RU2101353C1 - Thiomarinol b and a method of its preparing (variants) - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: thiomarinol B is prepared by culturing microorganism of species Alteromonas rova on nutrient medium containing carbon, nitrogen and mineral salts source under aerobic conditions followed by its isolation. Thiomarinol B is obtained by oxidation of thiomarinol. Thiomarinol B shows antibacterial and antimycoplasmial properties. EFFECT: improved method of preparing. 8 cl, 3 tbl

Description

 The invention relates to some new derivatives of thiomarinol and to methods for their preparation, describes methods and compositions for using them as antibacterial agents.

Его получают ферментацией микроорганизмов вида Alteromonas, в частности Alteromonas rava штамм SANK 73390. Мы обнаружили два производных тиомаринола, которые проявляют сходный тип бактерицидной активности, что и оригинальный тиомаринол.Thiomarinol is described, for example, in European Patent Application No. 512824. 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. We found two derivatives of thiomarinol, which exhibit a similar type of bactericidal activity as the original thiomarinol.

 Organisms of the Alteromonas species can be isolated from seawater and, as shown, some of them are capable of producing compounds that are potentially suitable for therapeutic use. For example, a compound known as besucaberin was obtained from one of the varieties of Alteromonas and has been shown to have antitumor activity (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. It includes pseudomonic acid A [produced by the species Pseudomonas fluorescens described in J. Chem. Soc. Perkin Trans. 1, 294 (1977)] pseudomonoic acid B [ibid., 318 (1977)] pseudomonoic acid C [ibid., 2827 (1982)] and pseudomonoic acid D [ibid., 2655 (1983)] Pseudomonoic acid A is sold under the name Bactroban (a trademark of Beecham) in the form of a 2% skin ointment for antibacterial use. However, these compounds known in the art have weaker antibacterial activity than the thiomarinol derivatives of the invention.

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

 Numerous studies have been conducted on derivatives of the compounds in these two groups, however, we do not have any information on the description of compounds having a molecular structure similar to the structure of thiomarinol, or having similar properties.

 A third group of compounds is described in publications such as Japanese Applications Kokai NN 52-102279, 54-12375, 54-90179, 54-103871 and 54-125672. It consists of derivatives of pseudomonic acid, 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 have less antibacterial activity than the original pseudomonoic acid.

The fourth group includes a compound similar to thiomarinol in that it is a physiological product derived from 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 carbonyl 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 seems that the closest known compound is pseudomonic acid A, and all of the thiomarinols, i.e., the initial thiomarinol, thiomarinol B and thiomarinol C, have significantly stronger antibacterial activity than pseudomonic acid A.

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

 A further and more specific objective of the 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.

 The invention provides a novel thiomarinol derivative, which is s, s-dioxo derivative, and is hereinafter referred to as “thiomarinol B”.

Тиомаринол B характеризуется следующими физико-химическими свойствами:
1) Внешний вид: порошок желтого цвета.It has now been found that the structure of thiomarinol B is as follows:

Thiomarinol B is characterized by the following physicochemical properties:
1) Appearance: yellow powder.

2) The molecular formula is C ₃₀ H ₄₄ N ₂ O ₁₁ S ₂ .

 3) Molecular mass: 672 (according to FAB mass spectrometry); FAB mass spectrometry fast atom bombardment mass spectrometry.

4) High resolution mass spectrometry;
C ₃₀ H ₄₄ N ₂ O ₁₁ S ₂ [(M + H) ⁺ ; according to FAB mass spectrometry]
Found 673.2468.

 Calculated 673.2465.

 5) Elemental analysis.

C ₃₀ H ₄₄ N ₂ O ₁₁ S ₂ + H ₂ O.

 Calculated, C 52.16; H 6.71; N, 4.06; S 9.28.

Found, C, 52.34; H 6.79; N, 3.92; S 9.02%
6) IR spectrum: ν _max cm ^-1 .

 The IR spectrum, measured by the method with a potassium bromide disk, is presented below: 3660, 3503, 3318, 3075, 2966, 2928, 2870, 1704, 1653, 1509, 1467, 1381, 1349, 1299, 1217, 1199, 1152, 1112, 1063 , 1047, 1019, 975, 949, 884, 839, 764, 730, 660, 609, 553.

7) UV spectrum: λ _max nm (s).

The UV spectrum measured in 1-propanol is presented below:
377 (2900), 301 (13000), 215 (21000)
The UV spectrum, measured in a mixture of 1-propanol + hydrochloric acid, is presented below:
377 (2900), 301 (13000), 223 (17000).

The UV spectrum, measured in a mixture of 1-propanol + sodium hydroxide, is presented below:
377 (2900), 301 (13000), 223 (19000).

