RU2089548C1 - Thiomarinol c and a method of its preparing - Google Patents

Abstract

FIELD: pharmacy. SUBSTANCE: product: derivative of thiomarinol that shows antibacterial and antimycoplasmic properties. Thiomarinol is prepared from microorganisms of genus Alteromonas as "thiomarinol C". EFFECT: improved method of preparing. 2 cl, 3 tbl

Description

 The invention relates to some new derivatives of thiomarinol, provides a method for their preparation and relates to methods and compositions for use as antibacterial agents.

Его получают ферментацией микроорганизмов вида Alteromonas, в частности Alteromonas rava штамм SANK 73390. Обнаружено два производных тиомаринола, которые проявляют сходный тип антибактериальной активности, что и первоначальный тиомаринол.Thiomarinol is described, for example, in European Patent Application No. 512824, which is published prior to the filing of this application, but later than its priority date. It can be represented by the following formula:

It is obtained by fermentation of microorganisms of the species Alteromonas, in particular Alteromonas rava strain SANK 73390. Two derivatives of thiomarinol were found that exhibit a similar type of antibacterial activity as the original thiomarinol.

 Organisms of the genus Alteromonas can be isolated from seawater and, as has been shown, some of them are capable of producing compounds that are potentially suitable for therapeutic use. For example, a compound known as bisucaberin was obtained from one species of the genus Alteromonas and has been shown to have antitumor activity (Kokai Japanese Patent Application No. Sho 63-27484).

 As for the structure of thiomarinol, several antibiotics are known that have a similar structure, which can be divided into four groups.

 The first group includes pseudomonoic acids, first isolated from microorganisms Pseudomonas spp. These include pseudomonic acid A [produced by the species Pseudomonas fluorescens described in J. Chem. Soc. Perkin Trans, 1, 294 (1997)] pseudomonic acid B [ibid., 318 (1977)] pseudomonoic acid C [ibid., 2827 (1982)] and pseudomonoic acid D [ibid., 2655 (1983)] Pseudomonoic acid A manufactured under the name "bactroban" (a Beecham trademark) in the form of a 2% skin ointment for antibacterial use. However, all of these compounds known in the art have weaker antibacterial activity than the thiomarinol derivatives of the present invention.

 The second group of substances that are similar in structure to the compounds of the present invention are antibiotics such as cholomycin (Helv. Chim, Acta, vol. 42, 563 (1959)] pyrrhotite [J. Am. Shem. Soc. Vol. 77, 2861 (1955)] thiolutin [Angew. Chem, Bd. 66, 745 (1954)] laureotricin [J. Am. Chem. Soc. Vol. 74, 6304 (1952)] and others. These antibiotics are usually produced by actinomycetes and are distinguished by that have a sulfur-containing chromophore in their composition Xenorhabdins IV are substances related to cholomycin and have also been isolated from bacteria (described in WO / 01775).

 Numerous studies of derivatives of these two groups of compounds have been carried out, however, we do not have any information about compounds having a molecular structure similar to the structure of thiomarinols, or having similar properties.

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

The fourth group includes a compound similar to thiomarinol in that it is a physiological waste product of marine bacteria [Abstracts of Articles 200 of the American Chemical Society Annual Conference (August 26-31, 1990), part 2, No. 139] However, a heterocyclic group attached to the terminal carboxyl the group of this compound is a 2-oxo-3-piperidyl group. As recently shown, it has antimicrobial activity [Experimentia, Vol 48, pp. 1165-169, 1992]
However, it is believed that the closest known compound is pseudomonoic acid A, and all thiomarinols, i.e. thiomarinol itself, thiomarinol B and thiomarinol C, have significantly stronger antibacterial activity than pseudomonoic acid A.

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

 A further and more specific objective of the present invention is the provision of compounds that have excellent antibacterial and antimycoplasmic activity.

