RU2444526C2 - Novel antibacterial compounds - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel purified compound PM 181104 of formula I

(with molecular weight 1514 and molecular formula C₆₉H₆₆N₁₈O₁₃S₅), pharmaceutically acceptable salts thereof, methods for synthesis via fermentation of a microorganism of the type Kocuria (ZMA B-1 / MTCC 5269), as well as pharmaceutical compositions.

EFFECT: high efficiency of using the composition to produce a medicinal agent for treating bacterial infections.

20 cl, 4 dwg, 4 tbl, 16 ex

Description

This invention relates to a new compound PM 181104 having antibacterial activity, which is obtained by fermentation of a microorganism belonging to species of Kocuria (ZMA B-1 / MTCC 5269). The invention also includes all stereoisomeric forms and all tautomeric forms of PM 181104, pharmaceutically acceptable salts and derivatives thereof. The present invention further relates to methods for producing a new antibacterial compound (s), for producing a microorganism belonging to Kocuria species (ZMA B-1 / MTCC 5269), pharmaceutical compositions containing the new compound (s) as an active ingredient, and its / their use in medicine for treatment and prevention of diseases caused by bacterial infections.

BACKGROUND OF THE INVENTION

The emergence of bacterial resistance to a number of antimicrobials, such as beta-lactam antibiotics, macrolides, quinolones and vancomycin, is becoming a major public health problem worldwide (Trends In Microbiology, 1994, 2, 422-425). The most significant problem in clinical practice is the expansion of Staphylococcus aureus methicillin-resistant infections (MRSA). Vancomycin alone is currently an effective treatment for the multi-resistant MRSA infection. However, there are a number of reports of vancomycin resistance in some MRSA isolates (Antimicrobial Agents and Chemotherapy, 1998, 42, 2188-2192). Another group of bacteria that are resistant to several clinically relevant drugs recently appeared is Enterococci, some of which also show resistance to vancomycin. The emergence of vanocycin-resistant Enterococci (VRE) infections puts physicians in a difficult position. Combinations of linezolid, an oxazolidinone compound, and streptogramin are the new drugs of choice for treating MRSA infections. However, resistance to the combination of oxazolidinone (Clinical Infectious Diseases, 2003, 36, supplement I, S11-S23; Annals of Pharmacotherapy, 2003, 37, 769-74) and streptogramin (Current Drug Targets Infectious Disorders, 2001, 1,215-25 ) and various glycopeptides (Clinical Infectious Diseases, 2003, 36, supplement 1, S11-S23) require the expansion of funds with alternative goals or modes of action. The increasing resistance of the important community of the acquired pathogen Streptococcus pneumoniae to penicillin and other antibacterial agents is also becoming a global public health problem. Multi-drug resistant Mycobacterium tuberculosis strains have appeared in some countries. The emergence and spread of persistent nosocomial and acquired pathogens poses a threat to the health of people around the world.

The discovery of new compounds that can be used as drugs to treat patients infected with bacteria, especially bacteria resistant to several drugs, such as MRSA and VRE, is urgently needed.

SUMMARY OF THE INVENTION

This invention relates to a new purified compound (designated here as PM 181104) isolated from a fermentation medium of a microorganism belonging to a species of Kocuria (ZMA B-1 / MTCC 5269) having antibacterial activity.

This invention also relates to all stereoisomeric forms, all tautomeric forms of PM 181104, a pharmaceutically acceptable salt (s), its ester derivative (s) and ether represented by formula I (as described herein).

Compound PM 181104, isomers, pharmaceutically acceptable salt (s) and its derivative (s) of ester and ether, are used to treat and prevent diseases caused by bacteria, especially bacteria resistant to a number of drugs, such as MRSA and VRE.

The present invention further relates to pharmaceutical compositions comprising the new compound PM181104, an isomer, a pharmaceutically acceptable salt (s), its derivative (es) of an ester and ether as an active ingredient for the treatment of medical conditions caused by bacteria that are particularly resistant to a number of drugs bacteria such as MRSA and VRE.

The present invention further relates to methods for producing the compound PM 181104 and / or its isomer (s) from a microorganism belonging to the species Kocuria (ZMA B-1 / MTCC 5269).

The present invention also relates to methods for producing a microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 5269), the cultivation of which produces the compound PM 181104 and its isomers.

The present invention also relates to methods for preparing derivatives of the ester and ether of compound PM 181104.

DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 shows the spectrum of ultraviolet absorption (UV) PM 181104.

Figure 2 shows the infrared absorption spectrum (IR) of PM 181104.

Figure 3 shows the ¹ H-NMR spectrum (500 MHz) of PM 181104 in DMSO-d ₆ (dimethyl sulfoxide-d ₆ ).

Figure 4 shows the ¹³ C-NMR spectrum (125 MHz) of PM 181104 in DMSO-d ₆ .

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a novel antibacterial compound PM 181104, and also includes all stereoisomeric forms, all tautomeric forms PM 181104, pharmaceutically acceptable salts and derivatives thereof, such as esters and ethers.

Therefore, this invention relates to new antibacterial compounds of the following formula I

where R = H (PM 181104), alkyl, alkylcarbonyl, (HO) ₂ PO-, alkyl-ORO (OH) -, (alkyl-O) ₂ PO-, cycloalkyl, cycloalkylcarbonyl, aryl, arylcarbonyl, heterocyclyl and heterocyclyl carbonyl.

As used herein, the term “alkyl” is either used alone or as part of a substituent group, means a radical of saturated aliphatic groups, including straight or branched chain alkyl groups. Moreover, unless stated otherwise, the term “alkyl” includes unsubstituted alkyl groups as well as alkyl groups which are substituted with one or more different substituents. In preferred embodiments, the straight or branched chain alkyl has 20 or fewer carbon atoms in its skeleton (for example, C ₁ -C ₂₀ for the straight chain, C ₃ -C ₂₀ for the branched chain). Examples of alkyl residues containing from 1 to 20 carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, heptadecyl and eicosyl, n-isomers of all these residues; isopropyl, isobutyl, 1-methylbutyl, isopentyl, neopentyl, 2,2-dimethylbutyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 2,3,4-trimethylhexyl, isodecyl, sec-butyl or tert-butyl. Substituted alkyl means an alkyl residue in which one or more hydrogen atoms, for example 1, 2, 3, 4 or 5 hydrogen atoms, are substituted with substituents, for example, halogen, hydroxyl, sulfonyl, alkoxyl, cycloalkyl, cyano, azido, amino, acyloxy, heterocyclo , aralkyl, aryl or fluorescent groups such as NBD [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] or BODIPY [4,4-difluoro-5,7-dimethyl- 4-bora-3a, 4a-diaza-s-indacene] group.

