ES2440791T3 - Antibacterial compounds - Google Patents

Abstract

Compounds of the following formula I; ** Formula ** in which R> = H, alkyl, alkylcarbonyl, (HO) 2PO-, alkyl-OPO (OH) -, (alkyl-O) 2PO-, cycloalkyl, cycloalkylcarbonyl, aryl, arylcarbonyl, heterocyclyl and heterocyclyl carbonyl; or stereoisomers or tautomers or pharmaceutically acceptable salts thereof.

Description

Antibacterial compounds

FIELD OF THE INVENTION

[0001] This invention relates to a novel compound PM181104, which has antibacterial activity, which is obtained by fermentation of the microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 5269). The invention also includes all stereoisomeric forms and all tautomeric forms of PM181104 and pharmaceutically acceptable salts and derivatives thereof. The present invention further relates to the processes for the production of the novel antibacterial compound or compounds, to the production of the microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 5269), and to pharmaceutical compositions containing the novel compound or compounds such as an active ingredient, and its use in medicines for the treatment and prevention of diseases caused by bacterial infections.

BACKGROUND OF THE INVENTION

[0002] The emergence of bacterial resistance to various antimicrobial agents, such as beta-lactam antibiotics, macrolides, quinolones and vancomycin is becoming a fundamental health problem worldwide (Trends in Microbiology, 1994, 2, 422-425). The most significant problem in clinical practice is the increase in the incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the only effective treatment for multiple MRSA infections is vancomycin. However, there are several reports on the occurrence of vancomycin resistance in some strains of MRSA isolated (Antimicrobial Agents and Chemotherapy, 1998, 42, 2188-2192). Another group of bacteria resistant to multiple clinically relevant drugs that has emerged recently are enterococci, some of which also show resistance to vancomycin. The appearance of vancomycin-resistant enterococcal infections (VRE) has forced a dilemma among doctors. The combinations of linezolid, an oxazolidinone and streptogramin compound are the new drugs of choice for the treatment of MRSA infections. However, resistance to these combinations of oxazolidinones (Clinical Infectious Diseases, 2003, 36, supplement 1, S11-S23; Annals of Pharmacotherapy, 2003, 37, 769-74) and streptogramins (Current Drug Targets Infectious Disorders, 2001, 1 , 215-25) and various glycopeptides (Clinical Infectious Disorders 2003, 36, supplement 1, S11-S23) require the expanded development of agents with alternative targets or modes of action. The growing resistance of the pathogen acquired in the important community Streptococcus pneumoniae to penicillin and other antibacterials is becoming a global health problem. Multiple drug resistant strains of Mycobacterium tuberculosis have emerged in several countries. The emergence and proliferation of nosocomial and community-acquired resistant pathogens is becoming a major threat to global public health. WO-A-94/14 838 refers to antibiotics GE 37468 A, B and C.

[0003] There is an urgent need to discover new compounds that can be used as drugs to treat patients infected with bacteria, particularly multi-drug resistant bacteria, such as MRSA and VRE.

SUMMARY OF THE INVENTION

[0004] The present invention relates to a novel purified compound (designated herein as PM181104), isolated from the fermented broth of the microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 5269), which has antibacterial activity .

[0005] The invention also relates to all stereoisomeric forms and all tautomeric forms of PM181104 and a pharmaceutically acceptable salt or salts, and an ester or ether derivative or derivatives thereof, represented by formula I (as described in This document).

[0006] The compound PM181104, and pharmaceutically acceptable isomers, salt or salts and derivative or derivatives of the ester and ether thereof, are useful for the treatment and prevention of diseases caused by bacteria, particularly multi-drug resistant bacteria, such as MRSA and VRE

[0007] The invention further relates to pharmaceutical compositions comprising the novel compound PM181104, an isomer, pharmaceutically acceptable salts, and the ester and ether derivatives thereof, as an active ingredient for the treatment of medical conditions caused by bacteria, particularly multi-drug resistant bacteria, such as MRSA and VRE.

[0008] The present invention further relates to processes for the production of compound PM181104 and / or its isomers from the microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 5269).

[0009] The present invention also relates to the processes for the production of the microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 5269), which in the culture produces the compound PM181104 and its isomers.

[0010] The present invention also relates to the processes for the production of the ester and ether derivatives of compound PM181104.

DESCRIPTION OF THE DRAWINGS 10

[0011]

Figure 1 shows the ultraviolet (UV) absorption spectrum of PM181104. Figure 2 shows the infrared (IR) absorption spectrum of PM181104. 15 Figure 3 shows the 1 H NMR spectrum (500 MHz) of PM181104 in DMSO-d6. Figure 4 shows the 13C (125 MHz) NMR spectrum of PM181104 in DMSO-d6.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a novel antibacterial compound PM181104 and also includes all stereoisomeric forms and all tautomeric forms of PM181104 and acceptable pharmaceutical salts and derivatives, such as esters and ethers thereof.

