Antibiotic compounds

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I and their pharmaceutically acceptable salts, method for preparing them by the fermentation of a microorganism of the species Streptomyces (PM0626271/MTCC 5447), and to their pharmaceutical compositions containing one or more compounds of formula I as an active ingredient.

EFFECT: compounds of formula I are used for treating and preventing the diseases caused by bacterial infections.

4 cl, 3 dwg, 6 tbl, 9 ex

 

The scope of the invention

The invention relates to new compounds of the formula I, characterized antibacterial activity. The compound can be obtained by fermentation of a microorganism belonging to the species Streptomyces (PM0626271/MTCC 5447). This invention also includes all stereoisomeric forms and all tautomeric forms of the compounds of formula I, and pharmaceutically acceptable salts and derivatives thereof. This invention also relates to methods for new antibacterial compounds and to pharmaceutical compositions containing one or more of the novel compounds as active ingredient, to their use in medicinal preparations for the treatment and prevention of diseases caused by bacterial infections.

Background of the invention

A sharp increase in the spread of antibiotic resistance among bacteria currently represents a serious threat to public health worldwide. Of particular concern are infections caused by methicillin-resistant Staphylococcus aureus (MRSA) resistant to penicillin Streptococcus pneumoniae (PRSP), vancomycin-resistant Enterococcus (VRE) (Clin. Environ. Infect., 2005, 11, Supplement 3: 22-28) and is resistant to multiple drugs (MDR) Mycobacterium tuberculosis (Eur. Respir. J., 2002, Supplement 36, 66S-77S).

Thiostrepton, an antibiotic isolated from Streptomyces azureus, as obsoles, an effective anti-infective drug, characterized by shared spectrum antibiotic, such as penicillin, and is used against gram-positive coccal infections (U.S. patent 2982689).

Siomycin, sulfur-containing peptide antibiotic isolated from Streptomyces sioyaensis, as reported, is active against gram-positive bacteria and mycobacteria with little or no activity against gram-negative bacteria (The Journal Of Antibiotics, 1969, 364-368).

There is a need to provide new compounds that can be used as drugs for the treatment of patients who are at risk of infection or infected with bacteria, especially resistant to multiple drugs by bacteria, such as MRSA, VRE and Mycobacterium tuberculosis.

Brief description of the invention

This invention relates to new compounds of formula I.

This invention also relates to novel purified compounds of the formula I, selected from the fermentation broth of a microorganism belonging to the species Streptomyces (PM0626271/MTCC 5447).

This invention also relates to all stereoisomeric forms and all tautomeric forms of the compounds of formula I and their pharmaceutically acceptable salts and derivatives.

The compounds of formula I and their isomers, pharmaceutics is acceptable salts and derivatives have antibacterial activity and are applicable for the treatment or prevention of diseases, caused by a bacterium that is extremely resistant to multiple drugs by bacteria, such as MRSA, VRE and Mycobacterium tuberculosis.

This invention also relates to pharmaceutical compositions comprising one or more of the new compounds of the formula I, their isomer, pharmaceutically acceptable salt or derivative as an active ingredient for the treatment of diseases caused by bacteria, especially resistant to multiple drugs by bacteria, such as MRSA, VRE and Mycobacterium tuberculosis.

This invention also relates to methods of preparing compounds of the formula I and/or their isomers from a microorganism belonging to the species Streptomyces (PM0626271/MTCC 5447).

A brief description of graphic materials

Figure 1 illustrates the1H NMR spectrum (500 MHz; Bruker Instrument) the compounds of formula I(a) in CDCl3:CD3OD (4:1).

Figure 2 illustrates the1H NMR spectrum (75 MHz; Bruker Instrument) the compounds of formula I(a) in CDCl3:CD3OD (4:1).

Figure 3 illustrates the1H NMR spectrum (500 MHz; Bruker Instrument) the compounds of formula I(b) in CDCl3:CD3OD (4:1).

Detailed description of the invention

Before describing this invention in detail it should be understood that this invention is not limited to the specific implementation options. Also it should be understood that the terminology used in this document is ente, merely serves the purpose of describing particular embodiments only and is not intended to limit.

As used in the Description and the claims, the singular number shall include references to the plural, unless the context otherwise indicated.

If not stated otherwise, all technical and scientific expressions used in this document, are characterized by the same meaning as commonly understood by a person skilled in the art to which this invention.

As used herein, the term “derivative” refers to a compound that is obtained from such connection, or connection, which, as you can imagine, comes from a different connection, if one atom is replaced by another atom or group of atoms.

As used herein, the term “stereoisomer” refers to all individual isomers of compounds that differ only in the orientation of their atoms in space. The expression stereoisomer includes isomers in mirror image (enantiomers), mixture of isomers in mirroring (racemates, racemic mixture, geometric (CIS/TRANS or SYN/anti, or E/Z isomers and isomers of compounds with more than one chiral center that are not mirror images of each other (diastereoisomer). Connection Yes is tion of the invention may have asymmetric centers and occur as racemates, racemic mixtures, individual diastereoisomer or the enantiomers or may exist as geometric isomers with all isomeric forms of these compounds, which are included in this invention.

As used herein, the expression “tautomer” refers to the co-existence of two (or more) compounds, which differ from each other only by the position of one (or more) moving atoms and the distribution of electrons, such as keto-enol and Imin-enaminone the tautomers.

As used herein, the term “fermentation broth” refers to a microbial suspension culture in a nutrient medium containing compounds obtained by microbes during their growth, as well as containing nepotreblenie nutrients.

As used herein, the term “mutant” refers to an organism or cell that carry the mutation, which is a phenotype alternative to wild type.

As used herein, the term “variant” refers to an individual organism, which is recognizably different from the conventional standard type among these types.

The new compounds of formula I is structurally represented by the following formula:

The new compounds of formula I(a) have the molecular formula C71H 83N19O18S5(molecular weight 1649,5). A new compound of formula I(b) has the molecular formula C71H85N19O18S5(molecular weight 1651,5). The new compounds of formula I(a) and formula I(b) can be characterized by one or more of their physico-chemical and spectral properties, such as high performance liquid chromatography (HPLC), mass spectrum (MS), infrared radiation (IR) and spectroscopic data of nuclear magnetic resonance (NMR), as discussed later in this document.