As used herein, the term “cycloalkyl” means a saturated mono- or bicyclic ring system containing 3-10 carbon atoms and more preferably having 3, 4, 5, 6, or 7 carbon atoms in the ring structure. Examples of cycloalkyl residues containing 3, 4, 5, 6 or 7 ring carbon atoms are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. Moreover, unless stated otherwise, the term “cycloalkyl” includes unsubstituted cycloalkyl and cycloalkyl substituted with one or more identical or different groups selected from halogen, hydroxyl, alkoxyl, alkyl, cycloalkyl, cyano, amino, aminoalkyl, acyloxy, heterocyclo, aralkyl and / or an aryl group.

As used herein, the term “aryl” means a monocyclic or polycyclic hydrocarbon group having up to 10 ring carbon atoms in which at least one carbocyclic ring has a conjugated π-electron system. Suitable examples of C ₆ -C ₁₀ aryl residues include phenyl, naphthyl or biphenyl, especially phenyl and naphthyl. Aryl residues, for example phenyl or naphthyl, can usually be optionally substituted with one or more substituents, up to five identical or different substituents selected from the groups including halogen, alkyl, hydroxyl, acyloxy, amino, substituted amino, cyano.

The term “heterocyclyl” means a saturated, partially unsaturated or aromatic monocyclic or polycyclic heterocyclic ring system containing 5, 6, 7, 8, 9 or 10 ring atoms, of which 1, 2 or 3 are identical or different heteroatoms selected from nitrogen, oxygen and sulfur. Suitable examples of such heterocyclyl groups are pyridinyl, piperazinyl, piperidinyl, imidazolyl, pyrrolidinyl and morpholinyl. In a polycyclic heterocyclic ring system, heterocyclyl may include either fused rings in which two or more carbons are common to two adjacent rings, or joined rings in which the rings are joined through non-adjacent atoms. In a polycyclic heterocyclic ring system, heterocyclyl preferably includes two fused rings (bicyclic), at least one of which is a 5- or 6-membered heterocyclic ring. Exemplary bicyclic heterocyclic groups include benzoxazolyl, quinolyl, isoquinolyl, carbazolyl, indolyl, isoindolyl, phenoxazinyl, benzothiazolyl, benzimidazolyl, benzoxadiazolyl and benzofurazanil. Heterocyclyl residues can usually be optionally substituted with one or more identical or different substituents selected from the groups consisting of halogen, alkyl, hydroxyl, acyloxy, amine, substituted amine, cyano.

According to a preferred embodiment of the invention, the group R in the formula I may be H, CH ₃ CH ₂ CH ₂ CO, CH ₃ (CH ₂ ) ₁₅ CH ₂ CO or

.

.

According to a more preferred embodiment, the new compound PM 181104 represented by the formula I above (where R = H) is isolated from a fermentation medium of a microorganism belonging to a species of Kocuria (ZMA B-1 / MTCC 5269) and then purified.

The new compound PM 181104 has the molecular formula C ₆₉ H ₆₆ N ₁₈ O ₁₃ S ₅ (molecular weight 1514) and can be characterized by any one or more of its physicochemical and spectral features, such as high performance liquid chromatography (HPLC) spectroscopic data ), mass spectrum (MS), ultraviolet radiation (UV), infrared radiation (IR), and nuclear magnetic resonance (NMR), as discussed below.

The structure of the new compound PM 181104 was explained and its complete characterization was performed using spectroscopic data from HPLC, MS, UV, IR and NMR. The structure was confirmed by three-dimensional (3D) NMR studies of bioactive ¹⁵ N and ¹³ C-labeled PM 181104.

The compound PM 181104 and its derivatives of ester and ether are new antibiotics that are active against bacteria, especially bacteria resistant to certain drugs, such as MRSA and VRE.

The microorganism that can be used to obtain compound PM 181104 is a strain of Kocuria species (ZMA B-1 / MTCC 5269), hereinafter referred to as ZMA B-1 culture isolated from a marine sample collected in Palk Bay, Tamil Nadu coast, India .

The present invention further provides a process for the preparation of compound PM 181104 from a ZMA B-1 culture, mutants and variants thereof, wherein a ZMA B-1 culture is grown under submerged aerobic conditions in a culture medium containing one or more carbon sources, one or more nitrogen sources, and optional nutritional inorganic salts and / or trace elements; the compound PM 181104 is isolated from the culture medium; purify compound PM 181104 using purification techniques commonly used in the art.

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

As used here, the expression "variant" means a separate organism that is markedly different from an arbitrary standard type in these species.

Preliminary identification of the ZMA B-1 culture, which produces PM 181104, was performed by examining the morphology of its colony, observing wet preparations, and the reaction to staining using the Gram method. Microscopic studies of the strain of isolated ZMA B-1 culture were performed in Zobell Marine 2216 nutrient medium (marine nutrient medium 2216) containing 1.5% agar, and observations were made on days 1, 2, and 3 of incubation at 25 ° C.

Growth in Zobell Marine 2216 nutrient medium (marine nutrient medium 2216), containing 1.5% agar, manifested itself in the form of a colony with a diameter of 2 mm with a smooth surface, yellow-orange pigmentation, regular border and soft consistency. Diffuse pigments were not observed in this medium. With contrast-phase light microscopy at a magnification of 400 x, cocci were observed. Cocci separate well and stand out. They are gram-positive and motionless. According to the observed morphology, this organism was classified as a member of the Micrococcaceae family. Isolates were identified by comparing their 16S rRNA polymerase chain reaction (PCR) with existing sequences available on the website of the National Center for Biotechnological Information (NCBI) (URL: http: //www.ncbi.nlm.nih.qov/). ZMA B-1 culture was deposited in the Microbiological Collection of Typical Cultures (MTCC) of the Institute of Microbiological Technology, sector 39-A, Chandigarh - 160 036, India, the International Depository Authority (IDA), recognized by the International Intellectual Property Organization (WIPO), and was awarded a number MTCC 5269.

In addition to the specific microorganism described herein, it should be understood that mutants, such as those obtained by the use of chemical or physical mutagens, including X-rays, UV rays, etc., and organisms genetically modified by molecular biology techniques, can also be cultured to obtain the compound PM 181104.

Examination of acceptable mutants and variants that can produce a compound of this invention can be performed using HPLC and / or determination of the biological activity of the active compounds accumulated in the culture medium, for example, by analyzing the compounds for antibacterial activity.

The medium and / or culture medium used to isolate and cultivate the ZMA B-1 culture, which produces the compound PM 181104, preferably contains carbon sources, nitrogen and inorganic inorganic salts. Sources of carbon are, for example, one or more of starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose or galactose. A preferred carbon source is glucose. Sources of nitrogen are, for example, one or more of soy flour, peanut flour, yeast extract, beef extract, peptone, malt extract, corn extract, gelatin or casamino 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, strontium chloride, potassium bromide, sodium fluoride, sodium acid phosphate, potassium acid phosphate, disodium phosphate, calcium carbonate, sodium bicarbonate, sodium silicate, ammonium nitrate, potassium nitrate, sodium sulfate, ammonium sulfate, magnesium sulfate, ferric iron citrate or boric acid. Calcium carbonate, sodium chloride and magnesium chloride are preferred.