[0013] Accordingly, the present invention relates to novel antibacterial compounds of the following formula I;

wherein R = H (PM181104), alkyl, alkylcarbonyl, (HO) 2PO-, alkyl-OPO (OH) -, (alkyl-O) 2PO-, cycloalkyl, cycloalkylcarbonyl, aryl, arylcarbonyl, heterocyclyl and heterocyclyl carbonyl.

[0014] As used herein, the term "alkyl" either used alone or as part of a group

substituent, refers to the radical of saturated aliphatic groups, including straight chain alkyl groups or

branched. In addition, unless otherwise indicated, the term "alkyl" includes unsubstituted alkyl groups, as well as alkyl groups, which are substituted with one or more different substituents. In preferred embodiments,

a straight chain or branched chain alkyl has 20 or less carbon atoms in its structure (by

example, C1-C20 for the linear chain, C3-C20 for the branched chain). Examples of alkyl residues containing

1 to 20 carbon atoms are: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,

undecyl, dodecyl, tetradecyl, heptadecyl and eicosyl, the n-isomers of all these residues; isopropyl, isobutyl, 140 methylbutyl, isopentyl, neopentyl, 2,2-dimethylbutyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 2,3,4-trimethylhexyl,

isodecyl, sec-butyl or tert-butyl. A substituted alkyl refers to an alkyl residue in which one or more atoms of

hydrogen, for example 1, 2, 3, 4 or 5 hydrogen atoms are replaced by substituents, for example, halogen,

hydroxyl, sulfonyl, alkoxy, cycloalkyl, cyano, azido, amino, acyloxy, heterocycle, aralkyl, aryl or groups

fluorescents, such as NBD [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] or the BODIPY [4,4-difluoro-5,7 group

dimethyl-4-bora-3a, 4a-diaza-s-indacene].

[0015] As used herein, the term "cycloalkyl" refers to a mono- or bicyclic saturated ring system containing from 3 to 10 carbon atoms, and more preferably having 3, 4, 5, 6 or 7 carbon atoms in the annular structure. Examples of cycloalkyl residues containing 3, 4, 5, 6 or 7 carbon atoms in the ring are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. In addition, unless otherwise indicated, the term "cycloalkyl" includes unsubstituted cycloalkyl and cycloalkyl which is substituted with one or more identical or different groups selected from halogen, hydroxyl, alkoxy, alkyl, cycloalkyl, cyano, amino, aminoalkyl, acyloxy, heterocycle, aralkyl and / or an aryl group.

[0016] As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having up to 10 carbon atoms in the ring, in which at least one carbocyclic ring having a conjugated electron system π. Suitable examples of C6-C10 aryl residues include phenyl, naphthyl or biphenyl, especially phenyl and naphthyl. The aryl residues, for example phenyl or naphthyl, in general may optionally be substituted with one or more substituents, up to five identical or different substituents selected from the groups consisting of halogen, alkyl, hydroxyl, acyloxy, amino, substituted amino and cyano.

[0017] The term "heterocyclyl" refers to 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 they are identical or different heteroatoms selected from: nitrogen, oxygen and sulfur. Suitable examples of said heterocyclyl groups are pyridinyl, piperazinyl, piperidinyl, imidazolyl, pyrrolidinyl and morpholinyl. In the heterocyclic polycyclic ring system, the heterocyclyl may comprise fused rings, in which two or more carbons are common to two adjacent rings, or bridged rings, in which the rings are joined through non-adjacent atoms. In the polycyclic heterocyclic ring system, the heterocyclyl preferably comprises two fused (bicyclic) rings, at least one of which is a 5- to 6-membered heterocyclic ring. Exemplary heterocyclic bicyclic groups include benzoxazolyl, quinolyl, isoquinolyl, carbazolyl, indolyl, isoindolyl, phenoxazinyl, benzothiazolyl, benzimidazolyl, benzoxadiazolyl and benzofurazanyl. Heterocyclyl residues, in general, may optionally be substituted with one or more identical or different substituents selected from the groups consisting of halogen, alkyl, hydroxy, alkoxy, acyloxy, amino, substituted amino and cyano.

[0018] According to a preferred embodiment of the present invention, the group R in formula I may represent H, CH3CH2CO, CH3 (CH2) 15CH2CO or

[0019] According to a more preferred embodiment, the novel compound PM181104 represented by formula I above (in which R = H) is isolated from the fermented broth of the microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 526 9) and is further purified.

[0020] The novel compound PM181104 has the molecular formula C69H66N18O13S5 (molecular weight 1514) and can be characterized by any one or more of its physicochemical and spectral properties, such as spectroscopic data of high performance liquid chromatography (HPLC), spectrum mass (MS), ultra violet (UV), infrared (IR) and nuclear magnetic resonance (NMR) as will be discussed hereinafter.