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

 In a general sense, the present invention provides two new derivatives of thiomarinol, one of which is an S, S-dioxo derivative and is hereinafter referred to as “thiomarinol B”, and is the subject of consideration in application N 93053043/13, from which this is isolated, and the second is a deoxy derivative hereinafter referred to as "thiomarinol C".

Изобретение предоставляет также способ получения тиомаринола C, который предусматривает культивирование продуцирующих тиомаринол микроорганизмов вида Alteromonas и выделение тиомаринола C из культуры.More specifically, the present invention provides a compound thiomarinol C, which may be represented by the following formula:

The invention also provides a process for the preparation of thiomarinol C, which comprises culturing thiomarinol producing microorganisms of the Alteromonas species and isolating thiomarinol C from the culture.

 From the above formula it is seen that thiomarinol C contains a number of asymmetric carbon atoms and several multiple bonds. In particular, isomerization in the α, b-unsaturated carbonyl fragment of thiomarinol is possible. Thus, thiomarinol can form various stereo and geometric isomers. Although they are all represented here in one molecular formula, the present invention includes both individual, isolated isomers and mixtures thereof, including racemates. In preparing a mixture of isomers, individual isomers can be obtained by conventional cleavage methods.

 However, since thiomarinols are usually obtained by fermentation or chemical manipulation of the fermentation products, they tend to acquire a standard optical configuration. Therefore, although other configurations are provided, a natural configuration is preferred.

Thiomarinol C can be characterized by the following physicochemical properties:
1. Character and appearance: yellow powder.

2. The molecular formula is C ₃₀ H ₄₄ N ₂ O ₈ S ₂ .

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

4. Elemental analysis:
Calculated for C ₃₀ H ₄₄ N ₂ O ₈ S ₂ + H ₂ O, C 56.05, H 7.21, N 4.36, S 9.97.

 Found, C 56.48, H 7.23, N 4.30, S 9.11.

5. IR spectrum: n _max cm ^-1 .

 IR spectrum recorded for the sample in the form of a tablet with potassium bromide: 3256, 3068, 2928, 2858, 1645, 1596, 1530, 1455, 1384, 1287, 1225, 1151, 1104, 1052, 974, 820, 712.

6. UV spectrum: λ _max , nm (ε).

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

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

7. Specific rotation of the plane of polarization:
[α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = -1.4 ^° (c 1.0, methanol).

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

 Solvent: 40 vol. aqueous acetonitrile.

 Eluent flow rate: 1.5 ml / min.

 Retention time: 11.3 minutes

9. ¹ H-NMR spectrum: (δ ppm).

 NMR spectrum (360 MHz) measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as the internal standard: 0.91 (3H, doublet, J 6.9 Hz), 0.95 (3H, doublet, J 6.3 Hz), 1 26 (2H), 1.29 (2H), 1.30 (2H), 1.51 (2H, multiplet), 1.56 (2H, multiplet), 1.63 (1H, multiplet), 2.06 (2H), 2.08 (1H), 2.11 (3H, singlet), 2.15 (1H), 2.34 (2H, triplet, J 7.3 Hz), 2.56 (1H, wide doublet , J 14.2 Hz), 3.18 (1H, broad multiplet), 3.35 (1H), 3.49 (1H), 3.58 (1H), 3.62 (1H), 3.67 ( 1H), 4.01 (2H, triplet, J 6.6 Hz), 4.32 (1H, doublet, J 4.3 Hz), 4.55 (1H, broad multiplet), 4.64 (1H, wide singlet), 5.33 (1H, multiplet), 5.37 (1H, multiplet), 5.68 (1H, singlet), 7.05 (1H, syn lithium), 9.81 (1H, singlet), 10.70 (1H, singlet).

10. ¹³ C-NMR spectroscopy: (d ppm).

 NMR spectrum (90 MHz) measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as the 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), 69.3 (doublet), 74.4 (doublet ), 110.4 (doublet), 115.3 (singlet), 116.5 (doublet), 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. Solubility. Soluble in alcohols such as methanol, ethanol, propanol and butanol, as well as in dimethyl sulfoxide, dimethylformamide, chloroform, ethyl acetate, acetone and diethyl ether. Insoluble in hexane and water.