8) Specific rotation of the plane of polarization:
[α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = +7.7 ^° (c = 1.0; 1 - propanol).
9) High Resolution Liquid Chromatography:
dividing column: Senshu-Pak ODS H-2151 (column diameter 6 mm, length 150 mm, trademark of Senshu Scientific Co. Ltd.);
solvent: 40 vol. aqueous acetonitrile;
eluent feed rate: 1.5 ml / min;
retention time: 8.4 min;
10) ¹ H-NMR spectrum: (δ ppm,);
NMR spectrum (360 MHz), measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as an internal standard, is presented below:
0.92 (3H, doublet, J = 6.9 Hz); 0.96 (3 H, doublet, J = 6.3 Hz);
1.25 (2H), 1.30 (2H), 1.32 (2H), 1.50 (2H), 1.58 (2H);
1.61 (1H), 2.02 (3H, singlet), 2.03 (1H), 2.09 (1H),
2.12 (1H), 2.43 (2H, triplet, J = 7.3 Hz), 3.35 (1H, doublet,
J = 10.9 Hz), 3.51 (1H), 3.54 (1H), 3.61 (1H), 3.64 (1H),
3.74 (1H, broad singlet), 4.02 (2H, triplet, J = 6.6 Hz),
4.18 (1H, doublet, J = 7.2 Hz), 4.28 (1H, doublet, J = 3.6 Hz),
4.43 (1H, doublet, J = 7.2 Hz), 4.61 (1H, broad singlet),
4.88 (1H, doublet, J = 7.5 Hz), 5.37 (2H, multiplet),
5.97 (1H, singlet), 7.23 (1H, singlet), 10.47 (1H, singlet)
11.28 (1H, broad singlet).

11) ¹³ C-NMR spectroscopy: (d ppm).

NMR spectrum (90 MHz), measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as an internal standard, is presented below:
15.6 (quartet), 15.7 (quartet), 20.0 (quartet),
24.5 (triplet), 25.3 (triplet), 28.1 (triplet),
28.2 (triplet), 28.3 (triplet), 31.9 (triplet),
34.7 (triplet), 42.2 (doublet), 43.2 (doublet),
63.0 (triplet), 63.9 (doublet), 64.3 (triplet),
69.3 (doublet), 69.6 (doublet), 72.4 (doublet),
76.2 (doublet), 109.4 (doublet), 114.4 (doublet),
115.2 (singlet), 123.3 (singlet), 127.8 (doublet),
134.2 (doublet), 143.2 (singlet), 160.8 (singlet),
165.7 (singlet), 166.1 (singlet), 173.5 (singlet).

 12) Solubility.

 It is 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.

 13) Thin layer chromatography.

The value of R _f 0,52
Adsorbent: silica gel (Merck Co. Inc. Art. 715).

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

 The invention also provides a method for producing thiomarinol B, which consists in culturing a thiomarin-producing microorganism of the genus Alteromonas and isolating thiomarinol B from the culture.

 The invention also provides a process for the preparation of thiomarinol B by oxidation of thiomarinol.

 Also described is a pharmacological composition comprising an antibacterial or antimycoplasmic agent in admixture with a pharmacologically acceptable carrier or diluent, in which the thiomarinol B is an antibacterial or antimycoplastic agent.

 A method of treating or preventing bacterial or mycoplasma infections using a compound of the invention is to administer to a mammal, which may be a person suffering from or exposed to such infections, an effective amount of an antibacterial or antimycoplasmic agent.

 From the presented formula it is seen that thiomarinol in contains a number of asymmetric carbon atoms and several multiple bonds. In particular, isomerization in the alpha, beta unsaturated carbonyl fragment of thiomarinols is possible. Thus, thiomarinol B can form various stereo and geometric isomers. Although all of them are presented here as a single molecule, the invention includes both individual, isolated isomers, and mixtures thereof, including racemates. If stereospecific synthesis is carried out or the starting compounds are optically active, individual isomers can be obtained directly from the synthesis; on the other hand, if a mixture of isomers is obtained, then individual isomers can be obtained by conventional cleavage methods.

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

 Thiomarinol B can be obtained by growing a thiomarinol producing organism of the genus Alteromonas, and then collecting the desired thiomarinol from the culture medium. Varieties of thiomarinol B with the required 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 this fermentation, or they can be obtained by direct chemical synthesis.

 In particular, it seems particularly preferable to use the species Alteromonas rava and the particularly 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 Coin coast, Minami-Izu Mati , Shizuoka Prefecture, Japan, and the strain is deposited at the Depository Institute, Fermentation Research Institute of the Agency for Applied Sciences and Technology of the Ministry of International Trade and Industry, Japan, April 30 In 1991, with registration number FERM BP-3381, in accordance with the Budapest Treaty.

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

 1. Morphological characteristics.

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

 2. Growth on marine agar.

SANK 73390 was grown 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 an environment 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: +
(11) Lipase Production: +
(12) Temperature for growth: slow growth at 4 ^o C, good growth in the range from 17 ^o C to 26 ^o C, termination of 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: in the basic nutrient medium described in J. Bact. vol. 107, 268 (1971), additionally containing 0.1 wt./about. Vitamin-free casamino acid when shaking the culture. The data are given in table. one.

 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 taxonomic characteristics given, Alteromonas rava strain SANK 73390 was compared with the strains described in Bergey's Manual of Systematic Bacteriology, vol. 1 (1984), as well as with the strains described in the latest issues of the International Journal of Bacteriology. Alteromonas rava strain SANK 73390 was found to have some similarities with Alteromonas citrea, another marine organism. The properties of SANK 73390 and Alteromonas citrea, ATCC 29719 (standard strain) were grown and compared.