Was known about the structure of the new compounds of formula I(a) and formula I(b), and its full characterization performed using HPLC, MS with high resolution (HRMS), IR and NMR spectroscopic data. The compounds of formula I(a) and formula I(b) are new antibiotics active against bacteria, especially resistant to multiple drugs to the bacteria, such as MRSA, VRE and Mycobacterium tuberculosis. The compounds of formula I(a) and formula I(b) are not presented earlier structures.

The microorganism which can be used to obtain the compounds of formula I(a) and formula I(b) is a strain of Streptomyces species (PM0626271/MTCC 5447), later in this document called culture No. PM0626271 isolated from soil sample collected from the Schirmacher Oasis in the Antarctic region.

This invention chrome is in represents methods of making compounds of formula I(a) and formula I(b) of culture No. PM0626271, which includes stages: cultivation of culture No. PM0626271 in aerobic conditions by immersion in a nutrient medium containing one or more carbon sources and one or more nitrogen sources, and optional nutrient inorganic salts and/or trace elements; selection of compounds of formula I(a) and formula I(b) of the fermentation broth; and purification of the compounds of formula I(a) and formula I(b) using the cleaning procedures, usually used in the respective field.

Preliminary identification of culture No. PM0626271, which produces the compounds of formula I(a) and formula I(b), was carried out by examining the morphology of the colonies, examination of wet preparations and reaction to gram staining. Microscopic examination of strain cultures No. PM0626271 was performed on agar for isolation of actinomycetes (AS-AIA; for details, see Examples) containing 1.5% agar, and observations were made on days 1, 2 and 3 of incubation at 25°C.

Growth on AS-AIA containing 1.5% agar, was manifested in the form colonies with a diameter of 1 mm with white sporulation, scanty yellow substrate mycelium, slightly elevated, no diffusible pigment and dark brown color of the reverse side. When fitokontrol luminous microsc the FDI at 400-fold increase observed wavy plexus slim mycelium with the tops of sporulation. They are gram-positive. Free spores are fixed. On the observed morphology of this organism is classified as a member of a family of Streptomycetes.

Culture No. PM0626271 were deposited in the Collection of standard microbiological cultures (MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh -160 036, India, recognized by the world intellectual property organization (WIPO) as an international Depositary authority (IDA), and received inventory number MTCC 5447.

In addition to the specific microorganism described herein, it should be understood that mutants, such as obtained by the use of chemical or physical mutagens, including x-rays, ultraviolet radiation, and so on, and organisms whose genome organization has been modified by molecular biology can also be cultivated to produce compounds of formula I(a) and formula I(b).

Screening at the applicable mutants and variants, which can produce compounds according to this invention can be confirmed using HPLC and/or by determination of the biological activity of the active compounds accumulated in the fermentation broth, for example, by testing compounds for antibacterial activity.

Environment and/or culture medium used for isolation and cultivation of culture No. PM062271, which produces the compounds of formula I(a) and formula I(b) preferably contains sources of carbon, nitrogen and inorganic nutrient salts. The carbon sources are, for example, one or more of starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose or galactose. Preferred carbon sources are soluble starch and glucose. The nitrogen sources are, for example, one or more of the soy flour, peanut flour, yeast extract, beef extract, peptone, malt extract, liquid corn extract, gelatin or Kazimirovich acids. Preferred nitrogen sources are peptone and yeast extract. Nutrient inorganic salts are, for example, one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride, strontium chloride, cobalt chloride, potassium bromide, sodium fluoride, sodium hydrogen phosphate, potassium hydrogen phosphate, dicale hydrogen phosphate, magnesium phosphate, calcium carbonate, sodium bicarbonate, sodium silicate, ammonium nitrate, potassium nitrate, ferrous iron sulfate, sodium sulfate, ammonium sulfate, magnesium sulfate, ferric citrate, boric acid or salts solution in trace concentrations. Preferred are calcium carbonate, sodium chloride and MAG the Oia chloride.

Maintaining culture No. PM0626271 can be performed at temperatures ranging from 22°C to 36°C and pH from about 7.5 to 8.0. Usually culture No. PM0626271 maintained at 25°C-27°C and pH from about 7.4 to 7.8. Well growing culture can be stored in the refrigerator at 4°C-8°C.

Cultivation of inoculated culture No. PM0626271 can be performed at a temperature ranging from 25°C to 36°C and pH from about 7.5 to 8.0 within 66 hours to 75 hours at 200 rpm (revolutions per minute) up to 280 rpm. Usually sown culture No. PM0626271 cultivated at 29°C-31°C and pH from about 7.4 to 7.8 for 72 hours at 230 rpm 250 rpm.

Obtaining compounds of formula I(a) and formula I(b) can be performed by culturing the culture No. PM0626271 fermentation at temperatures ranging from 26°C to 36°C and a pH of about 6.5-8.5 for 24 hours to 96 hours at 60 rpm 140 rpm and aeration of 100 liters per minute up to 200 liters per minute. Usually culture No. PM0626271 cultivated at 30°C-32°C and a pH of 7.4-7.8 for over 40 hours to 96 hours at 90 rpm and aeration 110 liters per minute.