[0021] The structure of the novel compound PM181104 has been elucidated and its complete characterization is done by spectroscopic data of HPLC, MS, UV, IR and NMR. The structure was confirmed by a three-dimensional (3D) NMR study of the bioactive PM181104 labeled with 15N and 13C.

[0022] The compound PM181104 and its ester and ether derivatives are novel antibiotics active against bacteria, particularly multi-drug resistant bacteria, such as MRSA and VRE.

[0023] The microorganism, which can be used for the production of compound PM181104, is a strain of the Kocuria species (ZMA B-1 / MTCC 5269), hereinafter referred to as the culture No. ZMA B-1, isolated from a marine sample collected in Palk Bay, on the coast of Tamil Nadu, India.

[0024] The present invention further provides a process for the production of compound PM181104 from the ZMA B-1 culture, its mutants and variants, comprising the steps of: growing the

No. ZMA B-1 culture under aerobic conditions submerged in a nutrient medium containing one or more carbon sources and one or more nitrogen sources and optionally inorganic nutrient salts and / or trace elements; isolate compound PM181104 from the culture broth; and purifying compound PM181104 using purification procedures generally used in the related art.

[0025] As used herein, the term "mutant" refers to an organism or cell that carries a mutation, which is an alternative phenotype to the natural type.

[0026] As used herein, the term "variant" refers to an individual organism that is recognized as being different from an arbitrary standard type in this species.

[0027] The preliminary identification of culture No. ZMA B-1, which is the producer of PM181104, was carried out by examining the morphology of its colony, observing the amount of moisture and the Gram staining reaction. Microscopic studies of the strain of the isolated culture No. ZMA B-1 were performed in Zobell Marine Broth 2216 broth (2216 marine broth), containing 1.5% agar and observations were made at 1, 2 and 3 days after incubation at 25 ° C.

[0028] The growth in the Zobell Marine Broth 2216 broth (2216 marine broth), which contains 1.5% agar, generates 2 mm diameter colonies with a smooth, yellow-orange pigmentation surface, a regular margin and a smooth consistency No diffusible pigments are observed in this medium. Under a phase contrast light microscope, coconuts are seen in a 400x magnification. Coconuts separate well and are isolated. They are Gram positive and without movement. The morphology observed classifies this organism as a member of the Micrococcaceae family. Isolates were identified by comparing their 16S rRNA polymerase chain reaction (PCR) with the existing sequences available on the National Center for Biotechnology Information (NCBI) website (URL: http: //www.ncbi.nlm. nih.gov/). Cultivation No. ZMA B-1 was deposited in the Microbial Type Crops Collection (MTCC), in the Institute of Microbial Technology, Sector 39-A, Chandigarh -160 036, India an International Depository Agency (IDA) recognized by the Organization World Intellectual Property (WIPO) and was granted access number MTCC 5269.

[0029] In addition to the specific microorganism described herein, it should be appreciated that mutants, such as those produced by the use of chemical or physical mutagens including X-rays, UV rays, and organisms whose genetic structure has been modified with techniques of Molecular biology can also be grown to produce the compound PM181104.

[0030] The search for suitable mutants and variants that can produce the compound according to the invention can be confirmed by HPLC and / or the determination of biological activity of the active compounds accumulated in the culture broth, for example by testing the compounds in search of antibacterial activity.

[0031] The nutrient medium and / or medium used for the isolation and purification of culture No. ZMA B-1, which produces compound PM181104, preferably contains sources of carbon, nitrogen and inorganic nutritive salts. Carbon sources are, for example, one or more of starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose or galactose. A preferred carbon source is glucose. Nitrogen sources are, for example, one or more of soybean meal, peanut flour, yeast extract, beef extract, peptone, malt extract, fermented corn liquor, gelatin or casaminoic acids. Preferred sources of nitrogen are peptone and yeast extract. Inorganic nutrient 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, sodium phosphate, calcium carbonate , sodium bicarbonate, sodium silicate, ammonium nitrate, potassium nitrate, sodium sulfate, ammonium sulfate, magnesium sulfate, ferric citrate or boric acid. Calcium carbonate, sodium chloride and magnesium chloride are preferred.

[0032] Culture No. ZMA B-1 can be maintained at a temperature ranging from 21 ° C to 35 ° C and a pH of approximately 6.5 to 8.5. Typically, culture No. ZMA B-1 is maintained at 27 ° C-29 ° C and a pH of approximately 7.4-7.8. Crops that have already grown can be stored in the refrigerator at 4 ºC-8 ºC.