Maintaining the culture of ZMA B-1 can be carried out in a temperature range from 21 ° C to 35 ° C and a pH from about 6.5 to 8.5. Typically, a ZMA B-1 culture is maintained at 27 ° C-29 ° C and a pH of about 7.4-7.8. Well-growing crops can be stored in the refrigerator at 4 ° C-8 ° C.

The cultivation of the seeded culture ZMA B-1 can be carried out in the temperature range from 25 ° C to 35 ° C and pH from about 6.5 to 8.5 for 20-55 hours at 200-280 rpm. Typically, a seeded ZMA B-1 culture is cultured at 29 ° C-31 ° C and a pH of from about 7.4 to 7.8 for 24-48 hours at 230-250 rpm.

The preparation of compound PM 181104 can be carried out by culturing a ZMA B-1 culture by fermentation in a temperature range from 26 ° C to 36 ° C and a pH from about 6.5 to 8.5 for 24-96 hours at 60-140 rpm and aeration 100 -200 liters per minute. Typically, a ZMA B-1 culture is cultured at 30 ° C-32 ° C and a pH of 7.4-7.8 for 40-72 hours at 90-110 rpm and aeration of 140-160 liters per minute.

The preparation of compound PM 181104 can be accomplished by culturing a ZMA B-1 culture in an acceptable nutrient medium under the conditions described herein, preferably under submerged aerobic conditions, for example, in agitated flasks, as well as in laboratory fermenters. The development of fermentation and the preparation of compound PM 181104 can be detected by high performance liquid chromatography (HPLC) and by measuring the bioactivity of the culture medium using Staphylococci and / or Enterococci species by a known method of microbiological analysis of agar diffusion in Petri dishes. The preferred culture is Staphylococcus aureus 3066, which is a methicillin-resistant strain, a β-lactam antibiotic reported in the literature, and Enterococcus faecium R2 (VRE), resistant to vancomycin. In the resulting culture medium, compound PM 181104 is present in the culture filtrate as well as in the cell mass and can be isolated by known separation techniques such as solvent extraction and column chromatography. Thus, the compound PM 181104 can be isolated from the culture filtrate by extraction at a pH of from about 5 to 9 with a water-immiscible solvent, such as petroleum ether, dichloromethane, chloroform, ethyl acetate, diethyl ether or butanol, or hydrophobic interaction chromatography using polymer resins, such as “Diaion HP-20®” (Mitsubishi Chemical Industries Limited, Japan), “Amberlite XAD®” (Rohm and Haas Industries USA), activated carbon, or ion exchange chromatography at pH 5-9. The active material can be isolated from the cell mass by extraction with a water-miscible solvent such as methanol, acetone, acetonitrile, n-propanol, or isopropanol, or with a water-immiscible solvent such as petroleum ether, dichloromethane, chloroform, ethyl acetate or butanol. Another option is to extract the whole nutrient medium with a solvent selected from petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol, acetone, acetonitrile, n-propanol, isopropanol or butanol. Typically, the active material is extracted with ethyl acetate from a complete culture medium. Concentration and lyophilization of the extracts gives the active crude material.

Compound PM 181104 of the present invention can be isolated from the crude material by fractionation using any of the following methods: normal phase chromatography (using alumina or silica gel as the stationary phase; eluents such as petroleum ether, ethyl acetate, dichloromethane, acetone, chloroform methanol or combinations thereof; and amine additives such as NEt ₃ ); reverse phase chromatography (using reverse phase silica gel such as dimethyl octadecylsilyl silica gel (RP-18) or dimethyl octyl silica 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® (Pharmacia 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 eluent system made of 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 applied repeatedly, separately or in combination. A typical method is reverse phase silica gel chromatography (RP-18).

Compound PM 181104 and its isomers can be converted into their pharmaceutically acceptable salts as provided by this invention. Salts can be obtained by standard methods known to a person skilled in the art, for example, salts such as sodium and potassium salts can be obtained by treating the compound PM 181104 and its isomers with acceptable sodium or potassium bases, for example, sodium hydroxide, potassium hydroxide.

Esters and ethers of compound PM 181104 represented by formula I can be prepared by methods described in the literature (Advanced Organic Chemistry, 1992, ^4th Edition, J. March, John Wiley & Sons). Esters can also be obtained by the method described in the literature (J. Med. Chemistry, 1992, 35, 145-151). In a preferred embodiment of the invention, compounds of formula I, wherein R is alkyl, cycloalkyl, aryl or heterocyclyl, are prepared by the reaction of PM 181104 with the corresponding acid of formula RCOOH; where R is alkyl, cycloalkyl, aryl or heterocyclyl, in the presence of a binding agent such as dicyclohexyl carbodimide (DCC) and catalytic amounts of a base such as dimethylaminopyridine (DMAP).

Phosphate esters can be prepared by a method known in the literature (Bioorganic & Medicinal Chemistry Letters, 1994, vol. 4, No. 21, 2567-2572). Ethers can be obtained by the method described in US patent 7022667, which is incorporated into this description by reference.

Compound PM 181104 has antibacterial activity against a wide range of bacterial strains. Compound PM 181104, stereoisomers, pharmaceutically acceptable salts and derivatives, such as their esters and ethers, separately or together, can be administered to animals such as mammals, including humans, as pharmaceutical preparations and in the form of pharmaceutical compositions. Therefore, the present invention also relates to the compound PM 181104, its stereoisomers, its pharmaceutically acceptable salts and its derivatives of ester and ether for use as pharmaceuticals and the use of the compound PM 181104, stereoisomers, pharmaceutically acceptable salts and its derivatives of ester and ether for obtaining drugs with antibacterial activity.

The present invention further relates to pharmaceutical compositions which comprise an effective amount of a compound PM 181104 and / or stereoisomers and / or one or more pharmaceutically acceptable salts and / or derivatives thereof, especially its esters and ethers, together with a pharmaceutically acceptable carrier. An effective amount of the compound PM 181104, or its stereoisomers, or its pharmaceutically acceptable salts, or its derivatives as active ingredient in pharmaceutical preparations, is usually from about 0.01 mg to 100 mg.

This invention also relates to a method for producing a medicament containing the compound PM 181104, and / or stereoisomers, and / or one or more pharmaceutically acceptable salts and / or derivatives of its ester and ether, for the treatment and prevention of diseases caused by bacterial infections.

The compounds of this invention are particularly useful as antibacterial agents. This invention, therefore, relates to the use of compound PM 181104, and / or stereoisomers, and / or one or more pharmaceutically acceptable salts and / or derivatives thereof, for the manufacture of a medicament for the prevention or treatment of diseases caused by bacterial infections. Bacterial infections for the treatment of which the compounds of this invention are used can be caused by bacteria belonging to the species Staphylococcus, Streptococcus, Enterococcus and Bacillus.