 12. Thin layer chromatography.

The value of R _f 0,66.

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

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

 Thiomarinol C can be obtained by culturing thiomarinol producing organisms of the Alteromonas species and isolating the desired thiomarinol from the culture medium. Varieties of thiomarinol C with the desired antibacterial activity can be obtained similarly from other strains or species of Alteromonas that produce the target compound, or they can be obtained by suitably modifying the compound isolated during the above fermentation, or they can be obtained by direct chemical synthesis.

 In particular, it seems to us more preferable to use the species Alteromonas rava and the especially recently isolated strain Alteromonas rava, which we gave the designation SANK 73390. Strain SANK 73390 is a marine organism that is isolated from sea water collected off the coast of Coin, Minamata, prefecture Sizuoka, Japan, and the strain is deposited with the Depository Institute, Fermentation Research Institute, Industrial Science and Technology Agency, Ministry of International Trade and Industry, Japan, April 30 I 1991 deposit number FERM BP-3381, according to the Budapest Treaty.

 The taxonomic characteristics of Alteromonas rava strain SANK 73390 are presented below.

 1. Morphological characteristics.

Alteromonas rava strain SANK 73390 was cultured at 23 ^° C for 24 hours on marine agar (Difco). Subsequent microscopic observations showed that the cells have a rod-shaped shape with a diameter of 0.8 to 1.0 μm and a length of 2.0 to 3.6 μm. This strain is gram-negative and is transported using bacterial flagella.

 2. Growth on marine agar.

SANK 73390 was cultured at 23 ^° C. for 24 hours on marine agar (Difco). The resulting colonies have a pale gray-green color, are opaque, flat, solid and have a ring shape.

 Water-soluble pigment is not formed.

 3. Physiological properties.

 (1) Sea water requirements: SANK 73390 requires sea water for its growth.

 (2) Oxidative-enzymatic test (Hugh-Leifson method [J. Bact. Vol. 66, 2426, 1953] in a medium prepared from artificial sea water does not affect the carbohydrate.

(3) Oxidase: +
(4) Catalase: +
(5) Oxygen Demand: Aerobic.

(6) Nitrate reduction: -
(7) Hydrolysis of starch: +
(8) Agar degradation: -
(9) Gelatin Liquefaction: +
(10) Production of diphosphopyridine nucleotidase (DNase): +
(11) Lipase Production: +
(12) Temperature for growth: poor growth at 4 ^o C, good growth in the range 17 26 ^o C, no growth at 35 ^o C.

 (13) Requirements for growth factors: on the main nutrient medium described in J. Bact. vol. 107, 268 (1971), SANK 73390, requires the addition of vitamin-free casamino acid.

 (14) Assimilation of carbon sources: on the main nutrient medium described in J. Bact. vol. 107, 268 (1971), additionally containing 0.1% weight. /about. vitamin-free casamino acid when shaking the culture (see table. 1).

 4. Chemotaxonomic characteristics.

 (1) Mol. guanine and cytosine (G + C content) in DNA: 43.4% (according to high resolution liquid chromatography).

 (2) Quinone system: ubiquinone Q-8.

 Given the above taxonomic characteristics, Alteromonas rava strain SANK 73390 was compared with the strains described in Bergey's Manual of Systematik Bacteriology, Vol. 1 (1984), as well as with the strains described in the latest issues of the International Journal of Bacteriology. It was found that Alteromonas rava strain SANK 73390 is somewhat similar to Alteromonas citrea, which is also a marine organism. The properties of SANK 73390 and Alteromonas citrea, ATCC 29719 (standard strain) were grown and compared.