[0033] Cultivation of culture seeds of culture No. ZMA B-1 can be carried out at a temperature ranging from 25 ° C to 35 ° C and a pH of approximately 6.5 to 8.5, for 20-55 hours at 200 -280 rpm Typically, the seed of culture No. ZMA B-1 is grown at 29 ° C-31 ° C and a pH of approximately 7.4-7.8, for 24-48 hours at 230 250 rpm.

[0034] The production of compound PM181104 can be carried out by culturing culture No. ZMA B-1 by fermentation at a temperature ranging from 26 ° C to 36 ° C and a pH of approximately 6.5 to 8.5, for 24-96 hours at 60-140 rpm and 100 to 200 lpm aeration. Typically, culture No. ZMA B-1 is incubated at 30 ° C-32 ° C and at

pH 7.4-7.8 for 40-72 hours at 90-110 rpm and 140-160 lpm aeration.

[0035] The production of compound PM181104 can be carried out by culturing culture No. ZMA B-1 in a suitable nutrient broth under the conditions described herein, preferably under submerged aerobic conditions, for example in shake flasks, as well as in laboratory fermenters. The progress of fermentation and production of compound PM181104 can be detected by high performance liquid chromatography (HPLC) and by measuring the bioactivity of the culture broth against staphylococcus and / or enterococcus species with the microbial agar plate diffusion test procedure known. The preferred culture is Staphylococcus aureus 3066, which is a strain resistant to methicillin, a β-lactam antibiotic indicated in the literature, and Enterococcus faecium R2 (VRE), which is resistant to vancomycin. In the resulting culture broth, compound PM181104 is present in the culture filtrate, as well as in the cell mass and can be isolated using known separation techniques, such as solvent extraction and column chromatography. Therefore, compound PM181104 can be recovered from the culture filtrate by extraction at a pH of about 5 to 9 with a water immiscible solvent, such as petroleum ether, dichloromethane, chloroform, ethyl acetate, diethyl ether or butanol, or by hydrophobic interaction chromatography using polymeric resins, such as "Diaion HP-20®" (Mitsubishi Chemical Industries Limited, Japan), "Amberlite XAD®" (Rohm and Haas Industries, United States), activated carbon, or by exchange chromatography ionic at pH 5-9. The active material can be recovered from the cell mass by extraction with a water-miscible solvent, such as methanol, acetone, acetonitrile, npropanol or iso-propanol, or with a water immiscible solvent, such as petroleum ether, dichloromethane, chloroform , ethyl acetate or butanol. Another option is to extract the whole broth with a solvent selected from petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol, acetone, acetonitrile, n-propanol, iso-propanol

or butanol. Typically, the active material is extracted with ethyl acetate from the whole broth. The concentration and lyophilization of the extracts produces the raw active material.

[0036] The compound PM181104 of the present invention can be recovered from the crude material by fractionation using any of the following techniques: 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 the additions of amines, such as NEt3); reverse phase chromatography (using reverse phase silica gel such as dimethyloctadecylsilyl silica gel, (RP-18) or dimethyloctylsilyl 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 (for example, 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 by countercurrent chromatography (using a biphasic eluent system made of two or more solvents such as water, methanol, ethanol, iso-propanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride, chloroform, ethyl acetate, ether of oil, benzene and toluene). These techniques can be used repeatedly, alone or in combination. A typical procedure is reverse phase silica gel chromatography (RP-18).

[0037] The compound PM181104 and isomers thereof, can be converted into their pharmaceutically acceptable salts, which are contemplated in their entirety by the present invention. The salts can be prepared by standard procedures known to one skilled in the art, for example, salts such as sodium and potassium salts, can be prepared by treating the compound PM181104 and isomers thereof, with a suitable sodium or potassium base. , for example sodium hydroxide, potassium hydroxide.

[0038] The esters and ethers of compound PM181104 represented by formula I can be prepared by the methods described in the literature (Advanced Organic Chemistry, 1992, 4th Edition, J. March, John Wiley & Sons). The esters can also be prepared by the procedure described in the literature (J. Med. Chemistry, 1992, 35, 145-151). In a preferred embodiment of the invention, the compounds of the formula I, wherein R is alkyl, cycloalkyl, aryl or heterocyclyl, are prepared by reacting the PM181104 with an appropriate acid having the formula RCOOH; wherein R is alkyl, cycloalkyl, aryl or heterocyclyl, in the presence of a coupling agent, such as dicyclohexyl carbodiimide (DCC) and catalytic amounts of a base such as dimethylaminopyridine (DMAP).

[0039] Phosphate esters can be prepared by a procedure indicated in the literature (Bioorganic & Medicinal Chemistry Letters, 1994, vol. 4, No. 21, 2567-2572). The ethers can be prepared by the process as described in US Patent No. 7,022,667.