The expression "Staphylococcus species" means gram-positive bacteria that look like clusters of grapes under a microscope, and when grown in Petri dishes on blood agar, they look like large, round, golden yellow colonies, often with β-hemolysis. Staphylococcus aureus, belonging to the species Staphylococcus, causes a number of purulent (pyogenic) infections, such as superficial skin lesions, such as boils, eyelids and furunculosis; more severe infections, such as pneumonia, mastitis, phlebitis, meningitis and urinary tract infections; and deep infections such as osteomyelitis and endocarditis. Staphylococcus aureus is the main cause of hospital-acquired (nosocomial) infections of surgical wounds and infections that inhabit medical devices. Staphylococcus aureus causes food poisoning by the release of enterotoxins into food and toxic shock syndrome due to the release of superantigens into the bloodstream.

The expression "Streptococcus species" means a genus of spherical gram-positive bacteria belonging to the type Firmicutes. Streptococci are lactic acid bacteria. Species of Streptococcus cause infectious diseases, such as meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotic fasciitis (carnivorous bacterial infections).

The expression "Enterococcus species" means a genus of lactic acid bacteria of the type Firmicutes. These are gram-positive cocci, which are often found in pairs (diplococci). Enterococci are facultative anaerobic organisms. Enterococci is among the most common causes of nosocomial infections. Enterococci develop resistance to antibiotics such as gentamicin and vancomycin.

The expression "species of Bacillus" means a large number of different rod-shaped gram-positive bacteria that move thanks to the cilia and are aerobic. They also belong to the Firmicutes type. Members of this genus are able to form endospores that are highly resistant to adverse environmental conditions. Bacillus cereus, belonging to the species of Bacillus, causes two types of food intoxication. One type is characterized by symptoms of nausea, vomiting, and abdominal cramps. The second type is manifested primarily in abdominal cramps and diarrhea. Infections caused by Bacillus subtilis, which belongs to the Bacillus species, include bacteremia, endocarditis, pneumonia and sepsis in patients with compromised immune statuses.

The compounds of this invention can be administered orally, nasally, topically, subcutaneously, intramuscularly, intravenously or by other means of administration.

Pharmaceutical compositions containing PM 181104, or a stereoisomer, or a pharmaceutically acceptable salt, or its derivatives of an ester or ether, with other pharmaceutically active substances can be obtained by mixing the active compounds with one or more pharmacologically tolerable excipients and / or excipients, such as moisturizers, a dissolving agent, such as surfactants, bases, tonic agents, fillers, colorants, taste masking agents, lubricants , disintegrating agents, diluents, binders, plasticizers, emulsifiers, ointment bases, emollients, thickeners, polymers, lipids, oils, cosolvents, complexing agents or buffering agents, and converting the mixture into an acceptable pharmaceutical form, such as, for example, tablets, coated tablets, capsules, granules, powders, creams, ointments, gels, syrups, emulsions, suspensions or solutions suitable for parenteral administration.

Examples of excipients and / or excipients that may be mentioned are cremophor, poloxamer, benzalkonium chloride, sodium lauryl sulfate, dextrose, glycerin, magnesium stearate, polyethylene glycol, starch, dextrin, lactose, cellulose, sodium carboxymethyl cellulose, agar, talc mineral oil, animal oil, vegetable oil, organic and mineral waxes, paraffin, gels, propylene glycol, benzyl alcohol, dimethylacetamide, ethanol, polyglycols, tween 80, HS 15 solutol and water.

It is also possible to use the active substances, as such, without bases or diluents in an acceptable form, for example in capsules.

As is generally accepted, the galenical formulation and method of administration, as well as the dosage range that are acceptable in a particular case, depend on the species to be treated and the level of the corresponding condition or disease, and can be optimized using methods known in the art. The average daily dose of the active compound in a patient is from 0.0005 mg to 15 mg per kg, usually from 0.001 mg to 7.5 mg per kg.

The following are illustrative examples of the present invention, not limiting its scope.

Example 1

Isolation of ZMA B-1 Culture from a Marine Source

a) Composition of isolation medium

Nutrient medium Zobell Marine 2216 (agarified with 1.5% agar-agar)

Pepsin digestion products of animal tissues 5.0 g, yeast extract 1.0 g, ferric iron citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1, 8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 80.0 mg, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, agar powder 15.0 g, twice distilled water 1.0 l, final pH (at 25 ° C) 7.4-7.8.

b) Methodology

Spirastrella inconstans var. digitata (Dendy) was taken to Palk Bay, the coast of Tamil Nadu, India, using scuba gear at a depth of three meters. A sponge sample was rinsed in sterile seawater and immediately transferred to sterile plastic containers. The containers were stored at -20 ° C and transported, keeping the temperature below 0 ° C, to the laboratory for further research. When entering the laboratory, the sponge samples were stored at a temperature below 0 ° C, and later warmed up to room temperature (25 ± 2 ° C) only before isolation of the culture. A sponge sample was cut under sterile conditions into 2 x 2 cm pieces and suspended in 5 ml of sterile sea water in a 25 ml sterile tube. The tube was shaken for 30 seconds; sea water was drained and fresh sea water was added. These actions were repeated three times. The sea water was finally drained, and the sponge pieces were placed in Petri dishes containing the aforementioned isolation medium [Zobell Marine 2216 nutrient medium (agarized 1.5% agar agar); HiMedia]. Petri dishes were incubated at room temperature (25 ± 2 ° C) until growth appeared in them. Colonies grown in Petri dishes were isolated based on the characteristics of the colonies and streaked in Petri dishes containing the aforementioned isolation medium [nutrient medium Zobell Marine 2216 (agarized 1.5% agar-agar); HiMedia]. The isolates were re-reseeded to obtain a pure ZMA B-1 culture. The ZMA B-1 culture is thus isolated from the grown microorganisms as a separate isolate.

Example 2

Purification culture ZMA B-1

a) the composition of the selection medium

Nutrient medium Zobell Marine 2216 (agarized 1.5% agar-agar)

Peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, agar 15.0 g, demineralized water 1.0 l, pH 7.4-7.8.

b) Methodology

The culture was obtained on a nutrient medium Zobell Marine 2216 (agarized 1.5% agar-agar) in a Petri dish with a diameter of 15 mm The culture growing on the Petri dish was streaked by streak on the Zobell Marine 2216 mowing medium (agarized 1.5% agar-agar). Mowed medium was incubated for 2 days at 25 ° C. One of the individual colonies in the upper part of the beveled medium was transferred to freshly beveled environments. Mowed media were incubated for 2 days at 25 ° C. Then they were used for fermentation in agitated flasks for an initial anti-infection examination.