Obtaining compounds of formula I(a) and formula I(b) can be performed by culturing the culture No. PM0626271 in the applicable nutrient broth under conditions described herein, preferably under aerobic conditions by submerged, e.g. in shake flasks, as well as in laboratory fermenters. Development of fermentation and the formation of compounds of the forms of the crystals I(a) and formula I(b) can be detected by high-performance liquid chromatography (HPLC) and measurement of the biological activity of fermentation broth by staphylococcal and/or Enterococcus species known method of microbial analysis diffusion in agar. The preferred culture is Staphylococcus aureus E710, which is a strain of methicillin-resistant, β-lactam antibiotic, which is confirmed in the literature, and Enterococcus faecium R2 (VRE), which is resistant to vancomycin. In the resulting fermentation broth of the compounds of formula I(a) and formula I(b) are present in the culture filtrate and cell mass, and can be selected using known separation techniques such as solvent extraction and column chromatography. Thus, the compounds of formula I(a) and formula I(b) can be extracted from the culture filtrate by extraction at a pH of from about 5 to 9 water-immiscible solvent such as petroleum ether, dichloromethane, chloroform, ethyl acetate, diethyl ether or butanol, or by chromatography with hydrophobic interaction with polymeric resins such as Diaion HP-20®" (Mitsubishi Chemical Industries Limited, Japan), "Amberlite XAD®" (Rohm and Haas Industries U. S. A.), activated charcoal, or by ion-exchange chromatography at pH 5-9. The active material can be extracted from the cell mass by extraction miscible with water solvent, such as methanol, acetone, acetonitrile, n-propanol or isopropanol, or water-immiscible solvent such as petroleum ether, dichloromethane, chloroform is, the ethyl acetate or butanol. Another option is the extraction of the whole broth by solvent is selected from petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol, acetone, acetonitrile, n-propanol, isopropanol or butanol. Usually active material is extracted with ethyl acetate from a whole broth. Concentration and lyophilization of the extracts gives active untreated material.

The compounds of formula I(a) and formula I(b) in this invention can be extracted from the crude material by fractionation using any of the following methods: normal-phase chromatography using alumina or silica gel as a stationary phase; eluents, such as petroleum ether, ethyl acetate, dichloromethane, acetone, chloroform, methanol or combinations thereof); reversed-phase chromatography (using reversed-phase silica gel, such as dimethyloctadecyl silica gel (RP-18) or dimethyloctadecyl silica gel (RP-8) as a stationary phase; and eluents such as water, buffers (e.g. phosphate, acetate, citrate (pH 2-8)], and organic solvents (e.g. methanol, acetonitrile, acetone, tetrahydrofuran, or combinations of these solvents); gel permeation chromatography (using resins such as Sephadex LH-20®(Parmacia Chemical Industries, Sweden), TSKgel®Toyopearl HW (TosoHaas, Tosoh Corporation, Japan) in solvents, such as methanol, chloroform, acetone, ethyl acetate, or combinations thereof, or Sephadex®G-10 and G-25 in water); or countercurrent chromatography (using a two-phase suantai system derived from two or more solvents such as water, methanol, ethanol, isopropanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride, chloroform, ethyl acetate, petroleum ether, benzene and toluene). These techniques can be used multiple times, separately or in combination. A typical method is chromatography on normal phase using silica gel.

The compounds of formula I(a) and formula I(b) and their stereoisomers can be converted into their pharmaceutically acceptable salts and derivatives, all of which are addressed by the present invention.

Salt compounds can be obtained by standard procedures known to the person skilled in the art, for example salts, such hydrochloric, and sulfate salts can be obtained by treating compounds of formula I(a) and formula I(b) and their isomers corresponding acid, for example hydrochloric acid, sulphuric acid.

The compounds of formula I(a) and formula I(b) have antibacterial activity against a wide range of bacterial strains. The compounds of formula I(a and formula I(b) are also characterized by protivokataralnoe activity to MDR strains of Mycobacterium tuberculosis, such as M. tuberculosis H37Rv; clinical isolate of M. tuberculosis are resistant to S (streptomycin), H (isoniazid or isonicotinohydrazide), R (rifampicin) and E (ethambutol); and a clinical isolate of M. tuberculosis sensitive to S, H, R, and E.

One or more of the compounds of formula I(a) and formula I(b), their stereoisomers and their pharmaceutically acceptable salts, separately or together, can be introduced animals, such as mammals, including humans, as pharmaceuticals and in the form of pharmaceutical compositions. One or more of the compounds of formula I(a) and formula I(b), their stereoisomers and their pharmaceutically acceptable salts, separately or together, can be entered prophylactically to a patient with the risk of being infected with a bacterial infection. The patient may be someone who may be exposed to the bacteria in a medical facility, such as a hospital or other institution where there may be bacteria.

Therefore, this invention also relates to compounds of formula I(a) and formula I(b), their stereoisomers and their pharmaceutically acceptable salts for use as pharmaceuticals and to the use of compounds of formula I(a) and formula I(b), their stereoisomers and their pharmaceutically acceptable salts for the manufacture of medical preparations with antibacterial activity is updated.

In addition, the present invention relates to pharmaceutical compositions that contain an effective amount of one or more compounds of formula I(a) and formula I(b) and/or stereoisomers and/or one or more of their pharmaceutically acceptable salts and/or derivatives thereof, together with at least one pharmaceutically acceptable excipient or carrier, applicable to prevent or treat bacterial infections.

An effective amount of compounds of formula I(a) and formula I(b) or stereoisomers, or pharmaceutically acceptable salts or derivatives thereof as the active ingredient in the pharmaceutical preparations normally is from about 0.01 mg to 1000 mg

This invention also relates to a method of treating or preventing a bacterial infection comprising the administration to a mammal in need, an effective amount of one or more of the compounds of formula I(a) and formula I(b), and/or stereoisomers and/or one or more pharmaceutically acceptable salts.

This invention also relates to a method of manufacture of a medical preparation containing one or more of the compounds of formula I(a) and formula I(b), and/or stereoisomers and/or one or more of their pharmaceutically acceptable salts, for the treatment or prevention of Zabol the requirements, bacterial infections.

Compounds according to this invention is particularly applicable as an antibacterial means. The invention therefore relates to the use of one or more of the compounds of formula I(a) and formula I(b) and/or stereoisomers and/or one or more pharmaceutically acceptable salts and/or derivatives thereof, for the manufacture of a medicinal product for preventing or treating diseases caused by bacterial infections.

Bacterial infections, treatable by the compounds according to this invention, can be caused by a bacterium belonging to the species Staphylococcus, Streptococcus, Enterococcus, Bacillus or Mycobacterium. Bacteria belonging to the Staphylococcus species may be resistant to methicillin or vancomycin. The bacterium belonging to Enterococcus species may be resistant to vancomycin. Bacteria belonging to the Mycobacterium species may be resistant to multiple drugs.