[0040] The compound PM181104 has antibacterial activity against a wide variety of bacterial strains. The compound PM181104, stereoisomers, pharmaceutically acceptable salts and derivatives such as esters and ethers thereof, alone or together, can be administered to animals, such as mammals, including

human beings, as pharmaceutical products and in the form of pharmaceutical compositions. Accordingly, the present invention also relates to compound PM181104, its stereoisomers, its pharmaceutically acceptable salts and its ester derivatives and ethers for use as pharmaceuticals and for the use of compound PM181104, stereoisomers, pharmaceutically acceptable salts and derivatives thereof. of ether for the production of drugs that have antibacterial activity.

[0041] The present invention further relates to pharmaceutical compositions containing an effective amount of compound PM181104 and / or stereoisomers and / or one or more pharmaceutically acceptable salts and / or derivatives, particularly esters and ethers thereof, together with a carrier. pharmaceutically acceptable. The effective amount of the compound PM181104, or its stereoisomers, or its pharmaceutically acceptable salts or its derivatives, as an active ingredient in pharmaceutical preparations is usually from 0.01 mg to 100 mg.

[0042] The present invention also relates to a process for the preparation of a medicament containing the compound PM181104 and / or stereoisomers and / or one or more pharmaceutically acceptable salts and / or ester and ether derivatives thereof, for the treatment and prevention of diseases caused by bacterial infections.

[0043] The compounds of the present invention are especially useful as antibacterial agents. The present invention also relates to the use of the compound PM181104 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 which the compounds of the present invention are used can be caused by bacteria belonging to the species staphylococci, streptococci, enterococci and bacilli.

[0044] The expression "Staphylococcus species" refers to a Gram-positive bacterium, which appears as clusters in the form of grapes when viewed through a microscope and as large, round colonies, golden-yellow colonies, often with β -hemolysis, when they grow on blood agar plates. Staphylococcus aureus belonging to the staphylococcus species causes a variety of infections that suppurate (form pus) such as superficial skin lesions such as boils, styes and furunculosis; more serious infections such as pneumonia, mastitis, phlebitis, meningitis and urinary tract infections; and entrenched infections, such as osteomyelitis and endocarditis. Staphylococcus aureus is the leading cause of hospital acquired infections (nosocomium) of surgical wounds and infections associated with the manipulation of medical devices. Staphylococcus aureus causes food poisoning by releasing enterotoxins in the food, and toxic shock syndrome from the release of superantigens into the bloodstream.

[0045] The expression "Streptococcus species" refers to a genus of spherical, Gram positive bacteria and a member of the Firmicutes division. Streptococci are lactic acid bacteria. The Staphylococcus species is responsible for infectious diseases such as meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis (bacterial infections that "eat meat").

[0046] The expression "Enterococcus species" refers to a genus of lactic acid bacteria of the Firmicutes division. They are Gram positive cocci that are often found in pairs (diplococci). Enterococci are anaerobic facultative organisms. Enterococci are among the most frequent causes of infections acquired in hospitals. Enterococci develop resistance to antibiotics such as gentamicin and vancomycin.

[0047] The expression "Bacillum species" refers to a large number of various rod-shaped bacteria, Gram positive, that have movement through perimeter flails and are aerobic. He is also a member of the Firmicutes division. Members of this genus are capable of producing endospores that are highly resistant to unfavorable environmental conditions. Bacillus cereus that belongs to the species Bacillum causes two types of food poisoning. One type is characterized by symptoms of nausea, vomiting and abdominal contractions. The second type is manifested primarily by abdominal contractions and diarrhea. Infections attributed to Bacillus subtilis belonging to the Bacillum species include bacteremia, endocarditis, pneumonia, and septicemia in patients with compromised immune system.

[0048] The compounds of the present invention can be administered orally, nasally, topically, subcutaneously, intramuscularly, intravenously or by other modes of administration.

[0049] Pharmaceutical compositions containing PM181104 or a stereoisomer or a pharmaceutically acceptable salt or an ester or ether derivative thereof, with other pharmaceutically active substances can be prepared by mixing the active compounds with one or more pharmacologically auxiliary

acceptable tolerances and / or excipients, such as wetting agents, solubilizers such as surfactants, vehicles, tonicity agents, fillers, colorants, taste masks, lubricants, disintegrants, diluents, binders, plasticizers, emulsifiers, ointment bases, emollients, agents thickeners, polymers, lipids, oils, co-solvents, complexing agents or buffering substances, and converting the mixture into a suitable pharmaceutical form, such as, for example, tablets, coated tablets, capsules, granules, powders, creams, ointments, gels, syrups , emulsions, suspensions or solutions suitable for parenteral administration.

[0050] Examples of auxiliaries 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 carboxymethylcellulose, talc agar, mineral oil, animal oil, vegetable oil, organic and mineral waxes, paraffin, gels, propylene glycol, benzyl alcohol, dimethylacetamide, ethanol, polyglycols, tween 80, solutol HS 15 and water.

[0051] It is also possible to administer the active substances as such, without carriers or diluents, in a suitable form, for example, in capsules.

