereo and geometric isomers. Although all of them are represented here one molecular formula, the present invention includes as an individual, isolated isomers and mixtures thereof, including racemates. Upon receipt of a mixture of isomers of the individual isomers may be obtained by conventional methods of splitting.

However, because tomaranai usually get by fermentation or chemical manipulation of the products of fermentation, they have a tendency to acquire standard optical configuration. Therefore, although there are other configurations, preferred is a natural configuration.

Tuamarina C can be characterized by the following physicochemical properties:

1. Character and appearance: yellow powder.

2. Molecular formula C30H44N2O8S2.

3. Molecular weight: 624 (according to FAB mass spectrometry).

4. Elemental analysis:

Calculated for C30H44N2O8S2+ H2O, C 56,05, H 7,21, N 4,36, S BECOMES 9.97.

is brazza in the form of tablets with potassium bromide: 3256, 3068, 2928, 2858, 1645, 1596, 1530, 1455, 1384, 1287, 1225, 1151, 1104, 1052, 974, 820, 712.

6. UV spectrum:maxnm ().

UV spectrum measured in methanol or methanol + hydrochloric acid: 388 (9600), 300 (2700), 215 (17000).

UV spectrum measured in methanol + sodium hydroxide: 386 (8600), 205 (49000).

7. Specific rotation of the plane of polarization:

[]2D5= -1,4(C 1.0, methanol).

8. Liquid chromatography high resolution:

Separation column: Senshu-Pak ODS H-2151 (column diameter 6 mm, length 150 mm, Senshu Scientific Co. Ltd.).

Solvent: about 40. aqueous acetonitrile.

The flow rate of eluent: 1.5 ml/min

Retention time: 11,3 minutes

9.1H-NMR spectrum: ( M. D.).

NMR spectrum (360 MHz), measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as an internal standard: of 0.91 (3H, doublet, J 6.9 Hz), of 0.95 (3H, doublet, J 6.3 Hz), 1.26 in (2H), 1,29 (2H), 1,30 (2H) and 1.51 (2H, multiplet), and 1.56 (2H, multiplet), and 1.63 (1H, multiplet), to 2.06 (2H), 2,08 (1H), 2,11 (3H, singlet), to 2.15 (1H), of 2.34 (2H, triplet, J and 7.3 Hz), of 2.56 (1H, broad doublet, J of 14.2 Hz), 3,18 (1H, broad multiplet), to 3.35 (1H), 3,49 (1H), to 3.58 (1H), 3,62 (1H), to 3.67 (1H), 4,01 (2H, triplet, J 6.6 Hz), 4,32 (1H, doublet, J 4.3 Hz), 4,55 (1H, broad multiplet), with 4.64 (1H, broad is ptx2">

10.13C-NMR spectroscopy: ( d m D.).

NMR spectrum (90 MHz), measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as an internal standard: 15,7 (Quartet), 18.6 (Quartet), 20,0 (Quartet), 24,9 (triplet), 25,2 (triplet), 28,1 (triplet), 28,3 (triplet), 28,4 (triplet), 32,1 (triplet), 34,6 (triplet), 42,0 (doublet), 42,5 (triplet), 43,1 (doublet), 63,0 (triplet), 64,0 (triplet), 68,1 (doublet), 69,2 (doublet) and 69.3 (doublet), 74,4 (doublet), 110,4 (doublet), 115,3 (singlet), 116,5 (doublet), uniforms, 127.6 (doublet), 133,6 (singlet), 133,9 (singlet), 134,2 (doublet), 157,9 (singlet), 165,7 (singlet), 167,9 (singlet), 171,8 (singlet).

11. The solubility. Soluble in alcohols, such as methanol, ethanol, propanol and butanol, and also dimethyl sulfoxide, dimethylformamide, chloroform, ethyl acetate, acetone and gatilova ether. Insoluble in hexane and water.

12. Thin-layer chromatography.

The value of Rf0,66.

Adsorbent: silica gel (Merck Co. Inc. Art. 5719).

Eluent: methylene chloride:methanol 85:15 by volume.

Tuamarina C can be obtained by culturing producing tuamarina organisms species Alteromonas and selection of the target thimerisol from the culture medium. Varieties thimerisol C with the required ant is irout target connection, or you can get them by modifying appropriately the compound isolated in the process of the above fermentation, or you can get them direct chemical synthesis.