The expression “species Staphylococcus” refers to gram-positive bacteria, which under the microscope look like bunches of grapes, and as large, round, Golden-yellow colonies, often with β-hemolysis when grown in cups with blood agar. Types of Staphylococus include Staphylococcus aureus.

The expression “Streptococcus species” refers to a genus of spherical gram the good bacteria and the member type Firmicutes. Streptococci are lactic acid bacteria. Types include Streptococcus bacteria, such as S. hemolyticus, S. mitis, S. salivarius, S. pneumoniae. Species of Streptococcus are responsible for infectious diseases such as meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis (“carnivorous” bacterial infection).

The expression “Enterococcus species” refers to a genus of lactic acid bacteria type Firmicutes. They are gram-positive cocci, often found in pairs (diplococci, such as Diplococcus pneumoniae). Enterococci are facultative anaerobic organisms.

Expression of the Bacillus species” refers to a large number of diverse, rod-shaped gram-positive bacteria, which are movable through the peritrichous flagella, and aerobic, such as B. anthracis, B. subtilis, or anaerobic, such as Clostridium spp., for example, C. difficile. These bacilli belong to the type Firmicutes.

The term “Mycobacterium species” refers to gram-positive, fixed, pleomorphism sticks, related actinomycetes. Tuberculosis in humans is called Mycobacterium tuberculosis. MDR-TB (Mycobacterium tuberculosis resistant to multiple drugs) outlines the strains of tuberculosis that are resistant to at least two TB drugs of the first line, isoniazid and rifampicin.

Connected to the I according to this invention can be administered orally, nasal, topically, parenterally, for example subcutaneously, intramuscularly, intravenously, or by other routes of administration.

Pharmaceutical compositions that contain one or more of the compounds of formula I(a) and formula I(b), or stereoisomer, or pharmaceutically acceptable salt, or a derivative thereof, optionally with other pharmaceutically acceptable excipient or carrier, can be obtained by mixing the active compounds with one or more pharmaceutically acceptable excipients and/or carriers, such as humectants, soljubilizatory, such as surfactants, environment, tools, regulatory toychest, fillers, dyes, tools, masking flavor, grease, disintegrant, diluents, binders, plasticizers, emulsifying agents, ointment bases, softeners, thickeners, polymers, fats, oils, joint solvents, agents for complexing or buffer substances, and the transformation mixture in an acceptable pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules, powders, creams, ointments, gels, syrups, emulsions, suspensions or solutions, acceptable for injection, used for parenteral administration.

Examples of excipients and/or carriers which may be mentioned are cremophor, poloxamer,benzalkonium chloride, sodium lauryl sulfate, dextrose, glycerin, magnesium stearate, polyethylene glycol, starch, dextrin, lactose, cellulose, carboxymethylcelluose sodium, talc, agar-agar, mineral oil, animal oil, vegetable oil, organic and mineral waxes, paraffin, gel, propylene glycol, benzyl alcohol, dimethylacetamide, ethanol, polyglycols, tween 80, solutol HS 15, water and brine. Also, the introduction of active substances by themselves, without media or diluents, in an acceptable form, such as capsules.

As usual, Galanov composition and method of administration, and the range of dosages that are applicable in a particular case depend on the species to be treated, and from the picture of the corresponding condition or disease and can be optimized using methods known in this field. The average daily dose of active compound in the patient is from 0.0005 mg to 50 mg per kg, usually from 0.001 mg to 20 mg per kg

Next are presented to illustrate examples of the present invention, which are not intended to limit its scope.

Example 1

Selection of culture No. PM0626271 from soil collected in the Antarctic area

a) the Composition environment selection

A modified agar with artificial sea water: peptone, 1.5 g, yeast extract 0.5 g, ferric chloride 0,007 g, 1.0 l of water (750 ml IP is umstvennoi sea water+250 ml of demineralized water), powder agar 15.0 g, final pH (at 25°C) 7,4-7,8.

Composition of artificial sea water: sodium chloride solution of 24.6 g of potassium chloride solution of 0.67 g of calcium chloride·2H2O 1,36 g magnesium sulfate·7H2O 6,29 g magnesium chloride·6H2O of 4.66 g of sodium bicarbonate 0.18 g, demineralized water 1.0 l, final pH (at 25°C) 7,8-8,2.

b) Procedure

In the area of the Schirmacher Oasis in the Antarctic region collected the soil surface layer and kept at -20°C for the expedition to Piramal Life Sciences Limited, Goregaon, Mumbai, India. The sample was stored at -20°C to -22°C, and then thawed to room temperature (25+2°C) for selection of microbes. Soil sample (~1 g) suspended in 25 ml of sterile water with 1% peptone, 100 ml of sterilized flask. The flask was shaken for 30 seconds. Serial dilution up to 10-5received in sterile water with 1% peptone. 100 μl of 10-5breeding distributed across the surface of the modified agar with artificial sea water. Cup incubated at room temperature (25±2°C) until such time has not yet been observed colonies. After incubation for one and a half months the colony, which appeared on the environment, were sown touches on Petri dishes containing agar for isolation of actinomycetes [Hi Media] prepared in 75% artificial sea water [AccumixTm] (AS-AIA). The isolate was purified and provide the culture with dentification number PM0626271. Thus, provided the culture No. PM0626271 of growing microorganisms as a separate isolate.

Example 2

Purification of culture No. PM0626271

a) the Composition environment for cleaning (agar for isolation of actinomycetes, agritechnology 1.5% agar-agar)

Glycerin 5.0 ml, sodium Caseinate 2.0 g, L-asparagine 0.1 g, sodium propionate 4.0 g, dicale phosphate, 0.5 g of magnesium sulfate, 0.1 g, ferrous sulfate, 0.001 g, 1.0 l of water (750 ml artificial sea water+250 ml of demineralized water), powder agar 15.0 g, final pH (at 25°C) 7,4-7,8.