[0052] As usual, the galenic formulation and the administration procedure, as well as the dosage range are suitable in a specific case, depend on the species to be treated and the condition of the respective condition or disease, and can be optimize using procedures known in the art. On average, the daily dose of the active compound in a patient is 0.0005 mg to 15 mg per kg, usually 0.001 mg to 7.5 mg per kg.

[0053] The following examples are provided as illustrative examples of the present invention:

Example 1

Isolation of culture No. ZMA B-1 from the marine source

a) Composition of the isolation medium:

Zobell 2216 marine broth (agarified with 1.5% agar-agar)

[0054] 5.0 g of animal tissue peptic digest, 1.0 g of yeast extract, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 8.8 g of magnesium chloride, 3, 24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium bicarbonate, 80.0 mg of potassium bromide, 34.0 mg of strontium chloride, 22.0 boric acid mg, 4.0 mg sodium silicate, 2.4 mg sodium fluorate, 1.6 mg ammonium nitrate, 8.0 mg disodium phosphate, 15.0 mg agar powder, 1.0 1 of double distilled water, final pH (at 25 ° C) 7.4 to 7.8.

b) Procedure

[0055] The sponge sample, Spirastrella inconstans var. Digitata (Dendy) was collected from Palk Bay on the coast of Tamil Nadu, India, by diving at a depth of three meters. The sponge sample was rinsed in sterile seawater and immediately transferred to sterile polyethylene containers. The containers were stored at 20 ° C and transported keeping the temperature below 0 ° C, to the laboratory for further studies. Upon arrival at the laboratory, the sponge samples were stored at less than 0 ° C and later defrosted at room temperature (25 ± 2 ° C) just before culture isolation. The sponge sample was cut aseptically into 2 x 2 cm pieces and suspended in 5 ml of sterile seawater in a 25 ml sterile test tube. The test tube was vortexed for 30 seconds; seawater was drained and clean seawater was added. The same process was repeated three times. Finally, the seawater was drained and the sponge piece was placed on petri dishes containing the insulating medium mentioned above [Zobell Marine Broth 2216 broth (agarified with 1.5% agar-agar); HiMedia]. The petri dish was incubated at room temperature (25 ± 2 ° C) until growth was observed in the plates. The colonies that grew on the plates were isolated based on the characteristics of the colony and seeded by striatum in petri dishes containing the isolation medium mentioned above [Zobell Marine Broth 2216 broth (agar agar agar agar 1.5%); HiMedia], The isolates were repeatedly subcultured until pure culture No. ZMA B-1 was obtained. Therefore, culture No. ZMA B-1 was isolated from among microorganisms grown in the form of a single isolate.

Example 2

Culture purification No. ZMA B-1 a) Composition of the isolation medium:

Zobel 2216 Marine Broth (agarified with 1.5% agar agar)

5 [0056] 5.0 g of peptone, 1.0 g of yeast extract, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 8.8 g of magnesium chloride, 3.24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium bicarbonate, 0.08 g of potassium bromide, 34.0 mg of strontium chloride, 22.0 mg of acid boric, 4.0 mg of sodium silicate, 2.4 mg of sodium fluorate, 1.6 mg of ammonium nitrate, 8.0 mg of disodium phosphate, 15.0 g of agar,

10 1.0 l of demineralized water, pH 7.4-7.8.

b) Procedure:

[0057] The culture was available in Zobell Marine Broth 2216 broth (agarified with 1.5% agar agar) in one

15 petri dish 15 mm in diameter. The petri dish culture was strewn on an inclined culture of Zobel Marine Broth 2216 broth (agarified with 1.5% agar agar). The inclined culture was incubated for 2 days at 25 ° C. One of the individual colonies of the upper part of the inclined bed was transferred to new inclined plates. The inclined plates were incubated for 2 days at 25 ° C. Then, they were used for fermentation in shake flasks for the purpose of the first anti-infective screening.

20 Example 3

Maintenance of the producing strain - culture No. ZMA B-1

25 [0058]

a) Composition of the medium (Zobel Marine Broth broth 2216): 5.0 g of peptone, 1.0 g of yeast extract, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 8.8 g of magnesium chloride, 3.24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of

30 sodium bicarbonate, 0.08 g of potassium bromide, 34.0 mg of strontium chloride, 22.0 mg of boric acid, 4.0 mg of sodium silicate, 2.4 mg of sodium fluorate, 1.6 mg of Ammonium nitrate, 8.0 mg disodium phosphate, 15.0 mg agar, 1.0 L demineralized water, pH 7.4-7.8. b) After dissolving the ingredients completely by heating, the resulting solution was distributed in test tubes and sterilized at 121 ° C for 30 min. The test tubes were cooled and

35 were allowed to solidify in an inclined position. The agar slopes were seeded by streaking with the ZMA B-1 culture by means of a wire handle and incubated at 27-29 ° C until good growth was observed. Crops that grew well were stored in the refrigerator at 4-8 ° C.