In particular, it seems to us preferable to use as the microorganism species Alteromonas rava and especially newly allocated strain Alteromonas rava, to whom we gave the designation SANK 73390. Strain SANK 73390 is a marine organism, isolated from seawater collected off the coast of the Coin, Minamata, Shizuoka Prefecture, Japan, and the strain deposited in the Depository institution, Research Institute of fermentation, the Agency of industrial science and technology, Ministry of international trade and industry, Japan, 30 April 1991, the Deposit number FERM BP-3381, in accordance with the Budapest Treaty.

Taxonomic characteristics of Alteromonas rava strain SANK 73390 presented below.

1. Morphological characteristics.

Alteromonas rava strain SANK 73390 was cultivated at 23oC for 24 h on marine agar (Difco). Consistent microscopic observations showed that cells have Blockoban shape with a diameter of 0.8 to 1.0 μm and a length of 2.0 to 3.6 μm. This strain is gramo the>SANK 73390 was cultivated at 23oC for 24 h on marine agar (Difco). The obtained colonies are pale gray-green, opaque, flat, solid and have an annular shape.

Water-soluble pigment is not formed.

3. Physiological properties.

(1) Requirements for sea water: SANK 73390 requires for its growth in sea water.

(2) Oxidative-fermentative test (method Hugh Leifson [J. Bact. vol. 66, 2426, 1953] in an environment prepared with artificial sea water, with no effect on carbohydrate.

(3) Oxidase: +

(4) Catalase: +

(5) the Need for oxygen: aerobic.

(6) Recovery of nitrates: -

(7) Hydrolysis of starch: +

(8) Decomposition of agar: -

(9) Liquefaction of gelatin: +

(10) the Production diphosphopyridine nucleotidase (DNase): +

(11) Production of lipase: +

(12) Temperature for growth: poor growth at 4oC good growth in the range of 17 of the 26oC, no growth at 35oC.

(13) Requirements for growth factors: basic nutrient medium described in J. Bact. vol. 107, 268 (1971), SANK 73390, requires adding free from vitamins casamino-acid.

(14) Assimilation of carbon sources: the OS is Minov, casamino acid, with shaking culture (see tab. 1).

4. Chemotaxonomic characteristics.

(1) Mol. guanine and cytosine (the content of G+C) in DNA: 43,4% ( according to liquid chromatography high resolution).

(2) Quinone system: ubiquinone Q-8.

Taking into account the above taxonomic characteristics, Alteromonas rava strain SANK 73390 compared with the strains described in Bergey''s Manual Systematik of Bacteriology, Vol. 1 (1984), and with the strains described in the latest editions of International Journal of Bacteriology. Discovered that Alteromonas rava strain SANK 73390 to some extent similarities with Alteromonas citrea, which also is a marine organism. Were cultured and comparison of properties SANK 73390 and Alteromonas citrea, ATSS 29719 (standard strain).

In comparison with pale grayish-yellow SANK 73390, the colony was ATSS 29719 had a greenish-yellow color. SANK 73390 differs from Alteromonas citrea growth rate at 4oC and the ability to assimilate trehalose and sodium propionate as carbon sources. Therefore, the strain Alteromonas rava SANK 73390 is a new strain of a new species Alteromonas rava and different in its essential characteristics from the nearest analogue, deposited under the number of ATSS 29719.

Bring the spp. subject to change, both natural and artificial means. The above characteristics determine the strain Alteromonas rava in the form in which it was deposited, but not necessarily typical of other species of the genus Alteromonas or strains of Alteromonas rava, which have the ability to produce tuamarina or naturally occurring variants. All these strains included in the scope of claims of the present invention.

It is clear that SANK 73390 or any other strain, capable of producing tuamarina or one of its variants, may be subjected to subculturing, changed or modified by means of biotechnology with the aim of producing an organism with different characteristics. The only requirement to receive the body is the ability to produce the desired connection.

Such changes or modifications can take any desired shape or may, for example, depend on the conditions of cultivation. Strains can thus change while growing and be chosen in such a way as to obtain samples with properties such as increased growth rate, growth at higher or lower temperatures.

Biotech modificat echeveste or susceptibility to bacteriostat or their combination, to maintain cleanliness, or to provide for a clean culture, especially the elite crops.

Other characteristics that can be introduced through genetic manipulation, are any of those that are valid in the case of Alteromonas spp. For example, you can type plasmids encoding resistance to, or remove any natural plasmids. The greatest benefits provide those plasmids that have the ability to form the mutant strains growing organism. Plasmids can be obtained from any convenient source or form by means of genetic engineering, highlighting the natural plasmid Alteromonas and giving it the desired gene or genes from another source. Natural plasmids can also modify any other desired manner.

In order to get tuamarina C from the culture of a suitable microorganism, microorganisms need to be fermented in a suitable medium. Such environments typically well known from the technical field and are often used in the production of other enzymatic products.