Compared with the pale grayish yellow SANK 73390, the ATCC 29719 colonies were greenish yellow. SANK 73390 differs from Alteromonas citrea in its growth rate at 4 ^° C and in its ability to absorb trehalose and sodium propionate as carbon sources. Therefore, the strain Alteromonas rava SANK 73390 is a new strain of the new species Alteromonas rava and differs in its most important characteristics from the nearest analogue, deposited under the number ATCC 29719.

 The above characteristics are typical for SANK 73390. However, as you know, the characteristics of the samples Alteromonas spp. can change both naturally and artificially. The above characteristics determine the strain of 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 thiomarinol or its naturally occurring variants. All such strains are included in the scope of the claims of the present invention.

 It is understood that SANK 73390 or any other strain capable of producing thiomarinol or one of its varieties can be subcultured, biotechnologically modified, or modified to produce an organism with different characteristics. The only requirement for the resulting organism is the ability to produce the desired compound.

 Such changes or modifications may take any desired form, or may, for example, depend on the growing conditions of the culture. Strains can be changed during cultivation and selected in such a way as to obtain samples with such properties as an increased growth rate, growth at higher or lower temperatures.

 Biotechnological modifications, as a rule, are carried out intentionally, and can introduce selective characteristics, such as, for example, resistance or susceptibility to a bacteriostat, or a combination thereof, to maintain purity, or to enable the purification of cultures, especially elite cultures.

 Other characteristics that can be introduced during genetic manipulation are any of those that are acceptable in the case of Alteromonas spp. For example, you can enter plasmids encoding resistance, or remove any natural plasmids. The greatest advantages are provided by those plasmids that have the ability to form mutant strains of a growing organism. Plasmids can be obtained from any convenient source or generated by genetic engineering by isolating the natural Alteromonas plasmid and introducing the desired gene or genes from another source into it. Natural plasmids can also be modified in any other desired manner.

 In order to obtain thiomarinol C from a culture of a suitable microorganism, the microorganisms must be fermented in a suitable medium. Such media are generally well known in the art and are often used in the manufacture of other enzymatic products.

 Typically, such a medium should contain any combination of a carbon source, a nitrogen source, and one or more inorganic salts that are absorbed by the corresponding microorganism. The minimum requirement for the environment is that it must contain such ingredients as are necessary for the growth of the microorganism.

 Suitable carbon sources are glucose, fructose, maltose, sucrose, mannitol, glycerin, dextrin, oat flour, rye, corn starch, potato, corn flour, soy 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. Typical amounts are 1 10% w / v. on the amount of medium, although this value may vary depending on the need and in accordance with the desired result.

 Suitable nitrogen sources can be any substance that contains, for example, protein. Typical examples of nitrogen sources are organic nitrogen sources obtained from animals and plants, these can be extracts extracted from natural sources such as soy flour, bran, peanut flour, cottonseed flour, casein hydrolyzate, fermine, 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 with carbon sources, they can be used alone or in combination with each other. Typical amounts are 0.1 to 6% w / v. on the amount of medium.

 Suitable nutritious inorganic salts are those that provide trace elements, as well as basic salt components. Salts should preferably contain ions such as sodium, potassium, ammonium, calcium, magnesium, iron, phosphate, sulfate, chloride and carbonate ions. Traces of metals (or trace elements) such as cobalt, manganese or strontium, or salts capable of producing ions such as bromide, fluoride, borate and silicate ions may also be present.

 Alteromonas rava is found in nature in sea water, therefore, unless expressly agreed, the conditions for the cultivation of these organisms should ideally correspond to the conditions of the marine habitat. So, ions that are contained in seawater in the form of traces are mainly included in any medium used to grow Alteromonas. In particular, 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 is fermented as a liquid culture, then it is desirable to use antifoam agents such as silicone oil or vegetable oil or other suitable surfactants.