Example 4 40 Fermentation of culture No. ZMA B-1 in shake flasks

[0059]

45 a) Composition of the seed medium (Zobel Marine Broth Broth 2216): 5.0 g of peptone, 1.0 g of yeast extract, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 8.8 g of magnesium chloride, 3.24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium bicarbonate, 0.08 g of potassium bromide, 34.0 mg of strontium chloride, 22.0 mg of boric acid, 4.0 mg of sodium silicate, 2.4 mg of sodium fluorate, 1.6 mg of ammonium nitrate, 8.0 mg of phosphate

50 disodium, 1.0 l of demineralized water, pH 7.4-7.8. b) The above medium was distributed in 40 ml portions in 500 ml Erlenmeyer flasks and autoclaved at 121 ° C for 30 min. The flasks were cooled to room temperature and each flask was inoculated with a colony of the good-growing strain (culture No. ZMA B-1) in the inclined medium and stirred on a rotary shaker for 24-48 hours at 230-250 rpm at 30 ° C (± 1 ° C) for

55 provide the seed crop. c) Composition of the production medium: 5.0 g of peptone, 1.0 g of yeast extract, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 8.8 g of magnesium chloride, 3 , 24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium bicarbonate, 0.08 g of potassium bromide, 34.0 mg of strontium chloride, 22, 0 mg boric acid,

60 4.0 mg of sodium silicate, 2.4 mg of sodium fluorate, 1.6 mg of ammonium nitrate, 8.0 mg of disodium phosphate, 1.0 l of demineralized water, pH 7.4-7.8 . d) 40 ml of the production medium in 500 ml Erlenmeyer flasks were autoclaved

capacity at 121 ° C for 30 min, cooled to 29 ° C-30 ° C and seeded with 2 ml of the seed culture which was mentioned in example 4b. e) Fermentation parameters Temperature 29 ° C-30 ° C; stirring 230-250 rpm; harvest time 48-72 hours.

[0060] The production of compound PM181104 in the fermentation broth was determined by testing the bioactivity against Enterococcus faecium R2 (VRE) and / or S. aureus strain 3066 MRSA using the agar diffusion procedure. The harvest pH of the culture broth was 7.0-8-0. The culture broth was harvested and the total broth was used for bioactivity tests, which is indicative of the presence of compound PM181104 in the broth.

10 fermented.

Example 5

Preparation of the seed crop in the shake flasks for fermentation 15

[0061]

a) Composition of the medium: 5.0 g of peptone, 1.0 g of yeast extract, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 8.8 g of

20 magnesium chloride, 3.24 g of sodium sulfate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium bicarbonate, 0.08 g of potassium bromide, 34.0 mg of Strontium chloride, 22.0 mg boric acid, 4.0 mg sodium silicate, 2.4 mg sodium fluorate, 1.6 mg ammonium nitrate, 8.0 mg disodium phosphate, 1.0 L demineralized water, pH 7.4-7.8. b) The above medium was distributed in amounts of 200 ml in 1L Erlenmeyer flasks and sterilized in

25 autoclave at 121 ° C for 30 min. The flasks were cooled to room temperature and each flask was inoculated with a colony of the producing strain that had already grown (culture No. ZMA B-1) in the inclined media and stirred on a rotary shaker for 24-48 hours at 230- 250 rpm at 29 ºC-31 ºC to give a seed crop.

30 Example 6

Cultivation of culture No. ZMA B-1 in the fermenter

[0062]

35 a) Composition of the production medium: 50.0 g of glucose, 11.0 g of yeast extract, 4.0 g of peptone, 4.0 g of beef extract, 5 g of calcium carbonate, 2 , 5 g of sodium chloride, 1 l of demineralized water, pH 7.6 (before sterilization).

40 b) 250 l of the production medium was sterilized in situ in a 300 l fermenter together with 80 ml of desmophene as antifoam agent, for 30 min at 121 ° C, cooled to 29 ° C-31 ° C and seeded with 6 l of the seed crop mentioned in example 5b. c) Fermentation parameters: Temperature 30 ºC-32 ºC, stirring 100 rpm; aeration 150 bpm, harvest time 44-66 hours.

[0063] The production of compound PM181104 in the fermentation broth was determined by testing the bioactivity against S. aureus 3066 (MRSA strain) and / or Enterococcus faecium R2 (VRE) using the agar diffusion procedure. The harvest pH of the culture broth was 7.0-8.0. The culture broth was harvested and the whole broth was used to isolate and purify the compound PM181104.