Composition of artificial sea water: sodium chloride solution of 24.6 g of potassium chloride solution of 0.67 g of calcium chloride·2H2O, 1,36 g magnesium sulfate·7H2O 6,29 g magnesium chloride·6H2O of 4.66 g of sodium bicarbonate 0.18 g, demineralized water 1.0 l, final pH (at 25°C) 7,8-8,2.

b) Procedure

Culture No. PM0626271 were sown strokes in a Petri dish with agar for isolation of actinomycetes (containing 75% of salts artificial sea water). The Petri dishes were incubated for 10 days at 25°C. One of the selected colonies from Petri dishes were transferred to fresh “shoals” agar for isolation of actinomycetes, cooked 75% artificial sea water. “Weeds” were incubated for 10 days at 25°C.

Example 3

Maintaining producer strain culture No. PM0626271

a) the Composition of the medium (agar for isolation of actinomycetes)

p> Glycerin 5.0 ml, sodium Caseinate 2.0 g, L-asparagine 0.1 g, sodium propionate 4.0 g, dicale phosphate, 0.5 g of magnesium sulfate, 0.1 g, ferrous sulfate, 0.001 g, 1.0 l of water (750 ml artificial sea water+250 ml of demineralized water), powder agar 15.0 g, final pH (at 25°C) 7,4-7,8.

Composition of artificial sea water: sodium chloride solution of 24.6 g of potassium chloride solution of 0.67 g of calcium chloride·2H2O 1,36 g magnesium sulfate·7H2O 6,29 g magnesium chloride·6H2O of 4.66 g of sodium bicarbonate 0.18 g, demineralized water 1.0 l, final pH (at 25°C) 7,8-8,2.

b) After dissolution of ingredients by heating the resulting solution was distributed in the analytical test tubes and sterilized at 121°C for 30 minutes. Analytical test-tubes were cooled and left to harden in an inclined position. Agar “weeds” were sown strokes growing culture No. PM0626271 wire loop and incubated at 27°C - 29°C, until a good growth. A well-grown culture was kept in a refrigerator at 4°C - 8°C.

Example 4

The fermentation culture No. PM0626271 in shake flasks

a) the Composition environment for seeding [AS-274 (1)]

Glucose 15 g, liquid corn extract, 5 g peptone 7.5 g, yeast extract 7.5 g, calcium carbonate, 2.0 g, sodium chloride 5.0 g, volume obtained from 750 ml of artificial sea water and 250 ml of demineralized water.

(b) Above who mentioned the medium was distributed in 40 ml quantities in the Erlenmeyer flask with a capacity of 500 ml and autoclaved at 121°C for 30 minutes. The flask was cooled to room temperature (25±2°C) and each flask was inoculable loop for sowing, growing well in “the post” producing strain (culture No. PM0626271) and were shaken in a rotary shaker for 72 hours at 230 rpm 250 rpm at 30°C (±1°C) to obtain a culture for inoculation.

c) the Composition environment to obtain [AS 36P (1)]

Soluble starch 20 g, glucose 15 g, yeast extract 2 g, peptone 3 g calcium carbonate 2 g ammonium sulfate 0.5 g, liquid corn extract, 2 g of sodium chloride 2 g magnesium phosphate 5 g of cobalt chloride and 1 ml/l of the starting solution 1 g/l solution of salts in trace concentrations, 1 ml/l, the volume was brought to 1 l using 75% artificial sea water and 25% demineralized water.

d) 40 ml of medium to get in Erlenmeyer flasks 500 ml autoclaved at 121°C for 30 minutes, cooled to 29°C - 30°C and seeded with 5% (volume/volume) of the culture for crops, referred to in Example 4b.

e) fermentation Parameters

Flask to obtain incubated on a shaker at 29°C and 220 rpm for 96 hours. The contents of the flasks to obtain collected and whole broth from each flask with the medium was extracted with an equal volume of methanol provided shaking for one hour at 29°C and centrifuged at 3500 rpm for half an hour. The supernatant was used for analysis of diffusion proteobacteria the social connections in agar for monitoring activity.

Obtaining compounds of formula I(a) and formula I(b) in the fermentation broth was determined by analysis of bioactivity against S. aureus E710 (MRSA strain), and/or Enterococcus faecium R2 (VRE) using the method of diffusion in agar. the pH of the harvested fermentation broth was 7.0 to 8.0. The fermentation broth was collected and whole broth was used for isolation and purification of the compounds of formula I(a) and formula I(b).

Example 5

Culture for planting in shake flasks for fermentation

a) the Composition environment [AS-274 (1)]

Glucose 15 g, liquid corn extract, 5 g peptone 7.5 g, yeast extract 7.5 g, calcium carbonate, 2.0 g, sodium chloride 5.0 g, volume obtained from 750 ml of artificial sea water and 250 ml of demineralized water.

b) the Above medium was distributed in 200 ml quantities in the Erlenmeyer flask with a capacity of 1000 ml and autoclaved at 121°C for 30 minutes. The flask was cooled to room temperature (25±2°C), each flask was inoculable loop for sowing, growing well in “the post” producing strain (culture No. PM0626271) and were shaken in a rotary shaker for 70-74 hours at 230 rpm 250 rpm at 29°C - 30°C to obtain a culture for inoculation.

Example 6

The cultivation of culture No. PM0626271 in the fermenter

a) the Composition environment to obtain

Artificial sea water (artificial sea salt in the s weighing 28.32 g) (75%), soluble starch 20 g, glucose 15 g, yeast extract 2 g, peptone 3 g calcium carbonate 2 g ammonium sulfate 0.05 g, liquid corn extract, 2 g of sodium chloride 2 g magnesium phosphate 5 g of cobalt chloride (demineralized water to 1.0 l with 1 g of cobalt chloride) 1 ml/l of a solution of salts in trace concentrations (copper sulfate 7 g, ferrous iron sulfate 1 g magnesium chloride 8 g of zinc sulfate 2 g of demineralized water 1.0 l) 1 ml/l, demineralized water 1.0 l, pH 6.5-7.5 (before sterilization).

b) 100 l medium to obtain a 150 l fermenter together with 30 ml of desmophen as protivovspenivayushchie tools were sterilized in situ for 30 minutes at 121°C, cooled to 29°C - 30°C and seeded with 2.5 l and 3.5 l of culture for inoculation obtained above (Example 5).

c) fermentation Parameters

Fermentation was performed at a temperature of 29°C - 30°C with agitation at 100 rpm, aeration of 60 liters per minute and collected after 70 hours 74 hours. Obtaining compounds of formula I(a) and formula I(b) in the fermentation broth was determined qualitatively by assessing the bioactivity against S. aureus E710 (MRSA strain), and/or Enterococcus faecium R2 (VRE) using the method of diffusion in agar. the pH of the harvested fermentation broth was 7.5 to 8.0. After collection of whole broth was subjected to solvent extraction.