In contrast to the pale grayish-green color of SANK 73390, ATCC 29719 colonies were yellow-green in color. SANK 73390 differs from Alteromonas citrea also in growth rate at 4 ^o C and in its ability to utilize trehalose and sodium propionate as carbon sources. Accordingly, Alteromonas rava strain SANK 73390 is a new strain of the new species Alteromonas rava and differs in its most important characteristics from the nearest known species deposited under registration number ATCC 29719.

 The characteristics given are typical for SANK 73390. However, it is well known that the characteristics of Alteromonas spp. are volatile in both natural and artificial ways. These characteristics determine the Alteromonas rava strain as it was deposited, but they are not necessarily typical of other Alteromonas species or Alteromonas rava strains that are capable of producing thiomarinol or its naturally occurring variants. All such strains are included in the scope of the claims of the invention.

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

 Such changes and modifications may take any desired form or may, for example, depend on the growing conditions of the culture. The strains can be modified by culture, and therefore are selected in such a way as to exhibit properties such as enhanced growth or growth at higher or lower temperatures.

 Biotechnological modifications are usually intentional and may introduce breeding characteristics, such as, for example, resistance or susceptibility to a bacteriostat, or a combination thereof, to maintain purity, or from time to time to allow the purification of crops, especially elite crops.

 Other characteristics that can be introduced through genetic manipulation are any that are valid for Alteromonas spp. For example, plasmids can be inserted that tint the resistance of plasmids, or any natural plasmids can be deleted. The greatest advantages are those plasmids that provide auxotrophy. Plasmids can be obtained from any suitable source or can be generated by genetic engineering by isolating the natural Alteromonas plasmid and incorporating the desired gene or genes from another source. Natural plasmids can also be modified in any other desired manner.

 In order to obtain thiomarinol B from a culture of a suitable microorganism, the microorganism 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 an environment should include 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 contains ingredients that are essential for the growth of the microorganism.

 Suitable carbon sources are glucose, fructose, maltose, sucrose, mannitol, glycerin, dextran, oat flour, rye, corn starch, potato, wheat flour, soy flour, cottonseed oil, molasses, citric acid and tartaric acid, any of which can be used alone or in combination with one or more other carbon sources. Typical amounts are from about 1 to 10% w / v. on the amount of medium, although this amount may vary, if necessary, and in accordance with the desired result.

 Suitable nitrogen sources can be any substance that, for example, contains protein. Typical examples of nitrogen sources are organic nitrogen sources obtained from animals and plants, these can be extracts from natural sources such as soybean flour, bran, peanut flour, cottonseed flour, casein hydrolyzate, farmin, 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 in the case of carbon sources, they can be used separately or in combination with each other. Typical amounts are 0.1-6 wt./about. on the amount of medium.

 Suitable nutritious inorganic salts are salts which, along with the basic components of the salt, contain traces of other elements. Salts should preferably contain ions such as sodium, potassium, ammonium, calcium, magnesium, iron, phosphate, sulfate, chloride and carbonation ions. Traces of metals such as cobalt, manganese and strontium, or salts capable of producing ions such as bromide, fluoride, borate or silications can 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 environment. So, traces of ions contained in seawater 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 the sea water.

 If the microorganisms are fermented as a liquid culture, it is preferable to use antifoam agents such as silicone oil or vegetable oil or other suitable surfactants.

 When using Alteromonas rava strain SANK 73390 to obtain thiomarinol B, the pH of the culture medium is preferably maintained in the range of pH 5.0-8.0, although the only requirement is that the pH of the medium does not interfere with the growth of the microorganism or have a harmful and irreversible effect on quality of the target product. To stop fermentation, an excess of acid or alkali can be added.

The strain Alteromonas rava SANK 73390 usually grows at 4-32 ^o C, and grows well at a temperature of 17-26 ^o C. Other temperatures that do not fall within this range may be applicable when a strain capable of growing at lower or more is developed high temperatures. To obtain thiomarinol B, the preferred temperature range is between 20 and 26 ^o C.

 Thiomarinol B is ideally obtained by aerobic cultivation and any suitable aerobic cultivation technique can be used, such as a solid culture, a shake culture or a stirred aerated culture.

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

To start the enzymatic cultivation, it is preferable to apply 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 inoculation is shaken in a thermostatic incubator at 23 ^o C for 1-3 days or until there is a noticeable growth. The resulting seed culture can be further used to inoculate a second seed culture or production culture. If repeated seeding is carried out, it can be carried out in the usual way, and part can be used to inoculate the production medium, the flask in which the seed is placed is shaken for a suitable period of time, for example, 1-3 days, or until maximum productivity obtained at a suitable temperature, for example, as described above. After the incubation period, the contents of the flask can be collected by centrifugation or filtration.

If the cultivation is carried out on a large scale, it may be preferable to culture in a suitable fermenter with aeration-mixing. In this case, the nutrient medium can be prepared directly in the fermenter. After sterilization at 125 ^° C., the medium is cooled and the inoculum previously prepared in a sterilized medium is seeded. Cultivation is carried out with stirring and aeration at 20-26 ^o C. This procedure is suitable for obtaining compounds in large quantities.