Example 3

Maintenance of the producing strain - ZMA B-1 culture

a) Composition of the medium (culture medium Zobell Marine 2216)

Peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, agar 15.0 g, demineralized water 1.0 l, pH 7.4-7.8.

b) After the ingredients are completely dissolved by heating, the resulting solution is distributed into tubes and sterilized at 121 ° C for 30 minutes. The tubes were cooled and the medium allowed to solidify in a beveled position. Mowed agar was streaked with a growing ZMA B-1 culture using a wire loop and incubated at 27-29 ° C until good growth was observed. Well-growing cultures were stored in a refrigerator at 4-8 ° C.

Example 4

Fermentation of ZMA B-1 culture in agitated flasks

a) The composition of the seed medium (nutrient medium Zobell Marine 2216)

Peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, demineralized water 1.0 l, pH 7.4-7.8.

b) The above medium was distributed 40 ml in 500 ml Erlenmeyer flasks and autoclaved at 121 ° C for 30 minutes. The flasks were cooled to room temperature, each flask was inoculated with a loop of a well-growing strain (ZMA B-1 culture) on a beveled medium and shaken on a rotary mixer for 24-48 hours at 230-250 rpm at 30 ° С (± 1 ° С ) to obtain a culture of sowing.

c) Production medium composition

Peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, demineralized water 1.0 l, pH 7.4-7.8.

d) 40 ml of production medium in Erlenmeyer flasks with a capacity of 500 ml were autoclaved at 121 ° C for 30 minutes, cooled to 29-30 ° C and seeded with 2 ml of the inoculum culture mentioned in Example 4b.

e) Fermentation parameters

Temperature 29-30 ° C; agitation 230-250 rpm; collection time 48-72 hours.

The production of compound PM 181104 in a fermentation medium was determined by analyzing the bioactivity compared to Enterococcus faecium R2 (VRE) and / or S. aureus 3066 MRSA strain by agar diffusion. The pH of the culture medium was 7.0-8.0. The culture medium was collected, and all of it was used for bioactivity analysis, which shows the presence of the compound PM 181104 in the fermentation medium.

Example 5

Obtaining culture of inoculation in agitated flasks for fermentation

a) the composition of the medium

Peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, demineralized water 1.0 l, pH 7.4-7.8.

b) The above medium was dispensed 200 ml into 1 L of an Erlenmeyer flask and autoclaved at 121 ° C for 30 minutes. The flasks were cooled to room temperature, each flask was inoculated with a loop of a well growing strain (ZMA B-1 culture) on a beveled medium and shaken on a rotary mixer for 24-48 hours at 230-250 rpm at 29-31 ° С to obtain a culture seeding.

Example 6

Cultivation of ZMA B-1 Culture in a Fermenter

a) the composition of the production medium

Glucose 50.0 g, yeast extract 11.0 g, peptone 4.0 g, beef extract 4.0 g, calcium carbonate 5 g, sodium chloride 2.5 g, demineralized water 1 l, pH 7.6 (before sterilization )

b) 250 L of production medium in a 300 L fermenter with 80 ml of desmophene as an anti-foam agent was sterilized in situ for 30 minutes at 121 ° C, cooled to 29-31 ° C and seeded with 6 L of the inoculum culture mentioned in Example 5b.

c) Fermentation parameters

Temperature 30-32 ° C; agitation 100 rpm; aeration of 150 liters per minute; collection time 44-66 hours.

The production of compound PM 181104 in a fermentation medium was determined by analyzing bioactivity compared to S. aureus 3066 (MRSA strain) and / or Enterococcus faecium R2 (VRE) by agar diffusion. The pH of the culture medium was 7.0-8.0. The culture medium was collected and all of it was used for bioactivity analysis, which shows the presence of the compound PM 181104 in the fermentation medium.

Example 7

Isolation and purification of compound PM 181104

The whole growth medium (240 L) of Example 6 was collected and extracted with ethyl acetate (240 L), stirring in a glass vessel. The organic layer was separated using a batch type disk separator (Alfa-laval, model LAPX404) and concentrated to obtain a crude extract (296 g). The resulting crude material was stirred, sonicated for 30 minutes using petroleum ether (3 × 1 L), and filtered to obtain an insoluble residue (38 g), which was chromatographed by vacuum liquid chromatography in the following manner.

The insoluble residue (35.5 g) was dissolved in a mixture of methanol and acetonitrile (3: 1, 400 ml) and pre-absorbed onto LiChroprep RP-18 [25-40 µ, 40 g] and a funnel with a porous filter (grade G-4) was used ; 10 cm x 10.5 cm) filled with LiChroprep RP-18 absorbent (25-40 μ, 110 g). Elution using a vacuum system (100-120 mm) was performed first with water (4 L), then with methanol (1: 1, 5 L), methanol (3 L), methanolacetonitrile (2.5 L) and acetonitrile. A purity test was performed by Ent bioassay. faecium R2 and / or S. aureus 3066 and / or analytical HPLC. Compound PM 181104 was determined in methanol and methanol fractions: acetonitrile and acetonitrile. These fractions were combined and concentrated to obtain a semi-material (1.826 g).

Final purification was performed by repeated preparative HPLC under the following conditions:

Column: Eurospher RP-18 (10 µ, 32 × 250 mm).

Eluent: acetonitrile: water (56:44).

Flow rate: 50 ml / min.

Definition: 220 nm.

Retention time: 12-14 minutes.

The purity of the fractions was measured by Ent bioassay. faecium R2 and / or S. aureus 3066 and / or analytical HPLC. Eluates containing compound PM 181104 were collected and concentrated under reduced pressure to remove the solvent to obtain 600 mg of pure compound.

Physicochemical and spectral features of the compound PM 181104

Appearance: white amorphous solid.

Melting point:> 300 ° C (decomposes).

Solubility: methanol, DMSO (dimethyl sulfoxide).

HPLC: at room temperature, 5.61 minutes.

Column: Kromasil C18 (5 µ; 150 x 4.6 mm inner diameter) (column temperature 40 ° C).

Mobile phase: acetonitrile: water (1: 1).

Injection volume: 10 µl (concentration in the mobile phase of 0.1 mg / ml).

Flow rate: 1 ml / min.

Definition: 220 nm.

HR-ESI (+) MS m / z: 1515.3733 (M + H).

Molecular Formula: C ₆₉ H ₆₆ N ₁₈ O ₁₃ S ₅ .

Molecular mass: 1514.

UV (MeOH): 205.2, 220.6 and 306.2 nm (according to Figure 1).

IR (KBr): 3368, 2981, 1654, 1516, 1429, 1314, 1199, 1269, 1074, 1034, 805, 580 cm ^-1 (according to Figure 2).

¹ H NMR: according to table 1 and Figure 3.

¹³ C NMR: according to table 2 and Figure 4.