Example 7

Isolation and purification of compounds of formula I(a) and forms the crystals I(b)

Whole broth (party of 10 l) was extracted using ethyl acetate (1:1). Separated organic and aqueous layers. The organic layer was treated with evaporating the solvent to obtain an ethyl acetate crude extract (1.5 g). The crude extract was further processed by flash chromatography (silica gel, 30 g, solvent: stepwise gradient of methanol/chloroform, flow: 15 ml/min). The active compound was suirable 1% methanol - 5% methanol in chloroform, which was concentrated to obtain semifinished compound (250 mg). Further purification was performed repeated normal-phase preparative HPLC.

Conditions preparative HPLC

ColumnEurospher silicon oxide (10 µ, 20x250 mm)
Eluentmethanol:chloroform (5:95)
The flow velocity20 ml/min
Detection (UV)245 nm
Retention timethe compound of formula I(a) (5-6 minutes)
the compound of formula I(b) (8-10 minutes)

The purity of the fractions was checked by bioanalysis against E. faecium R2 and/and and S. aureus 3066, and/or analytical HPLC. Eluate containing the compounds of formula I(a) and formula I(b), were combined and concentrated under reduced pressure to remove solvent to obtain compounds of formula I(a) (40 mg) and the compounds of formula I(b) (3 mg).

The analytical conditions HPLC

ColumnEurospher RP-18 (3 µ, 4,6x125 mm)
The solvent systema gradient from 0% acetonitrile to 100% in 15 minutes in relation to water, followed by 100% acetonitrile for 5 minutes)
The flow velocity1 ml/min
Detection (UV)245 nm
Retention timethe compound of formula I(a) (12-13 minutes), and the compound of formula I(b) (11-12 minutes)

A. Physical and spectral properties of the compounds of formula I(a)

Appearancewhite powder
Melting point240°C (decomposition)
Solubilitysoluble in chloroform, ethyl acetate, methanol and Nera the solubility in water
MS [HR-ESI(+) MS)] mass/charge1650,4858 (M+H)
Molecular weight1649,5
Molecular formulaC71H83N19O18S5
IR (KBr)3386, 2927, 1648, 1507, 1206, 756, 666 cm-1
1H NMRsee Table 1 and Figure 1
13C NMRsee Table 2 and Figure 2

B. Physical and spectral properties of the compounds of formula I(b)

Appearancewhite powder
Solubilitysoluble in chloroform, ethyl acetate, methanol and insoluble in water
MS [HR-ESI(+) MS)] m/h1674,4787 (M+Na)
Molecular weight1651,50
Molecular formulaC71H85N19O18S5
1H NMRsee Table 3 and Figure 3

That is person 1

1H NMR of compounds of formula I(a) in CDCl3:CD3OD (4:1) at 500 MHz

PeakδPeakδPeakδ
1of 0.7(d, 3H)18to 3.67(d, 1H)356,91(s, 1H)
2to 0.74(d, 3H)19of 3.7(q, 1H)366,94(s, 1H)
3of 0.95(d, 3H)20to 4.33(d, 1H)37to 7.2(s, 1H)
4the 1.04(s, 3H)21to 4.33(d, 1H)38the 7.43(s, 1H)
5a 1.08(d, 3H)22to 4.62 (q, 1H)39was 7.45(s, 1H)
6 1,2(d, 3H)23a 4.86(dd, 1H)40the 7.65(s, 1H)
7of 1.28(d, 3H)245,19 (s, 1H)417,87(s, 1H)
8of 1.34(d, 3H)255,19(s, 1H),
5,67 (s, 1H)
42with 8.05(s, 1H)
9to 1.37(m, 1H)26to 5.2(t, 1H)438,17(s, 1H)
10of 1.5(d, 3H)275,6(d, 1H)44of 8.2 (s, 1H)
11of 1.6(d, 3H)285,62(s, 1H), 6,44(s, 1H)45of 8.5 (s, 1H)
122,1(m, 1H)29the 5.65(d, 2H)468,67 (s, H)
132,2 (m, 1H)305,72(s, 1H), is 6.61(s, 1H)478,99 (s, 2H)
142,2(m, 1H)
to 3.99(m, 1H)
316,1(q, 1H)489,72 (s, 1H)
15the 2.8(d, 1H)32of 6.25(m, 1H)49of 9.8 (s, 1H)
163,05(t, 1H) 3,5(t, 1H)336,28(d, 2H)
17to 3.49(d, 2H)34to 6.8(d, 1H)

Table 2

13C NMR of compounds of formula I(a) in CDCl3:CD3OD (4:1) at 75 MHz

The signalδThe signalδThe signalδ
112,112564,4549144,61
213,742664,7350148,17
314,12765,6951148,45
414,82865,9552151,89
516,482970,2753152,76
617.11 per bbl3077,154155,45
717,2731101,4555157,9
817,5532101,4556159,0
920,933102,657160,04
1023,1334116,5258160,36
1127,5635120,6359161,01
1227,5636121,5960163,75
1329,0437123,2861164,46
1433,2438123,8762164,5
15 46,1739123,8763166,6
1650,1440125,4864167,13
1751,341126,0365167,95
1853,9142126,6966168,47
1953,9143128,2467168,67
2055,844130,3468170,35
2157,3245130,9869170,35
2258,8 46131,1470171,63
2362,4247132,3971172,0
2462,7348141,85