 The amount of thiomarinol B produced by the culture can be controlled from time to time by, for example, high-performance liquid chromatography. Typically, the amount of thiomarinol B produced reaches a maximum after a period of 19-200 hours.

 After a suitable growing time, thiomarinol B can be isolated and purified by any known means. For example, any amount of thiomarinol B remaining in the culture broth can be isolated by filtering off solids, for example using diatomaceous earth as a filter material, or by centrifugation followed by extraction of the supernatant and purification in accordance with the physicochemical properties of thiomarinol B. For example , thiomarinol B, which is present in the filtrate or in the residue after centrifugation, can be extracted with water-immiscible organic solvents such as ethyl acetate, chlorofor m, ethylene chloride, methylene chloride or a mixture of these solvents under neutral or acidic conditions, and then clean.

 Alternatively, activated carbon or an adsorption resin 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 B through a thin layer of adsorbent; thiomarinol B 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 B can be purified by extraction with a suitable solvent, for example, aqueous acetone or aqueous methanol, which preferably has a concentration of 50-90 vol. and removing the organic solvent, followed by extraction, as described previously for the filtrate or supernatant.

The resulting thiomarinol B can be further purified 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 in the art, these methods of isolation and purification can be carried out separately or in any convenient combination, and, if necessary, and repeatedly in order to isolate and purify the target product.

 Thiomarinol B can also be obtained by oxidation of thiomarinol, and this method is preferred since only small amounts of thiomarinol B are formed during fermentation.

 The oxidation is preferably carried out by allowing the oxidizing agent to act on thiomarinol in the presence of a solvent and in the presence or absence of a base.

 The nature of the oxidizing agent used is not very significant for the process of the invention, and with the same success, any commonly used oxidizing agent can be used in this case. Examples of such oxidizing agents are: potassium permanganate; chromates such as potassium dichromate (potassium dichromate), sodium dichromate (sodium dichromate), chromium oxide (YI), chromyl chloride and tert-butyl chromate; ruthenium tetroxide; halogens such as chlorine, bromine and iodine; ozone; oxygen; hydrogen peroxide; organic peroxides such as bis (trimethylsilyl) peroxide, cumene hydroperoxide and tert-butyl hydroperoxide; dioxiranes such as dioxirane, methyldioxirane, dimethyldioxirane, diethyl dioxirane, ethylmethyldioxirane, methylpropyldioxyran, butylmethyldioxirane, fluorodioxirane, methylfluorodioxyran, difluorodioxirane, bis (trifluoromethyl) dioxirane, methyl trifluoromethyldimethyldioxi tri di; organic peracids and their salts, such as peracetic acid, formic acid, m-chloroperbenzoic acid; and peroxysulfuric acids and their salts, such as peroxymonosulfuric acid, potassium peroxydisulfate and potassium peroxymonosulfate (especially those sold by the company Aldrich Chemical Co. under the trade name "Oxon"). Preferred examples of oxidizing agents include hydrogen peroxide, organic peroxides and their salts, organic peroxides, dioxiranes or peroxysulfuric acids and their salts, and most preferred examples are hydrogen peroxide, dimethyldioxirane and peroxysulfuric acids and their salts.

 Similarly, there are no particular restrictions on the nature of the bases used in this reaction, except that they should not adversely affect the course of the reaction and the reagents involved. Preferred examples of such bases are inorganic salts, for example, alkali metal carbonates (such as sodium carbonate, potassium carbonate or lithium carbonate), acidic alkali metal carbonates (such as sodium bicarbonate, potassium bicarbonate and lithium bicarbonate), alkali metal hydrides (such as hydride lithium, sodium hydride or potassium hydride), alkali metal hydroxides (such as sodium hydroxide, potassium hydroxide, barium hydroxide or lithium hydroxide) and alkali metal fluorides (such as sodium fluoride, potassium fluoride I or cesium fluoride); organic salts, for example alkali metal alkoxides (such as sodium methoxide, sodium ethoxide, potassium tert-butoxide or lithium methoxide), alkali metal alkyl sulfides (such as sodium methyl sulfide or sodium ethyl sulfide); and nitrogen compounds (such as triethylamine, tributylamine, di-iso-propylethylamine, N-methylmorpholine, pyridine, 4- (N, N-dimethylamino) pyridine, N, N-dimethylaniline, N, N-diethylaniline, 1,5-diaaabicylo [4.3.0] non-5-en (DBN), 1,4-diazabicylo [2.2.2] octane (DABCO) and 1,8-diazabicylo [5.4.0] undec-7-ene (DBU) Most preferred are alkali metal carbonates and acidic alkali metal carbonates.

 The reaction is usually and preferably carried out in the presence of a solvent, the nature of which does not play a big role, except that it should not have a harmful effect on the course of the reaction, but should dissolve the reagents at least to some extent. Examples of suitable solvents are: water; alcohols such as methanol, ethanol or propanol; ketones such as acetone or methyl ethyl ketone; organic acids such as acetic acid or formic acid; esters such as ethyl acetate; ethers such as diethyl ether and tetrahydrofuran; amides, such as dimethylformamide or dimethylacetamide; as well as mixtures of any two or more of these solvents. Alcohols and mixtures of water with ketones are preferred, with a mixture of water and acetone being most preferred.