Table 1: ¹ H NMR of the compound PM 181104 in DMSO-d ₆ Peak 8 Peak 8 Peak 8 one 1.33-1.34 (d, 3H) 21 5.33 (m, 1H) 41 8.57-8.58 (d, 1H) 2 1.94 (m, 4H) 22 5.52 (s, 1H) 42 8.65 (s, 1H) 3 2.05-2.06 (brm, 1H) 23 5.63 (s, 1H) 43 8.81 (t, 2H) four 2.12 (brm, 1H) 24 5.83 (s, 1H) 44 9.07 (s, 1H) 5 2.25-2.28 (brd, 1H) 25 5.92 (s, 1H) 45 9.16 (s, 1H) 6 2.40-2.42 (brm, 1H) 26 6.07 (s, 1H) 46 9.48 (s, 1H) 7 2.68 (s, 3H) 27 6.50 (s, 1H) 47 10.03 (s, 1H) 8 2.76-2.82 (m, 2H) 28 6.59-6.61 (d, 2H) 9 2.97-2.99 (d, 1H) 29th 6.86 (s, 1H) 10 3.17-3.21 (m, 1H) thirty 7.04-7.06 (d, 2H) eleven 3.24-3.26 (m, 1H) 31 7.17-7.18 (m, 1H) 12 3.62 (t, 2H) 32 7.26 (d, 4H) 13 3.69 (m, 1H) 33 7.30 (s, 1H) fourteen 3.77 (s, 2H) 34 7.33-7.35 (d, 1H) fifteen 4.49-4.50 (d, 1H) 35 7.53 (s, 1H) 16 4.69-4.71 (t, 1H) 36 7.69 (s, 1H) 17 4.80 (m, 1H) 37 7.90 (s, 1H) eighteen 4.89 (m, 1H) 38 7.95 (s, 1H) 19 4.93-4.97 (t, 1H) 39 8.27-8.29 (d, 2H) twenty 5.23 (t, 1H) 40 8.49-8.50 (d, 1H)

Table 2: ¹³ C NMR of compound PM 181104 in DMSO-d ₆ signal 8 signal 8 signal 8 one 12.04 22 115.46 * 43 151.04 2 16.88 23 116.92 44 152.08 3 25.16 * 24 118.77 45 153.43 four 29.42 25 122.84 46 154.20 5 33.09 26 123.39 47 156.27 6 36.67 27 124.33 48 156.45 7 37.00 28 127.08 49 159.06 8 38.62 29th 127.36 fifty 161.27 9 38.73 thirty 128.71 * 51 161.54 * 10 47.22 31 129.18 52 162.79 eleven 47.76 32 129.76 * 53 163.35 12 47.88 33 130.11 54 165.58 13 48.88 34 130.99 * 55 167.95 fourteen 52.69 35 133.99 56 169.68 fifteen 54.43 36 135.17 57 170.39 16 59.92 37 136.87 58 171.03 17 60.56 38 137.67 59 171.77 eighteen 77.80 39 140.92 60 171.96 19 103.89 40 147.97 61 173.51 twenty 104.94 41 149.73 62 174.14 21 107.52 42 150.83 63 174.90 * two carbon

BIOLOGICAL ASSESSMENT OF COMPOUND RM 181104

In vitro experience

Example 8

In vitro activity was determined by the lowest inhibitory concentration (MIC) of compound PM 181104 against bacterial strains using the method of diluting the nutrient macroenvironment according to the National Committee for Clinical Laboratory Standards (2000) [Journal of Anti-microbial Chemotherapy, 2002, 50, 125-128 ( Cross Reference 8: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically-Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA)]. Unless stated otherwise, Mueller-Hinton culture medium was used as a culture medium for analysis. Linezolid (manufactured by Glenmark Pharma Ltd; Part No. K2005028) was used as a known standard in all in vitro experiments. To obtain a mother liquor, the compound PM 181104 was dissolved in chloroform (5% of the total required volume) and diluted with methanol (95% of the total required volume).

results

The results obtained are presented in Table 3 below, and it is shown that the compound PM 181104 is acceptable for the treatment of bacterial infections.

Table 3: MIC compound PM 181104 by bacterial strains Body to be analyzed MIC (μg / ml) Body to be analyzed MIC (μg / ml) S. aureus KEM-MRSA1 0,03125 S. epidermidis 32965 0,03125 S. aureus KEM-MRSA2 0,03125 S. haemolytica 809 0.0625 S. aureus KEM-MRSA7 0.01563 Enterococci KEM-VRE26 0,00781 S. aureus KEM-MRSA8 0.01563 Enterococci KEM-VRE27 0,00391 S. aureus KEM-MRSA11 0.0625 Enterococci KEM-VRE28 0,00781 S. aureus KEM-MRSA12 0.01563 Enterococci KEM-VRE29 0,00391 S. aureus KEM-MRSA18 0.01563 E. faecium (R-2) 0,00781 S. aureus KEM-MRSA19 0,00781 E. faecium (VR-1) 0,00781 S. aureus KEM-MRSA20 0.01563 E. faecium D-59 0,00781 S. aureus KEM-MRSA21 0.01563 E. faecium D 18F 0,00781 S. aureus KEM-MRSA22 0,00781 E. faecium (02-D3IP1) 0,00391 S. aureus KEM-MRSA23 0.01563 E. faecium 4045H 0,00781 S. aureus KEM-MRSA24 0,00781 E. faecalis (FH-1) 0,00391 S. aureus KEM-MRSA25 0.01563 E. faecalis (uD.8b) 0,00391 S. aureus lilavati-mrsa3 0.01563 E. faecalis 4073H 0,00781 S. aureus rehaja-mrsal 0,03125 E. faecium ATCC 51559 0,00781 S. aureus bom-mrsa2 0.0625 E. faecalis, ATCC 51299 0.01563 Staphylococcus aureus subsp.aureus ATCC 0,03125 E. faecalis ATCC 51575 0,03125 S. aureus (789) 0,00781 E. faecalis ATCC BAA 472 0.01563 S. aureus (20666) 0.01563 B. cereus 0,00391 S. aureus (3066) 0,03125 B. subtilis ATCC 6633 0,00781 S. aureus SG511 0.0625 B. subtilis 0.01563 S. aureus wien 8 0.01563 B. megaterium FH 1127 0,00781 S. aureus wien 13 0.0625 B. firmus 0.01563 S. aureus Cl 3184 0.0625 Streptococcus hirae 55 0,00391 S. aureus (E710) 0,03125 Streptococcus equinus 02D5Grl 0.01563 S. aureus ATCC29213 0,03125 Streptococcus durans 4939 (1) H 0,00781 S. epidermidis 5744IW 0.01563 Streptococcus durans 0,00781 S. epidermidis pat 01 IV 0.0625 Salmonella typhi para a > 1 S. epidermidis 823 0,03125 Pse.aeruginosa (M-35) > 1 S. epidermidis 6098 0.0625 Klebsiella pneumoniae > 1 S. epidermidis 649311 (2) 0.0625 Citrobacter diversus 2046 E > 1 S. epidermidis 1022111 W 0,00781 E.coli SS > 1

Abbreviations Used in Table 3

S: Staphylococcus

E: Enterococci

B: Bacillus

In vivo experience

In vivo activity was evaluated using protective dose (PD) determinations of compound PM 181104 by its antibiotic activity in three animal models using either male or female Balb / C mice. The models used were traditional models for analyzing target efficacy and organ / tissue specific efficacy. The traditional models used to analyze target efficacy were the systemic infection model (sepsis) and the localized infection model (hip neutropenia model). The organ / tissue-specific infection models used for efficacy analysis were kidney and lung infection and a skin abscess model.