When using the Alteromonas rava SANK 73390 strain for the production of thiomarinol, the acidity of the growth medium should preferably be maintained at 5.0-8.0 pH, although the only requirement is that the acidity of the medium does not interfere with the growth of the microorganism or adversely and irreversibly affect the yield of the target product. To stop fermentation, an excess of acid or alkali can be added. Alteromonas rava SANK 73390, as a rule, grows at a temperature of 4 32 ^o C, grows well at a temperature of 17 26 ^o C. You can work at other temperatures that do not fall in the specified interval, if a strain capable of growing at lower or higher temperatures. To obtain thiomarinol C, the temperature range should preferably be 20 26 ^o C.

 Thiomarinol C is ideally obtained by aerobic cultivation, and any aerobic cultivation technique can be applied, for example, solid culture, shake culture or air-mixed culture.

If the cultivation is carried out on a small scale, then a shake culture, which is fermented for several days at a temperature of 20 26 ^o C., is usually preferred.

To begin the enzymatic growth, it is preferable to use the method using the initial inoculum prepared in one or two stages, for example, in an Erlenmeyer flask. For the culture medium, a combination of a carbon source and a nitrogen source can be used. The flask with inoculum is shaken in a thermostatic incubator at 23 ^o C for 1 to 3 days or until a tangible growth begins. The resulting seed crop can then be used to plant a second seed crop or production crop. If repeated seeding is carried out, it can be carried out in the usual way, and some can be used to inoculate a production medium. The medium in which the seed is placed is shaken at the appropriate temperature for the required amount of time, for example from 1 to 3 days, or until a noticeable formation of the product begins, as described above. After the incubation period, the contents of the flask are separated by centrifugation or filtration.

If the cultivation is carried out on a large scale, it is preferable to use a culture mixed with air in a fermenter. In this case, the nutrient medium can be prepared directly in the fermenter. After sterilization at 125 ^° C., the resulting medium is cooled and inoculated with inoculum previously prepared in a sterile medium. Cultivation is carried out with stirring and aeration at a temperature of 20 26 ^o C. This technique is suitable for the preparation of compounds in large quantities.

 The amount of thiomarinol produced by the culture can be controlled from time to time by, for example, high-performance liquid chromatography. Typically, the amount of thiomarinol C produced reaches a maximum in a time interval of 19,200 hours, and the amount of thiomarinol C produced reaches a maximum in a time interval of 19 to 96 hours.

 After a suitable growing time, thiomarinol C can be isolated and purified by any known means. For example, either the amount of C left in the culture broth can be obtained by filtering off solids, for example, using diatomaceous earth as a filter material, or by centrifugation followed by extraction of the liquid over the precipitate and purification in accordance with the physicochemical properties of thiomarinol C. For example, thiomarinol C, which is in the filtrate or in the residue after centrifugation, can be extracted with water-immiscible organic solvents such as ethyl acetate, chloroform, ethyl chloride n, methylene chloride or a mixture of these solvents under neutral or acidic conditions, and then cleaned.

 Alternatively, activated carbon or adsorbent resins such as Amberlit (trademark) XAD -2, XAD -4 (Rohm Haas) or Diaion (trademark) HP-10, Hp-20, CHP-20, HP can be used as adsorbent. -50 (Mitsubishi Kasei Corporation). Impurities can be removed after adsorption by passing a liquid containing thiomarinol C through a thin layer of adsorbent, thiomarinol C can also be purified after adsorption by elution with a suitable solvent, for example, aqueous methanol, aqueous acetone or a mixture of butanol / water.

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

Further purification of thiomarinol C can be carried out using well-known methods, namely:
adsorption column chromatography using carriers such as silica gel or magnesium silica gel, for example, marketed under the trade name "Florisil";
distribution column chromatography using adsorbents such as Sephadex LH-20 (trade name for the product from Pharmacia);
high performance liquid chromatography using normal or reverse phase columns.

 As is well known from the technical field, these methods of isolation and purification can be carried out separately or in any convenient combination, and if necessary, and repeatedly for the purpose of isolation and purification of the target product.