 In order to accelerate the reaction, it can be carried out in the presence of an inorganic catalyst, such as platinum oxide or vanadium oxide, although the reaction proceeds without a catalyst.

The reaction can be carried out in a wide temperature range, and the selected specific temperature value does not play a big role for this invention. It was found that in the General case, the reaction is conveniently carried out in the temperature range from -78 ^o C to 100 ^o C, most preferably from -10 ^o C to room temperature. The time required to complete the reaction may also vary depending on many factors, including the process temperature and the nature of the reagents, in particular the oxidizing agent and base used. However, in most cases, a period of from 15 minutes to 30 hours is sufficient, and more preferably from 15 minutes to 2 hours.

 Upon completion of the reaction, the target compound can be isolated from the reaction mixture by conventional means. For example, a convenient isolation procedure includes the following operations: pour the mixture into water; extracting the aqueous phase with a water-immiscible solvent such as an aromatic hydrocarbon (e.g. benzene), ether (e.g. diethyl ether), an organic acid ester (e.g. ethyl acetate) or a halogenated hydrocarbon (e.g. methylene chloride); and removing the solvent from the extract. The target compound obtained in this way, if necessary, is subjected to further purification by conventional methods, for example, using various chromatographic methods or preparative thin layer chromatography.

 Thiomarinol, which is the starting compound in this oxidation reaction, can be obtained by fermenting microorganisms of the Alteromonas species, in particular the Alteromonas rava SANK 73390 strain, previously described with respect to the preparation of thiomarinol B.

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

 When the compounds of the invention are intended to be used for therapeutic purposes, they can be administered alone or in the form of a pharmaceutical formulation containing, in addition to the active compound, one or two standard diluents, carriers, excipients or excipients. The nature of the finished formulation of course depends on the method of taking the medicine. For oral administration, the compound is preferably formed in the form of powders, granules, tablets, capsules or syrups. For parenteral administration, it is preferably formulated as injections (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, solubilizing agents, flavoring agents, perfumes, suspending agents or coating agents 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, and the method of prescribing the drugs, in the case of oral administration to an adult patient, the compounds of the invention can usually be taken as a daily dose of 20 to 2000 mg Compounds can be taken in a single dose or in divided doses, for example, two or three times a day.

 The preparation of the compounds of the invention is further illustrated by the following examples, which do not limit the invention, and the biological activity of these compounds is illustrated further in the test examples. Example 6 shows a method for producing thiomarinol, which is used as a starting compound in the oxidative method for the preparation of thiomarinol B.

 Example 1. Obtaining thiomarinol B by growing in tenka.

 A) Culture.

Alteromonas rava strain SANK 73390 was grown for 3 days at 22 ^° C on mown sea agar (Difco product). The resulting culture is suspended in 3 ml of sterilized artificial sea ox. 0.1 ml of the resulting suspension is inoculated into each of two Erlenmeyer flasks with a capacity of 500 ml each, containing 100 ml of sterilized medium having the following composition:
Sea broth (Difco) 37.4 g
Deionized Water 1000 ml
pH Not set.

The culture is then incubated for 24 hours at 23 ^° C. with shaking at a speed of 210 rpm using a rotary shaker. The whole culture was then inoculated into a 600-liter aerated pan equipped with a stirrer and containing 200 L of medium having the same composition as above, which was sterilized separately. The mixture was then cultured for 26 hours at 23 ^° C., while the aeration of the culture was carried out with air at a speed of 0.5 v / v. min (that is, "volume per volume per minute": 1 rpm.min. means that the amount of air supplied in 1 min is equal to the volume of air in the tenk), and the rotation speed is set in the range 82.5-170 rpm so as to maintain a dissolved oxygen concentration of 5.0 ppm.

 C) Isolation.

 A sufficient amount of hydrochloric acid solution is added to 230 L of the resulting culture solution in order to bring the pH to 2.5. Then, 200 L of acetone was added and the mixture was extracted with stirring for 0.5 h. 4.0 kg of Celite 545 (trade name of the product from Johnes Manvill Project Corporation, USA) was added to the mixture as a filter medium, and the mixture was filtered. The filtrate (430 L) was extracted once with 200 L of ethyl acetate, and then twice with 100 L of ethyl acetate. The combined ethyl acetate extracts were washed with 200 L of a 5% aqueous solution of sodium bicarbonate and then with 100 L of a saturated aqueous solution of sodium chloride, after which they were dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to obtain approximately 80 g of an oil.

 All the oil obtained in the previous step is dissolved in methylene chloride, and the solution is placed in a column containing 1.1 kg of silica gel saturated with methylene chloride. The solution is then eluted with a mixture of methylene chloride and ethyl acetate (1: 1), then with ethyl acetate alone and finally with a mixture of ethyl acetate and methanol (9: 1) (the polarity of the solvent increases in this order). After the eluate was fractionated into 500 ml portions each, a fraction containing thiomarinol B, which was eluted with a mixture of ethyl acetate and methanol, was collected and evaporated to dryness under reduced pressure to obtain 60 g of an oil.