Example 9

Systemic infection model

Animals were intraperitoneally infected from ~ 10 ⁸ to 10 ⁹ CFU with a fast-growing culture resistant to Staphylococcus aureus E710 methicillin (MRSA) suspended in isotonic solution (0.85% sodium chloride). A solution of PM 181104 was obtained in the formulation of cremophor and ethanol as described in Example 14. The solution was administered intravenously at doses of 5 mg, 2.5 mg and 1.25 mg / kg immediately after infection. Each experimental group contained ten animals. PD ₁₀₀ PM 181104 was determined for the sepsis model, which was 5 mg / kg compared to the standard antibiotic linezolid (manufactured by Glenmark Pharma Ltd; lot no. K2005028), which showed PD ₁₀₀ at 25 mg / kg.

Example 10

Hip neutropenia model

Neutropenia was induced in mice by cyclophosphamide (150 mg and 100 mg / kg) 96 hours and 24 hours, respectively, before infection with S. aureus E-710. The animals were injected intramuscularly with thigh infection at ~ 10 ⁷ CFU of a rapidly growing culture of S. aureus E-710 suspended in isotonic solution (0.85% sodium chloride). Each experimental group contained six animals. A solution of PM 181104 was obtained in the formulation of cremophor and ethanol as described in Example 14. The solution was administered intravenously at a dose of 5 mg 2 hours after infection. Animals were killed at different points in time and femoral tissues were taken to determine the number of viable microorganisms. A decrease of approximately 1 logarithm was observed with PM 181104 at a dose of 5 mg / kg, at a time point of 6 hours, which was compared with the standard antibiotic linezolid (manufactured by Glenmark Pharma Ltd; lot no. K2005028) at a dose of 25 mg / kg.

Example 11

Kidney infection model

0.2 ml of 2% λ carrageenan was administered intravenously to Balb / C mice 7 days before infection. A culture of Enterococcus faecium ATCC 47077 in a logarithmic growth phase, adjusted to approximately 10 ⁹ CFU / ml, was injected intravenously into 0.2 ml mice. A solution of PM 181104 obtained in the formulation of cremophor and ethanol as described in Example 14 was intravenously administered to mice at time points 4 hours, 24 hours and 48 hours after infection. 72 hours after infection, the animals were killed and the kidneys were taken to determine the bacterial load. A decrease in the number of viable bacteria by approximately 1 logarithm was observed with PM 181104 at a dose of 5 mg / kg, which was compared with the standard antibiotic linezolid (manufactured by Glenmark Pharma Ltd; lot no. K2005028) at a dose of 25 mg / kg and vancomycin hydrochloride (manufactured by HiMedia; No. B catalog RM217-500 mg; batch No. 06-0350) at a dose of 150 mg / kg.

Example 12

Lung infection model

In Balb / C mice, neutropenia was induced by intraperitoneal administration of 200 mg / kg cyclophosphamide four days and two days before infection. On the day of infection, mice were anesthetized and infected with a suspension of S. aureus E-710 culture in a logarithmic growth phase with a bacterial density of approximately 10 ⁶ to 10 ⁷ CFU / ml. 24 and 36 hours after infection, the first and second appropriate doses of the drug were administered intravenously. 48 hours after infection, the animals were humanely sacrificed and lungs were collected aseptically to determine the number of viable bacteria. In this model, PM 181104 obtained in the formulation of cremophor and ethanol, as described in Example 14, was analyzed at a dose of 5 mg / kg. Two standard antibiotics were used as a positive control, namely linezolid (in a single dose of 80 mg / kg 24 hours after infection) and vancomycin (110 mg / kg, two doses 24 and 48 hours after infection). PM 181104 showed bacteriostatic activity, which was compared with the standard linezolid (manufactured by Glenmark Pharma Ltd; lot no. K2005028). The vancomycin hydrochloride standard (manufactured by HiMedia; Catalog No. RM217-500 mg; Lot No. 06-0350) showed a bactericidal profile. This amounted to a difference of approximately 2 logarithms in terms of the number of viable bacteria in the lungs of animals treated with PM 181104, compared with that in untreated control animals.

Example 13

Skin abscess model

Balb / C mice were subcutaneously infected with approximately 10 ⁸ CFU of a fast-growing methicillin resistant S. aureus E710 (MRSA) culture. Bacteria were suspended in a 1: 1 mixture of 2% cetodex granules in isotonic solution (0.85% sodium chloride). PM 181104 was obtained in the formulation of cremophor and ethanol as described in Example 14. The solution was administered intravenously at doses of 2.5 mg, 5 mg and 10 mg / kg two hours after infection. Each experimental group consisted of six animals. After the formation of the abscess, the animals were killed and an abscess was collected to determine the number of viable bacteria. A decrease in the number of viable bacteria by approximately 1 logarithm was observed with PM 181104 at a dose of 5 mg / kg compared to the standard antibiotic used, namely linezolid (manufactured by Glenmark Pharma Ltd; lot no. K2005028) at a dose of 50 mg / kg.

FORMULATION OF COMPOUND PM 181104

Example 14

Formulations for injection were prepared by the following general method.

Ethanol and Cremophor EL were mixed in a ratio of 1: 1 (by weight). PM 181104 was added and shaken. This mixture was sonicated at 25 ° C. This mixture (regarded as a 10% component) was diluted by adding water (90%) and shaken to obtain an injection formulation.

DERIVATIVE COMPOUNDS RM 181104

Example 15

Derivative of PM 181104 butyric acid ester

To a solution of PM 181104 (0.13 g, 0.085 mmol) in dichloromethane (2 ml) was added butyric acid (0.008 μl, 0.085 mmol), DCC (dicyclohexylcarbodiimide) (0.018 g, 0.085 mmol) and a catalytic amount of DMAP (dimethylaminopyridine) (0.002 g, 0.016 mmol), and the reaction mixture was stirred for 18 hours under a nitrogen atmosphere. Cold water was added to the reaction mixture and the organic layer was separated; the aqueous layer was extracted with dichloromethane (3 × 50 ml), the organic extracts were combined and washed with water (2 × 30 ml). The organic layer was dried over sodium sulfate and concentrated. The crude product was purified by column chromatography [silica gel (60-120 mesh), 4% methanol in chloroform] to obtain the title compound as a white solid. Yield: 0.11 g (81%); MS m / z (ESI): 1585 (M + H).