 Since thiomarinol C has an antibacterial effect on gram-positive and gram-negative bacteria and mycoplasmas in animal organisms (in particular humans, dogs, cats and rabbits), it can be used in a variety of ways, depending on the nature of the infection, to treat or prevent bacterial or mycoplasma infections.

 In the event that the compound of the present invention is intended to be used for therapeutic purposes, it can be administered alone or in the form of a pharmaceutical composition containing, in addition to the active compound, one or two standard diluents, carriers, inert excipients or auxiliary compounds. The form of the composition will naturally depend on the method of administration of the drug. For oral administration, the compound is advantageously converted into the form of a preparation in the form of powders, granules, tablets, capsules or syrups. For parenteral administration, it is predominantly prepared in the form of injectable preparations (which may be intravenous, intramuscular or subcutaneous), drops, suppositories, ointments or liniment.

 These formulations can be prepared by known methods by adding additives such as carriers, binders, disintegrants, lubricants, stabilizers, modifiers, wetting agents, flavoring agents, perfumes, dispersants or coating materials to the active compound. Although the dose may depend on the symptoms of the disease and the age of the patient, the nature and severity of the infection, as well as the method of prescribing the drugs, in the case of oral administration to an adult patient, the compound of the present invention can usually be used as a daily dose of 20 2000 mg. The compound can be taken in a single dose or in portions, for example, two or three times a day.

 The preparation of the compound of the present invention is further illustrated by the following example, which does not limit the present invention, and the biological activity of the compound is illustrated further in the test examples.

 Example. Obtaining thiomarinol C cultivation in tenka.

 A) Culture.

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

A 30-liter jug-type fermenter containing 15 liters of the same sea broth is sterilized by heating, and then the entire amount of the bacterial suspension is seeded and grown for 24 hours at a temperature of 23 ^° C and an air supply rate of 7.5 l / min. The initial mixing speed is 100 rpm, and then it is selected so that the concentration of dissolved oxygen is 5 ppm.

In each of the 600-liter tenks placed 300 l of nutrient medium having the following composition,
Glucose 1.5
Bactopeptone (Difco) 1.5
Bacter Yeast Extract (Difco) 0.2
NaCl 3.89
MgCl ₂ • 6H ₂ O 2.52
Na ₂ SO ₄ 0.648
CACl ₂ • 2H ₂ O 0.4767
KCl 0.11
Na ₂ CO ₃ 0,038
Iron Citrate 0.02
pH 7.6 before sterilization.

Tenki are sterilized by heating, and then 3 l of culture are sown in each of them and grown for 29 hours at 23 ^° C and an air supply rate of 150 l / min. The initial mixing speed is 82 rpm, and then it is selected so that the concentration of dissolved oxygen is 5 ppm.

 B) Isolation.

 A sufficient amount of aqueous hydrochloric acid solution is added to 700 L of the resulting culture solution in order to bring the pH to 3. Then, 700 L of acetone is added to the resulting mixture and the mixture is extracted with stirring for 1 hour. The obtained extract, in turn, is extracted with one times 700 l of ethyl acetate, and then twice 300 l of ethyl acetate. The combined ethyl acetate extracts are washed first with 300 L of a 5% aqueous sodium bicarbonate solution and then with 300 L of a saturated aqueous sodium chloride solution, after which they are dried over anhydrous sodium sulfate. The solvent was evaporated in vacuo. During the evaporation process, 540 g of silica gel is added, and then continue to evaporate to dryness.

 The residue thus obtained was suspended in methylene chloride, and the resulting suspension was placed in a column with 4 kg of silica gel saturated with methylene chloride. First, it is eluted with a mixture of methylene chloride and ethyl acetate (1: 1 by volume), then with ethyl acetate alone, and finally with a mixture of ethyl acetate and methanol (9: 1), and the polarity of the eluents increases in this order. After the eluate was divided into 2 L portions each, a fraction containing thiomarinol C, which was eluted with a mixture of ethyl acetate and methanol, was collected and evaporated to dryness under reduced pressure. During the evaporation process, 50 g of silica gel is added, and then continue to evaporate to dryness.