Table 3

1H NMR of compounds of formula I(b) in CDCl3:CD3OD (4:1) at 500 MHz

PeakδPeakδPeakδ
1to 0.88(d, 3H)234,59(t, 1H)457,80(s, 1H)
2of 0.95(d, 3H)24br4.61(dd, 1H)46 with 8.05(s, 1H)
30,99(d, 3H)254,74(t, 1H)478,11(s, 1H)
41,0(m, 1H)26of 4.95(t, 1H)48to 8.14(s, 1H)
5of 1.16(s, 3H)275,19(s, 1H), 5,78(s, 1H)49compared to 8.26(s, 1H)
6of 1.18(d, 3H)285,19(s, 1H)50charged 8.52(t, 1H)
7of 1.32(d, 3H)295,3(d, 1H)518,65(d, 1H)
8of 1.35(d, 3H)305,52(s, 1H), 6,65(s, 1H)52of 9.2(s, 1H)
9of 1.43(d, 3H)31 the 5.65(s, 1H)539,87(s, 1H)
10of 1.5(d, 3H)325,72(d, 1H)549,92(s, 1H)
11to 1.61(d, 3H)33of 5.8(d, 1H)
121,72(d, 3H)346,1(q, 1H)
132,02(m, 1H)356,34(m, 1H)
14of 2.28(m, 1H)366,36(d, 1H)
15the 2.8(d, 1H)37of 6.73(d, 1H)
163,17(t, 1H) 3,69(t, 1H)38 6,91(d, 1H)
17of 3.43(d, 1H)396,94(s, 1H)
18of 3.46(d, 1H)407,14(s, 1H)
19to 3.58(d, 1H)41at 7.3(s, 1H)
20of 3.8(q, 2H)427,46(s, 1H)
214,06(m, 1H)43rate of 7.54(d, 1H)
22of 4.45(d, 1H)44the 7.65(d, 1H)

Biological evaluation of the compounds of formula I(a) and formula I(b)

In-vitro tests

Example 8

The effectiveness of in-vitro by definition the minimum inhibitory concentration (MIC) of the compounds of formula I(a) and formula I(b) against bacterial strains using the method of makroyavleny broth according to the directions of the National Committee for clinical laboratory standards (2000) (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically-Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA). The broth Mueller Hinton was used as nutrient medium for analysis if not stated otherwise. PM181104 (PCT publication No. WO2007119201) was used as a known standard in all in vitro experiments. To prepare the initial solution of the compounds of formula I(a) and formula I(b) was dissolved in chloroform (5% of the total required volume) and diluted using methanol (95% of required volume).

Results

The results obtained are shown in Table 4 and Table 5 and demonstrate that the compounds of formula I(a) and formula I(b) are used to treat bacterial infections.

Table 4. MIC of the compounds of formula I(a) against bacterial strains

The analyzed organismMIC (mcg/ml)The analyzed organismMIC (mcg/ml)
S. aureus C1 MRSA 20,25S. aureus E712, MRSA ErythroR, 590,25
S. aureus C1 MRSA 30,25S. aureus 503, MRSA, 62 >1
S. aureus C1 MRSA 50,25S. aureus SG 511, MRSA, 631
S. aureus C1 MRSA 70,125S. aureus 789, MRSA, 64>1
S. aureus C1 MRSA 80,25S. aureus 209 P, MSSA0,063
S. aureus C1 MRSA 90,25S. epidermidis 823, TeichoR, 2300,25
S. aureus C1 MRSA 100,25S. epidermidis 6098, ErythroR, 233>1
S. aureus C1 MRSA 130,25S. epidermidis 6729 II W, ErythroR, 2360,125
S. aureus C1 MRSA 160,25S. epidermidis 2361 W, 2460,25
S. aureus C1 MRSA 170,125S. epidermidis 4264 I (1) W, 2470,125
S. aureus C1 MRSA 200125 S. epidermidis Pat 01 IV, 2510,25
S. aureus C1 MRSA 210,125E. faecium C1 VRE 260,25
S. aureus C1 22 MRSA0,25E. faecium C1 VRE 270,25
S. aureus C1 MRSA 230,016E. faecium C1 VRE 280,125
S. aureus C1 MRSA 240,25E. faecium C1 VRE 310,5
S. aureus C1 MRSA 250,15E. faecium C1 VRE 330,125
S. aureus KEM MRSA 10,25E. faecium C1 VRE 340,25
S. aureus KEM MRSA 20,25E. faecium KEM VRE 10,25
S. aureus KEM MRSA 30,25E. faecium KEM VRE 20,5
S. aureus KEM MRSA 40,125 E. faecium KEM VRE 30,25
S. aureus KEM MRSA 50,125E. faecium KEM VRE 40,5
S. aureus MRSA 3 lilavati0,125E. faecium KEM VRE 50,25
S. aureus Misk MRSA 350,125E. faecium R-2 (VRE)0,25
S. aureus Misk MRSA 370,125Enterococcus faecium, VSE (322)0,125
S. aureus Misk 38 MRSA0,125Bacillus cereus (121)0,031
S. aureus E710, MRSA0,125Bacillus subtilis ATCC 6633 (123)0,031
S. aureus ATCC 33591, MRSA0,125B. megaterium Bacillus FH1127 (124)0,125

Table 5. MIC of the compounds of formula I(a) and formula I(b) against bacterial strains

Analyzed culture Minimum inhibitory concentration (mcg/ml)
The compound of formula I(a)The compound of formula I(b)
E. faecium, R-2 (VRE)0,1252
S. aureus E710, (MRSA)0,1252
S. aureus 209P (MSSA)0,064-0,1250,25-0,5

Abbreviations used in Table 4 and Table 5, are

S: Staphylococcus;

E: The Enterococci.

Example 9

Assessment protivokataralnoe activity

The analysis was performed, as reported in J. Clin. Environ., 1999, 37, 1144.