Derived PM 181104 butyric acid ester showed a MIC value of 2.5 μg / ml against the bacterial strain E.faecium R-2 (VRE).

Example 16

Derivative of PM 181104 stearic acid ester

To a solution of PM 181104 (0.12 g, 0.079 mmol) in dichloromethane (2 ml) was added stearic acid (0.022 μl, 0.079 mmol), DCC (0.016 g, 0.079 mmol) and a catalytic amount of DMAP (0.002 g, 0.016 mmol), and the reaction mixture was stirred for 6 hours under nitrogen. Cold water was added to the reaction mixture and the organic layer was separated; the aqueous layer was extracted with dichloromethane (3 x 50 ml), the organic extracts were combined and washed with water (2 x 30 ml). The organic layer was dried over sodium sulfate and concentrated. The crude product was purified by column chromatography [silica gel (60-120 mesh), 4% methanol in chloroform] to obtain the title compound as a white solid. Yield: 0.1 g (71%); MS m / z (ESI): 1781 (M + H).

The stearic acid ester derivative PM 181104 showed a MIC value of 1.25 μg / ml against the bacterial strain E.faecium R-2 (VRE).

Example 17

Derived PM 181104 Niacin ester

To a solution of PM 181104 (0.02 g, 0.013 mmol) in N, N-dimethylformamide (1 ml) were added nicotinic acid (0.008 g, 0.065 mmol), DCC (0.014 g, 0.065 mmol) and a catalytic amount of DMAP (0.0008 g, 0.0065 mmol), and the reaction mixture was shaken for 18 hours under nitrogen. The solvent was removed and 10 ml of dichloromethane was added to the residue. Insoluble urea was filtered off, and the filtrate was washed with water (2 x 10 ml). The organic layer was dried over sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography [C-18 reverse phase column (Eurosphere, 20 nm), 55% acetonitrile in water] to obtain the title compound as a white solid. Yield: 0.011 g (56%); MS m / z (ESI): 1621 (M + H).

The nicotinic acid ester derivative PM 181104 was analyzed by bacterial strains. The results are shown in table 4 below, and show that the derivative PM 181104 of the nicotinic acid ester is acceptable for the treatment of bacterial infections.

Table 4: MIC of the PM181104 derivative of nicotinic acid ester by bacterial strains Organism to be analyzed MIC (μg / ml) Organism to be analyzed MIC (μg / ml) E. faecalis ATCC 51299 0.312 E. faecium R-2 (VRE) 0.312 E. faecalis ATCC 51575 0.312 S. aureus MRS A ATCC 33591 > 10 E. faecalis ATCC BAA472 0.312 S. aureus M RSA E710 > 10 E. faecium ATCC 51559 0.156

Claims (20)

1. The compound of the following formula I:

where R = H, C ₁ -C ₂₀ alkylcarbonyl and

or a pharmaceutically acceptable salt (s) thereof. 2. The compound according to claim 1, where R is H, named PM181104, or a pharmaceutically acceptable salt thereof. 3. The compound PM181104 according to claim 2, having antibacterial activity, the compound isolated from the fermentation medium of a microorganism belonging to the species Kocuria (ZMA B-1 / MTCC 5269), and is characterized by:
(a) a molecular weight of 1514,
(b) the molecular formula C ₆₉ H ₆₆ N ₁₈ O ₁₃ S ₅ ,
(c) UV spectrum as shown in Figure 1,
(d) IR spectrum as shown in Figure 2,
(e) ¹ H NMR spectrum as shown in Figure 3,
(f) ¹³ C NMR spectrum as shown in Figure 4. 4. The method of obtaining compounds PM181104 according to claim 2 or 3, in which:
(a) cultivating a microorganism of the species Kocuria (ZMA B-1 / MTCC 5269) or one of its variants or mutants under submerged aerobic conditions in a nutrient medium containing carbon and nitrogen sources to obtain the compound PM181104,
(b) isolate compound PM181104 from the fermentation medium, and
(c) purify the compound PM181104. 5. A method of obtaining a pharmaceutically acceptable salt of compound PM181104 according to claim 2 or 3, wherein:
(a) cultivating a microorganism of the species Kocuria (ZMA B-1 / MTCC 5269) or one of its variants or mutants under submerged aerobic conditions in a nutrient medium containing carbon and nitrogen sources to obtain the compound PM181104,
(b) isolate compound PM181104 from a fermentation culture medium,
(c) purify the compound PM181104, and
(d) converting the compound PM181104 into a pharmaceutically acceptable salt thereof. 6. The compound according to claim 1, where R = CH ₃ CH ₂ CH ₂ CO or CH ₃ (CH ₂ ) ₁₅ CH ₂ CO. 7. A pharmaceutical composition adapted for the treatment of a bacterial infection, comprising an effective amount of the compounds according to claim 1, with at least one pharmaceutically acceptable excipient, or additive, or excipient. 8. The pharmaceutical composition according to claim 7, where the pharmaceutical composition is in the form of a tablet, coated tablets, capsules, granules, powder, cream, ointment, gel, emulsion, suspension or injection. 9. The pharmaceutical composition according to claim 7, wherein said bacterial infection is caused by bacteria belonging to the species Staphylococcus, Streptococcus, Enterococcus or Bacillus. 10. The pharmaceutical composition according to claim 9, where these bacteria belong to species of Staphylococcus or Enterococci. 11. The pharmaceutical composition of claim 10, wherein said bacterium belonging to a species of Staphylococcus is methicillin resistant. 12. The pharmaceutical composition of claim 10, wherein said bacterium belonging to a species of Staphylococcus is resistant to vancomycin. 13. The pharmaceutical composition of claim 10, where the specified bacterium,
belonging to the species Enterococci, is resistant to vancomycin. 14. A method of treating a bacterial infection in which an effective amount of a compound according to claim 1 is administered to a mammal, if necessary. 15. The method according to 14, where the bacterial infection is caused by a bacterium belonging to the species Staphylococcus, Streptococcus, Enterococci and Bacillus. 16. The method according to clause 15, where the bacterial infection is caused by a bacterium belonging to the species Staphylococcus or Enterococci. 17. The method according to clause 16, where the specified bacterium belonging to the species of Staphylococcus, is resistant to methicillin. 18. The method according to clause 16, where the specified bacterium belonging to the species of Staphylococcus, is resistant to vancomycin. 19. The method according to clause 16, where the bacterium belonging to the species Enterococci, is resistant to vancomycin. 20. The use of compounds according to claim 1, to obtain a medicinal product for the treatment of bacterial infections.

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