Typically, this environment must contain any combination of carbon source, nitrogen source and one or more inorganic salts, which urate ingredients such which is necessary for growth of the microorganism.

Suitable carbon sources are glucose, fructose, maltose, sucrose, mannitol, glycerol, dextrin, oat flour, rye, corn starch, potatoes, corn flour, soybean flour, cottonseed oil, molasses, citric acid and tartaric acid, each of which can be used alone or in combination with one or more other carbon sources. A typical number is 1 to 10% wt./about. the amount of protection, although this value may vary depending on the needs and in accordance with the desired result.

Suitable nitrogen sources may be any substances which contain, for example, protein. Typical examples of nitrogen sources are organic sources of nitrogen derived from animals and plants, it can be extracts isolated from such natural sources as soy flour, bran, peanut flour, flour from the seeds of the cotton plant, a hydrolysate of casein, feramin, fish meal, corn extract, peptone, meat extract, yeast, yeast extract, malt extract, and inorganic nitrogen sources such as sodium nitrate, ammonium nitrate and ammonium sulfate. As if history is Aut 0.1 to 6 wt%./about. the amount of environment.

Suitable nutrient inorganic salts are those that provide micronutrients, as well as the major salt components. Salt should preferably contain ions such as ions of sodium, potassium, ammonium, calcium, magnesium, iron, phosphate, sulfate, chloride and carbonate ions. May also contain traces of these metals (or elements) as cobalt, manganese or strontium, or salt, is able to give ions such as bromide, fluoride, borate and silicate ions.

Alteromonas rava occurs naturally in sea water, therefore, unless otherwise stated, conditions for growing these organisms should match the conditions of the marine environment. Thus, the ions are in the form of traces contained in sea water, mainly included in any medium used for the cultivation of Alteromonas. In particular, the microorganisms should preferably be grown in the presence of sea water, artificial sea water, or components, corresponding to the composition of sea water.

If the microorganism fermentation in the form of liquid culture, it is desirable to use defoamers, such as silicone oil or vegetable oil or Draginja thimerisol pH for cultivation should preferably be maintained at 5.0 to 8.0 pH, although the only requirement is that the pH does not hinder the growth of the microorganism or harmful and irreversible did not affect the yield of the target product. To stop the fermentation, you can add an excess of acid or alkali. Alteromonas rava SANK 73390, as a rule, grows at a temperature of 4 32oC, grows well at a temperature of 17 of the 26oC. you Can operate at other temperatures that do not fall within the specified range, if the selected strain able to grow at lower or higher temperatures. To obtain thimerisol C temperature interval should preferably be 20 26oC.

Tuamarina C is ideally obtained by aerobic cultivation, and you can apply any technique aerobic cultivation, for example, solid culture, shake culture or mixed air culture.

If the cultivation is carried out in small scale, it is usually preferable shake a culture that fermentation for several days at a temperature of 20 of the 26oC.

To start the enzymatic growing preferable to use the method using the original inoculum cooked in one the ka of carbon and nitrogen source. The flask seeds shaken out in a cooled incubator at 23oC for 1 to 3 days or until until the start of significant growth. The obtained seed culture can then be used to seed the second seed culture or producing culture. If re-seeding, it can be done in the usual way, and some to use for inoculation producenti environment. The environment in which you place the seed material, shaken at the appropriate temperature for the required amount of time, for example from 1 to 3 days, or until such time until the start of the appreciable formation of the product as described above. After the end of the incubation period the contents of the flask separated by centrifugation or filtration.

If the cultivation is carried out in large scale, it is preferable to use culture, stir air into the fermenter. In this case, the nutrient medium can be prepared directly in the fermenter. After sterilization at 125oC the resulting medium is cooled and inoculated with AMF inoculum, pre-prepared in a sterile environment. The cultivation is conducted under stirring and aeration at a temperature of 20 of the 26oC. This method Predoctoral, you can from time to time be controlled by, for example, liquid chromatography high resolution. Typically, the amount of biogas produced thimerisol C reaches its maximum in the time interval of 19 200 h, and the amount of biogas produced thimerisol reaches its maximum in the time interval 19 96 hours

After a suitable time growing tuamarina C can be extracted and cleaned by any known means. For example, either the amount of C remaining in the broth culture, can be obtained by filtering the solids, for example, by using as the material for filtration diatomaceous earth, or by centrifugation with subsequent extraction of the liquid above the sediment and cleaned in accordance with the physico-chemical properties thimerisol C. for Example, tuamarina C, located in the filtrate or residue after centrifugation, can be extracted immiscible with water, organic solvents such as ethyl acetate, chloroform, ethylene chloride, methylene chloride or a mixture of these solvents at neutral or acidic conditions, and then to clear.