 All the resulting oil was dissolved in 6 L of a 50% aqueous methanol solution, and the resulting solution was placed in a 2.3 L column filled with Diaion HP-20 support (trade name) saturated with water, after which the oil solution was subjected to gradient elution with an aqueous solution methanol, the concentration of which is increased from 30 to 90 vol. More specifically, after 4 L of each of an aqueous solution containing 30% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol is applied to the column, the column is eluted with a 90% aqueous methanol solution. Elution is continued until the elution of the target compound is no longer observed, which is controlled by high-performance liquid chromatography, while the total amount of 90% methanol used in the elution is 10 l. The fraction eluted with 90% methanol was collected and evaporated to dryness under reduced pressure to obtain 3.8 g of a yellow powder. The powder was subjected to column chromatography with 320 g of a Sephadex LH-20 support saturated with a mixture of methylene chloride, ethyl acetate and methanol (19: 19: 2 by volume), eluting with a mixture of the same composition for purification.

 The resulting product was further purified by high-resolution liquid chromatography using a reverse phase column [Senshu-Pak ODS H-5251 (Column size: diameter 20 mm, length 250 mm), a trademark of Senshu Scientilic Co. Ltd. and 40 vol. a solution of acetonitrile in water as an eluent at a flow rate of 15 ml / min; the control of the cleaning method is carried out by absorption at 220 nm. Since thiomarinol B emerges as a peak with a retention time of 13-14 minutes, this fraction is collected and evaporated to dryness under reduced pressure, isolating 130 mg of the target compound, which has the physicochemical properties described above.

 Example 2. Obtaining thiomarinol B by oxidation of thiomarinol.

 100.9 mg of thiomarinol (prepared as described in Example 5) was dissolved in a mixture of 5 ml of acetone and 5 ml of water, and the resulting solution was cooled in an ice bath. 112.2 mg of oxone (trade name of a product from Aldrich Chemical Co. Inc.) was added and the mixture was stirred while cooling in an ice bath for 40 minutes. At the end of the indicated period, 1.2 ml of a saturated aqueous sodium bicarbonate solution are added, and the mixture is again stirred under cooling in an ice bath for 30 minutes, 5 ml of water are added and extracted with a mixture of methylene chloride and tetrahydrofuran 10: 1. The extract is dried over anhydrous sulfate sodium and the solvent are then removed by distillation under reduced pressure. The residue was separated and purified by reverse phase high-resolution liquid chromatography (using a Senshu Pak ODS-5251-N column and a 40 vol. Acetonitrile aqueous solution as eluent) to obtain 79.5 mg (75% yield) of light yellow thiomarinol B, which has the physico-chemical parameters given above.

 Example 3. Obtaining thiomarinol B by the oxidation of thiomarinol.

 100 mg of thiomarinol (prepared as described in Example 5) is dissolved in a mixture of 15 ml of acetone and 7.5 ml of water, and then 0.074 ml of a 35% aqueous solution of hydrogen peroxide and 2 drops of diluted aqueous sodium bicarbonate are added. The resulting mixture was stirred for 5 minutes and acetone was removed by distillation under reduced pressure. Acetonitrile was added to the residue, the formation of thiomarinol B was confirmed by reverse phase high-resolution liquid chromatography (using a Senshu Pak ODS-H-5251 column and 40 vol. Acetonitrile aqueous solution as eluent). The solvent was removed by distillation under reduced pressure, and the residue was dissolved in 40% aqueous acetonitrile. It is then separated and purified by reverse phase high-resolution liquid chromatography (using a Senshu Pak ODS-5251-N column and 40 vol. Acetonitrile aqueous solution as eluent) to give 43.2 mg (41% yield) of light yellow thiomarinol B , which has the physico-chemical parameters given above.

 Example 4. Obtaining thiomarinol B by oxidation of thiomarinol.

 100 g of thiomarinol (obtained as described in Example 5) is dissolved in 40 ml of acetone and then, while cooling in an ice bath and with stirring, 134 mg of m-chloroperbenzoic acid are added to the resulting solution. The mixture was further stirred at room temperature for 1.5 hours, extracted with methylene chloride and washed three times with water and then once with a saturated aqueous solution of sodium sulfate. The extract was dried over anhydrous sodium sulfate, and the solvent was then removed by distillation under reduced pressure. The residue was separated and purified by reverse phase high-resolution liquid chromatography (using a Senshu Pak ODS-5251-N column and a 40 vol. Acetonitrile aqueous solution as eluent) to give 8.5 mg (8% yield) of light yellow thiomarinol B, which has the physico-chemical parameters given above.

 Example 5. Fermentation of thiomarinol in a vessel.

 A) Culture.