 The residue was suspended in a mixture of hexane and acetone, the resulting suspension was placed on a column with 200 g of silica gel saturated with hexane, and eluted with a mixture of hexane and acetone (1: 1). The eluate was divided into 500 ml portions each, obtaining fractions 1 and 2 containing thiomarinol C. Fraction 1 was evaporated under reduced pressure. During the evaporation process, add 25 g of silica gel, and then continue to evaporate to dryness.

 The resulting residue was suspended in a mixture of hexane, acetone and ethyl acetate (1: 1: 2 by volume), the resulting suspension was placed in a column with 200 g of silica gel saturated with hexane, and eluted with a mixture of hexane, acetone and ethyl acetate (1: 1: 2). The eluate was divided into 500 ml portions each and fractions containing thiomarinol C were isolated. This fraction was combined with fraction 2 obtained above and evaporated under reduced pressure. During the evaporation process, 20 g of silica gel is added, and then continue to evaporate to dryness.

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

 All of the resulting oily substance was subjected to further chromatographic purification using a 200 ml Sephadex LH-20 column saturated with a mixture of methylene chloride, ethyl acetate and methanol (19:19: 2 by volume), eluting with the same solvent mixture. A fraction containing thiomarinol C was collected, which was evaporated under reduced pressure. During the evaporation process, add 25 g of silica gel, and then continue to evaporate to dryness.

 The residue thus obtained was suspended in methylene chloride, and the resulting suspension was placed on a column of 250 g of silica gel saturated with 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 gradually increases. The eluate is divided into portions of 1 l each, fractions containing thiomarinol C are collected and evaporated to dryness under reduced pressure to obtain 150 mg of thiomarinol C, which has the physicochemical parameters described above.

 The biological activity of thiomarinol C is confirmed by the following test examples, in which they are compared with pseudomonic acid A and thiomarinol.

 Test Example 1. Antibacterial activity of thiomarinol C.

 The minimum inhibitory concentration (MIC) of thiomarinol, thiomarinol C and pseudomonoic acid A (indicated by A, C and P, respectively), expressed in μg / ml, in relation to gram-positive and gram-negative bacteria is determined by agar dilution using nutrient agar medium ( product of Eiken Chemical Co. Ltd.).

 The results are presented in table. 2.

 Test Example 2. Antimycoplasmic activity of thiomarinol C.

 By a similar methodology described in Test Example 1, the activity of thiomarinol, thiomarinol C, and pseudomonoic acid A (quantified as A, C, and P, respectively) with respect to various mycoplasma samples is quantified. The results are presented in table. 3.

 Inoculum: 0.005 ml 10 CFU / ml.

 Analysis medium: for all types of microorganisms, the tests of thiomarinol C and pseudomonoic acid A are carried out in Chenok's medium (obtained according to the procedure described in P. N. A. S. v. 48, 41 49 (1962)) with the addition of 20% horse serum.

Thiomarinol is analyzed in the following environments:
M. Bovis and M. gallisepticum: Chanock medium (prepared as described above).

 M. hoysynoviae: agar medium with mucin PPLO (pleuro-pneumonia-like organism) supplemented with 15% horse serum.

PPLO broth without CV (Difco) 21 g
Bacterial mucin (Difco) 5 g
Distilled water 800 ml
Noble agar (Difco) 12 g
Horse serum 150 ml
25% fresh yeast extract 50 ml
The culture growing conditions: 37 ^o C, 5 days, slightly aerobic environment (gas wrapping method, growing in a disposable CO ₂ generator from Becton Dickinson Microbiology Systems, Coquisville, Maryland, USA).

 From the presented results it is clear that thiomarinol C exhibits excellent antibacterial and antimycoplasmic activity, which is at least no worse than that of thiomarinol, and in the general case is significantly higher than the activity of pseudomonoic acid A.

Claims (1)

1. Thiomarinol C Formulas

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

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