Alternatively the adsorbent is possible to use activated carbon or adsorbent resin, Takamasa can be removed after adsorption, flowing liquid containing tuamarina C, through a thin layer of adsorbent, tuamarina C can also be cleaned after adsorption by elution with a suitable solvent, e.g. aqueous methanol, aqueous acetone or a mixture of butanol/water.

Intracellular tuamarina C can be cleaned by extraction with a suitable solvent, for example, aqueous acetone or aqueous methanol, which preferably has a concentration of about 50 90. and removing the organic solvent, followed by extraction as described previously for the filtrate or liquid above the sediment.

Further purification thimerisol C can be implemented using well-known methods, namely:

adsorption column chromatography, using devices such as silica gel or magnesium-silica gel, for example, supplied to the market under the trade name "Florisil";

distribution column chromatography using such adsorbents as Sephadex LH-20 (trade name of product of the company Pharmacia);

liquid chromatography high resolution using speakers with normal or reversed phase.

As is well known from the field of technology, these methods of isolation and purification can assests is on the product.

Because tuamarina C has an antibacterial effect on gram-positive and gram-negative bacteria and Mycoplasma in animals (particularly humans, dogs, cats and rabbits), it can be a variety of ways depending on the nature of the infection to use for the treatment or prevention of bacterial or mycoplasmal infections.

In that case, if the connection of the present invention is to be used in therapeutic purposes, it can be assigned independently or in the form of pharmaceutical compositions that contain, besides the active compound, one or two standard diluent, carrier, an excipient or auxiliary connections. The form of the composition, of course, will depend on the method of taking the medicine. For oral destination connection is mainly converted into the form of the drug in the form of powders, granules, tablets, capsules or syrups. For parenteral purposes it is mainly prepared in the form of injectable preparations (which may be intravenous, intramuscular or subcutaneous), drops, suppositories, ointments or liniments.

These forms of drugs can be prepared by a renowned SPO shall catalysts, modifiers, wetting, fragrances, perfumes, dispersing agents or materials to cover the shell. Although the dose may depend on the symptoms and age of the patient, nature and severity of the infection, as well as the method of prescribing, in the case of oral destination adult patient the compound of the present invention may typically be applied in the form of a daily dosage of 20 to 2000 mg of the Compound can be taken as a single dose or portions, for example two or three times a day.

Obtaining the compounds of the present invention is further illustrated by the following example which does not limit the present invention, and the biological activity of the compounds illustrated in the examples below the test.

Example. Getting thimerisol C growing in Tinqueux.

A) Culture.

One bevel marine agar (Difco), on which are planted and grown strain Alteromonas rava SANK 73390, placed in 10 ml of sterilized marine broth (Difco) to obtain a suspension of bacteria.

30-liter fermenter jug type containing 15 liters of the same marine broth is sterilized by heating, and then sow it all the amount of bacterial suspension and grown in accordance with what is 100 rpm, and then pick it up so that the dissolved oxygen concentration was 5 ppm.

In each of the 600-liter tankov placed in 300 liters of a nutrient medium having the following composition

Glucose 1,5

Bactopeptone (Difco) 1,5

Backdragging extract (Difco) and 0.2

NaCl 3,89

MgCl26H2O 2,52

Na2SO40,648

CACl22H2O 0,4767

KCl 0,11

Na2CO30,038

Citrate of iron 0.02

pH 7.6 to sterilization.

Tenki sterilized by heating, and then in each of them are sown in 3 l of culture and grown for 29 h at 23oC and the speed of air flow 150 l/min Initial stirring speed is 82 rpm, and then pick it up so that the dissolved oxygen concentration was 5 ppm.

B) Allocation.

To 700 l of the resulting solution with culture add a sufficient quantity of an aqueous solution of hydrochloric acid to bring the pH to 3. Then to the resulting mixture add 700 l of acetone and extracted the mixture with stirring for 1 h the extract Obtained, in turn, extracted once with 700 l of ethyl acetate and then twice with 300 l of ethyl acetate. United utilizating solution of sodium chloride, then dried over anhydrous sodium sulfate. The solvent is evaporated in vacuum. In the process of evaporation type 540 g of silica gel, and then continue was evaporated to dryness.