The strain Alteromonas rava SANK 73390 was grown for 3 days at 22 ^° C on mown sea agar (Difco product). The resulting culture is suspended in 3 ml of artificial sea water. 0.1 ml of the resulting suspension is aseptically added to a 500 ml Erlenmeyer flask containing 100 ml of sterilized medium [37.4 g of sea broth (product of Difco) in 1 l of deionized water, pH is not established]
The flask is then incubated for 24 hours at 23 ^° C. with shaking at a speed of 200 rpm (rotation radius 70 mm) using a rotary shaker. After this time, 15 ml of culture taken aseptically from an Erlenmeyer flask are inoculated into each of the four jug-type fermenters with a capacity of 30 L, each containing 15 L of the sterile medium described above. The fermenters are incubated for 23 hours at 23 ^° C. and stirred at a speed of 100 rpm; the aeration rate is 7.5 l / min.

 C) Isolation.

 After 23 hours, the contents of the fermenters are combined to give 60 L of culture fluid. The pH of the liquid was adjusted to 3 with hydrochloric acid, and then 60 L of acetone was added and the mixture was extracted with stirring for 30 minutes. The solution was filtered using 1.2 kg of Celite 545 (a trademark of Johns Manville Co.) as a filter aid. 110 L of the obtained filtrate is then extracted once with 60 L of ethyl acetate, and then twice with 30 L of ethyl acetate. The extracts in ethyl acetate are combined, washed with 30 l of a 5% aqueous solution of sodium bicarbonate, and then with 30 l of a saturated aqueous solution of sodium chloride, after which they are dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to obtain 14 g of an oily substance.

 All the oily substance obtained in the previous step is dissolved in methylene chloride and the solution is placed in a column containing 200 g of silica gel saturated with methylene chloride. The solution is eluted first with a mixture of methylene chloride and ethyl acetate (1: 1), then with ethyl acetate alone and finally with a mixture of ethyl acetate and methanol (9: 1), the polarity of the solvent increases in this order. The eluent was collected as 18 ml fractions and fractions eluted with a mixture of ethyl acetate and methanol, which contained thiomarinol, were retained.

 The separated fractions were evaporated in vacuo to give 7 g of an oily substance, which was dissolved in 400 ml of a 50% aqueous methanol solution, and the resulting solution was placed in a 600 ml column filled with Diaion HP-20 support (trademark of Mitsubishi Chem. Ind .) saturated with water. The column is washed with a 50% aqueous methanol solution and the target product is eluted with a 90% aqueous methanol solution. By evaporation under reduced pressure, 1 g of a yellow powder is obtained from the fraction. The yellow powder was further eluted on a Sephadex LH-20 chromatographic column with a mixture of methylene chloride, ethyl acetate and methanol (19: 19: 2). 750 mg of thiomarinol are isolated from the active fractions in the form of a yellow powder.

 The obtained thiomarinol has the following characteristics.

 1) Appearance: yellow powder.

2) Melting point: 84-89 ^o C.

3) The molecular formula is C ₃₀ H ₄₄ N ₂ O ₉ S ₂ .

 4) Molecular mass: 640 (according to FAB mass spectrometry). FAB mass spectrometry fast atom bombardment mass spectrometry.

5) High-resolution mass spectrum:
C ₃₀ H ₄₄ N ₂ O ₉ S ₂ [N + H] ⁺ ; according to FAB mass spectrometry).

 Found 641.2567.

 Calculated 641.2585.

 6) Elemental analysis.

 Calculated, C 56.23; H 6.92; N, 4.37; S 10.01.

 Found, C, 55.92; H, 6.82; N, 4.23; S 9.90.

7) IR spectrum: in the IR spectrum (method with a KBr disk, n _max cm ^-1 ) the following peaks are observed:
3394, 2930, 1649, 1598, 1526, 1288, 1216, 1154, 1102, 1052.

8) UV spectrum: λ _max nm (s).

The UV spectrum, measured in methanol or a mixture of methanol + hydrochloric acid, is presented below:
387 (12000), 300 (3500), 214 (26000).

The UV spectrum measured in methanol + sodium hydroxide mixture is presented below:
386 (9600), 306 (3200), 206 (25000).

9) Specific rotation:
[α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = + 4.3 ^° (c = 1.0; methanol).
10) 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.

 The flow rate of the eluent: 1.5 ml / min.

 Wavelength: 220-350 nm (detector photodiode array).

 Retention time: 5.9 minutes

11) ¹ H-NMR spectroscopy (δ ppm).

NMR spectrum (270 MHz), measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as an internal standard, is presented below:
0.91 (3H, doublet, J = 6.8 Hz), 0.95 (3H, doublet, J = 5.9 Hz),
1.30 (6H, broad multiplet), 1.55 (5H, wide multiplet), 2.03 (3H, singlet), 2.09 (3H, multiplet),
2.34 (2H, triplet, J = 7.3 Hz), 3.33 (1H, doublet, J = 10.7 Hz),
3.52 (2H, multiplet), 3.64 (2H, multiplet), 3.73 (1H, doublet of doublets), 4.02 (2H, triplet, J = 6.6 Hz), 4.18 (1H, wide doublet, J = 7.3 Hz), 4.30 (1H, doublet, J = 4.4 Hz), 4.44 (1H, doublet, J = 7.8 Hz), 4.63 (1H, doublet , J = 3.4 Hz), 4.89 (1H, doublet, J = 7.3 Hz), 5.37 (2H, multiplet), 5.97 (1H, broad singlet), 7.04 (1H, singlet), 9.80 (1H, broad singlet), 10.68 (1H, broad singlet).