50 μl of bacterial (as mentioned in Table 6) suspension, equivalent to the standard MacFarlands No. 2 (equivalent to>5x107CFU/ml) (Remel, Lenexa, Kan.) added to 400 µl G7H9 with and without the compounds of formula I(a) and formula I(b) (analyzed at 0.5 mg/ml, 5 μg/ml and 10 μg/ml) and incubated for 72 hours at 37°C. After incubation, all the vessels were added 50 μl of phage reporter luciferase with a high titer (phAE 129) and 40 ál of 0.1 M calcium chloride (CaCl2), and the system was incubated for 4 hours at 37°C. After incubation took 100 ál of the mixture from each vessel in a luminometer cuvette and add an equal amount of work is about the solution of D-luciferin (0.3 mm in 0.05 M sodium citrate buffer, pH 4.5). Relative light units (RLU) was measured after 10 seconds of integration in a luminometer (Monolight 2010).

For each specimen were recorded reading in replications and calculated the average. The reduction percentages in RLU was calculated for each of the analyzed sample and compared with the control. The experiment was repeated, if the average RLU of control was less than 1000. The criterion for activity is protivokataralnoe activity, shows a fifty percent reduction in RLU in the presence of the compound compared to the control, nesteriak connection.

Table 6: Protivokataralnoe activity of the compounds of formula I(a) and formula I(b)

StrainConnection% reduction in RLU
0.5 mg/ml5 mg/ml10 mg/ml
M. tuberculosis H37RvThe compound of formula I(a)36,3677,1783,76
The compound of formula I(b)28,8754,83 76,73
Clinical isolate: resistant to S, H, R and EThe compound of formula I(a)to 20.8878,1188,42
The compound of formula I(b)5,6770,983,74

Conclusion: the compounds of formula I(a) and formula I(b) are active against a standard strain of TB (H37RV) and the strain of MDR Mycobacterium tuberculosis [resistant to 4 standard antibiotics: S (streptomycin), H (isoniazid or isonicotinoyl hydrazine), R (rifampicin) and E (ethambutol)].

1. A compound selected from the compounds of formula I(a) or the compounds of formula I(b):


or their pharmaceutically acceptable salts.

2. Connection on p. 1, where the compound isolated from the fermentation broth of a microorganism belonging to the species Streptomyces (PM0626271/MTCC 5447).

3. The compound of formula I(a) under item 1, where the connection specified by:
(a) molecular weight 1649,5,
(b) the molecular formula C71H83N19O18S5,
(c)1H NMR spectrum, as shown in Figure 1, and
(d)13C NMR spectrum, as shown in Figure 2.

4. The compound of formula I(b) under item 1, where the decree is Noah compound is characterized by:
(a) molecular weight 1651,5,
(b) the molecular formula C71H85N19O18S5and
(c)1H NMR spectrum, as shown in Figure 3.

5. The method of obtaining the compounds of formula I(a) and the compounds of formula I(b) under item 1 or a pharmaceutical salt, which cultivate the microorganism Streptomyces species (PM0626271/MTCC 5447) in aerobic conditions by immersion in a nutrient medium containing sources of carbon and nitrogen, to obtain the compounds of formula I(a) and the compounds of formula I(b) in a fermentation broth.

6. The method according to p. 5, in which additional
(a) include a compound of formula I(a) and the compound of formula I(b) of the fermentation broth obtained after cultivation of a microorganism of the species Streptomyces (PM0626271/MTCC 5447); and
(b) purified compound of formula I(a) and the compound of formula I(b).

7. The method according to p. 6, further comprising a stage on which make the compound of formula I(a), or compound of formula I(b), or both in their pharmaceutically acceptable salt.

8. Pharmaceutical composition for preventing or treating a bacterial infection comprising an effective amount of the compounds of formula I(a) or the compounds of formula I(b) under item 1 and at least one pharmaceutically acceptable excipient or carrier.

9. The pharmaceutical composition under item 8, wherein the pharmaceutical composition is in the form of tablets, pokr is the pill capsules, granules, powder, cream, ointment, gel, emulsion, suspension or solution for injection.

10. The pharmaceutical composition under item 8, wherein the bacterial infection is caused by bacteria belonging to the species Staphylococcus, Streptococcus, Enterococcus, Bacillus or Mycobacterium.

11. The pharmaceutical composition according to p. 10, where the bacterium belonging to the species of Staphylococcus that is resistant to methicillin, vancomycin-resistant or both.

12. The pharmaceutical composition according to p. 10, where the bacterium belonging to the species of enterococci, is resistant to vancomycin.

13. The pharmaceutical composition according to p. 10, where the bacteria belonging to the Mycobacterium species is resistant to multiple drugs.

14. The use of the compounds of formula I(a) or the compounds of formula I(b) p. 1 to prevent or treat bacterial infections.

15. Application under item 14, where the bacterial infection is caused by bacteria belonging to the species Staphylococcus, Streptococcus, enterococci, Bacillus or Mycobacterium.

16. Application under item 15, where the bacterium belonging to species of Staphylococcus that is resistant to methicillin, vancomycin-resistant or both.

17. Application under item 15, where the bacterium belonging to species enterococci is resistant to vancomycin.

18. Application under item 15, where the bacteria belonging to the Mycobacterium species is resistant to several of Carstensen tools.

19. The use of the compounds of formula I(a) or the compounds of formula I(b) under item 1 for the manufacture of a medicinal product for preventing or treating diseases caused by a bacterial infection.

20. A method of treating or preventing a bacterial infection, when administered to a mammal in need, an effective amount of the compounds of formula I(a) and the compounds of formula I(b) under item 1.

21. The method according to p. 20, where the bacterial infection is caused by bacteria belonging to the species Staphylococcus, Streptococcus, enterococci, Bacillus or Mycobacterium.

22. The method according to p. 21, where the bacterium belonging to species of Staphylococcus that is resistant to methicillin, vancomycin-resistant or both.

23. The method according to p. 21, where the bacterium belonging to species enterococci is resistant to vancomycin.

24. The method according to p. 21, where the bacteria belonging to the Mycobacterium species is resistant to multiple drugs.



 

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