Thus obtained residue is suspended in methylene chloride, and the resulting suspension is placed in the column with 4 kg of silica gel saturated with methylene chloride. Elute first with a mixture of methylene chloride and ethyl acetate (1: 1 by volume), then one with ethyl acetate and finally with a mixture of ethyl acetate and methanol (9:1), while the polarity of the eluents increases in this order. After the eluate is divided into portions of 2 l each, collect the fraction containing tuamarina C, which is eluted with a mixture of ethyl acetate and methanol, and evaporated it to dryness under reduced pressure. In the process of evaporation add 50 g of silica gel, and then continue was evaporated to dryness.

The residue is suspended in a mixture of hexane and acetone, the resulting suspension is placed in a column of 200 g of silica gel saturated with hexane, and elute with a mixture of hexane and acetone (1:1). The eluate is divided into portions of 500 ml each, getting fractions 1 and 2, containing tuamarina C. Fraction 1 evaporated under reduced pressure. In the process of evaporation will add 25 g of silica gel, and then the ATA (1: 1: 2 by volume), the resulting suspension is placed in a column of 200 g of silica gel saturated with hexane, and elute with a mixture of hexane, acetone and ethyl acetate (1: 1:2). The eluate is divided into portions of 500 ml each, and allocate fractions containing tuamarina C. This fraction is combined with the fraction 2 obtained above, and evaporated under reduced pressure. In the process of evaporation add 20 g of silica gel, and then continue was evaporated to dryness.

The resulting residue is suspended in a mixture of hexane, acetone and ethyl acetate (1: 1: 1 by volume), the resulting suspension is placed in a column of 200 g of silica gel saturated with hexane, and elute with a mixture of hexane, acetone and ethyl acetate (1:1:1). The eluate is divided into portions of 500 ml each, and allocate fractions containing tuamarina C, and evaporated under reduced pressure, obtaining an oily substance.

All the obtained oily substance is subjected to further chromatographic purification using a column of Sephadex LH-20 with a capacity of 200 ml, saturated with a mixture of methylene chloride, ethyl acetate and methanol (19:19:2 by volume), elwira the same mixture of solvents. Collect the fraction containing tuamarina C, which evaporated under reduced pressure. In the process of evaporation will add 25 g of silica gel, and then continue upassende placed in a column of 250 g of silica gel, saturated methylene chloride. Elute with a mixture of methylene chloride and acetone in a ratio varying from 9:1 to 1:9 so that the polarity of the eluent was gradually increased. The eluate is divided into portions of 1 liter each, the fractions containing tuamarina C, collected and evaporated to dryness under reduced pressure, receiving 150 mg thimerisol C, which has a physico-chemical parameters listed above.

Biological activity thimerisol C the following examples of trials where they are compared with Pseudomonas acid a and tuomarila.

Example test 1. Antibacterial activity thimerisol C.

Minimum inhibitory concentration (MIC) thimerisol, thimerisol C and Pseudomonas acid A (denoted, respectively, through A, C and P), expressed in µg/ml against gram-positive and gram-negative bacteria is determined by the method of agar dilution, using nutrient agar medium (product of Eiken Chemical Co. Ltd.).

The results obtained are presented in table. 2.

Example of test 2. Antimycoplasma activity thimerisol C.

According to a similar method described in example test 1, conducted number is public, as A, C and P) in relation to different patterns of Mycoplasma. The results are presented in table. 3.

The AMF inoculum: 0,005 ml 10 CFU/ml

A framework for analysis: for all types of microorganisms tested thimerisol C and Pseudomonas acid (A) is carried out in the environment Chanaka (produced according to the methods described in P. N. A. S. v. 48, 41 49 (1962)) supplemented with 20% horse serum.

Tuamarina subject to the analysis in the following environments:

M. Bovis and M. gallisepticum: environment Chanaka (produced as above).

M. hoysynoviae: agar medium with mucin PPLO (pleuro-pneumonectomy body) supplemented with 15% horse serum.

PPLO broth without CV (Difco) 21 grams

The bacterial mucin (Difco) 5 g

Distilled water 800 ml

Agar noble (Difco) 12 grams

Horse serum 150 ml

25% fresh yeast extract 50 ml

Conditions of cultivation: 37oC, 5 days, slightly aerobic environment (gas method wraps, growing in the generator CO2disposable firm Becton Dickinson Microbiology Systems, Cockeysville, MD, USA).

From the presented results it is clear that tuamarina C shows excellent antibacterial and antimycoplasmal activity, which is not at least x 1. Tuamarina With formulas

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2. The method of obtaining thimerisol, characterized in that the microorganism strain Alteromonas rava FERM BP-3381 cultivated in a nutrient medium containing sources of carbon, nitrogen and inorganic salts under aerobic conditions, followed by separation of the target product.

 

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