12) ¹³ C-NMR spectrum: (dpm).

NMR spectrum (68 MHz), measured in hexadeuterated dimethyl sulfoxide using tetramethylsilane as an internal standard, is presented below:
16.3 (quartet), 16.6 (quartet), 20.3 (quartet),
26.7 (triplet), 27.0 (triplet), 29.7 (triplet),
30.0 (triplet), 30.1 (triplet), 33.4 (triplet),
36.6 (triplet), 43.9 (doublet), 45.3 (doublet),
64.9 (triplet), 65.7 (doublet), 66.0 (triplet),
71.8 (doublet), 72.1 (doublet), 74.4 (doublet),
77.6 (doublet), 113.7 (doublet), 115.8 (singlet),
116.3 (doublet), 129.8 (doublet), 135.1 (singlet),
135.7 (doublet), 137.9 (singlet), 161.1 (singlet),
168.6 (singlet), 170.4 (singlet), 174.3 (singlet).

 13) 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.

 14) Color reactions.

 Positive with sulfuric acid, iodine and potassium permanganate.

 15) Thin layer chromatography.

The value of R _f 0,57.

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

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

 Biological activity.

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

 Test Test Example 1.

 Antibacterial activity of thiomarinol B.

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

 The results are presented in table. 2.

 Test Test Example 2.

 Antimycoplasmic activity of thiomarinol B.

 By a similar methodology described in test test example 1, the activity of thiomarinol, thiomarinol B, and pseudomonoic acid A (designated “A”, “B”, and “P”, respectively) with respect to various mycoplasma samples is quantified. The results are presented in table. 3.

Inoculum: 0.005 ml 10 ⁵ CFU / ml.

 The environment for analysis.

 For all microorganisms, tests of thiomarinol B and pseudomonoic acid A are carried out in Chenok's medium (obtained by the method described in P.N.A.S. v. 48, 41-49 (1962) with the addition of 20% horse serum).

 Thiomarinol is analyzed in the following media.

 M. bovis and M. gallisepticum: Chanock medium (prepared as described above).

M. hyosynoviae: agar medium with PPLO mucin (*) (supplemented with 15% horse serum),
(*) PPLO pleuro-pneumonia-like organism
PPLO broth without CV (Difco) 21 g
Bacteriological mucin (Difco) 5 g
Distilled water 800 ml
Noble agar (Difco) 12 g
Horse serum 150 ml
25% fresh yeast extract 50 ml
Culture conditions: 37 ^° C., 5 days, slightly aerobic medium (BBL gas wrap method [single-use CO ₂ generator from Becton Dickinson Microbiology Sysyems, Kokkisville, Maryland, USA]).

 From the presented results it is clear that thiomarinol B 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 (8)

1. Thiomarinol B Formulas

2. A method of producing thiomarinol B, characterized in that the microorganism of the species Alteromonas rava is cultured in a nutrient medium containing sources of carbon, nitrogen and inorganic salts, under aerobic conditions, followed by isolation of the target product.  3. The method according to claim 2, characterized in that the strain Alteromonas rava SANK 73390 is cultivated, registered under depository number FERM BP-3381. 4. The method of obtaining thiomarinol B according to claim 1, characterized in that the thiomarinol of the formula

subjected to reaction with an oxidizing agent.  5. The method according to claim 4, characterized in that the oxidation is carried out using an oxidizing agent selected from the group consisting of potassium permanganate, chromates, ruthenium tetroxide, halogens, ozone, oxygen, hydrogen peroxide, organic peroxides, dioxiranes, organic peracids and their salts and supra-sulfuric (peroxysulfuric) acids and their salts.  6. The method according to claim 4, characterized in that they use an oxidizing agent selected from the group consisting of hydrogen peroxide, organic peracids and their salts, organic peroxides, dioxiranes and sulfuric acids and their salts.  7. The method according to claim 4, characterized in that they use an oxidizing agent selected from the group consisting of hydrogen peroxide, dimethyldioxirane and sulphurous acids and their salts.  8. The method according to claim 4, characterized in that they use an oxidizing agent selected from the group consisting of potassium permanganate, potassium dichromate, sodium dichromate, chromium oxide (VI), chromyl chloride, tert-butyl chromate, ruthenium tetraoxide, chlorine, bromine , iodine, ozone, oxygen, hydrogen peroxide, bis (trimethylsilyl) peroxide, cumyl hydroperoxide, tert-butyl hydroperoxide, dioxirane, methyldioxirane, dimethyldioxirane, diethyl dioxirane, ethylmethyldioxirane, methylpropyldioxirane, butyloxydio-dioxydio-dioxydio-dioxydio-dioxydio-dioxydio-dioxydio-dioxydio-dioxydio-dioxydio-dioxydio ftordioksirana, bis (trifluoromethyl) dioxirane, metiltriftormetildioksirana, triftormetilhlordiftormetildioksirana, peracetic acid, performic acid, m-chloroperbenzoic acid, peroxymonosulfuric acid, potassium peroxydisulphate and potassium peroxymonosulphate. Priority on points:
09/18/92 according to claims 1 3.  11/05/92 according to claims 4-8.

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