50 Example 7

Isolation and purification of compound PM181104

[0064] The whole broth (240 L) of Example 6 was harvested and extracted using ethyl acetate (240 L) by stirring in a glass vessel. The organic phase was separated using a disk stack separator (Alfa-laval, model No. LAPX404) and concentrated to obtain the crude extract (296 g). The crude material obtained was stirred and sonic for 30 min using petroleum ether (3 x 1 L) and filtered to obtain the insoluble residue (38 g), which was subjected to chromatography by vacuum liquid chromatography using the following procedure. .

[0065] 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 applied to a sintered filter funnel (G-4 quality; 10 cm

x 10.5 cm) filled with LiChroprep RP-18 adsorbent (25-40 μ, 110 g). Elution was performed using a vacuum network (100-120 mm) initially with water (4 l) followed by water: methanol (1: 1, 5 l), methanol (3 l), methanol: acetonitrile (2.5 l ) and acetonitrile. The purification control was performed by bioassay against Ent. faecium R2 and / or S. aureus 3066 and / or analytical HPLC. Compound PM181104 was detected in the methanol, methanol: acetonitrile and acetonitrile fractions. Such fractions were combined and concentrated to obtain the semi-pure material (1,826 g).

[0066]

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

Eluent column Flow Rate Detection (UV) Retention time

: Eurospher RP-18 (10 μ, 32 x 250 mm): acetonitrile: water (56:44): 50 ml / min: 220 nm: 12-14 min

[0067]

The purity of the fractions was checked by bioassay against Ent. faecium R2 and / or S. aureus 3066

and / or analytical HPLC. Eluates containing compound PM181104 were combined and concentrated under reduced pressure to remove the solvent to obtain 600 mg of the pure compound.

[0068] Physicochemical and spectral properties of compound PM181104

Appearance: white amorphous solid

Melting point:> 300 ° C (decomposes)

Solubility: Methanol, DMSO

HPLC: Tr 5.61 min

Column: Kromasil C18 (5 μ; 150 x 4.6 mm ID) (column temperature 40 ºC)

Mobile phase: water: acetonitrile (1: 1)

Volume of: 10 μl (concentration 0.1 mg / ml in the mobile phase)

injection

Flow rate: 1 ml / min

Detection: 220 nm

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

Molecular Formula: C69H66N18O13S5

Molecular Weight: 1514

UV (MeOH): 205.2, 220.6 and 306.2 nm (see Figure 1)

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

1H NMR: see Table 1 and Figure 3

13C NMR: see Table 2 and Figure 4

Table 1: 1H NMR of compound PM181104 in DMSO-d6

Peak

δ  Peak δ  Peak δ

one

1.33-1.34 (d, 3H) twenty-one 5.33 (m, 1 H) 41 8.57-8.58 (d, 1H)

2

1.94 (m, 4H) 22 5.52 (s, 1 H) 42 8.65 (s, 1 H)

3

2.05-2.06 (m a, 1H) 2. 3 5.63 (s, 1 H) 43 8.81 (t, 2H)

4

2.12 (m a, 1H) 24 5.83 (s, 1 H) 44 9.07 (s, 1 H)

5

2.25-2.28 (d a, 1H) 25 5.92 (s, 1 H) Four. Five 9.16 (s, 1 H)

6

2.40-2.42 (m a, 1H) 26 6.07 (s, 1 H) 46 9.48 (s, 1 H)

7

2.68 (s, 3 H) 27 6.50 (s, 1 H) 47 10.03 (s, 1 H)

8

2.76-2.82 (m, 2H) 28 6.59-6.61 (d, 2H)

9

2.97-2.99 (d, 1H) 29 6.86 (s, 1 H)

10

3.17-3.21 (m, 1H) 30 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, 1 H) 33 7.30 (s, 1 H)

14

3.77 (s, 2H) 3. 4 7.33-7.35 (d, 1H)

fifteen

4.49-4.50 (d, 1H) 35 7.53 (s, 1 H)

16

4.69-4.71 (t, 1H) 36 7.69 (s, 1 H)

17

4.80 (m, 1 H) 37 7.90 (s, 1 H)

18

4.89 (m, 1 H) 38 7.95 (s, 1 H)

19

4.93-4.97 (t, 1H) 39 8.27-8.29 (d, 2H)

twenty

5.23 (t, 1 H) 40 8.49-8.50 (d, 1H)

Table 2: 13C NMR of compound PM181104 in DMSO-d6

signal

δ  signal δ  signal δ

one

12.04 22 115.46 * 43 151.04

2

16.88 2. 3  116.92 44  152.08

3

25.16 * 24  118.77 Four. Five  153.43

4

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 29  127.36 fifty  161.27

9

38.73 30  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 3. 4 130.99 * 55 167.95

14

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

18

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

twenty-one

 107.52  42 150.83 63 174.90

* two carbons

BIOLOGICAL EVALUATION OF COMPOUND PM181104 5 In vitro test

Example 8

[0069] In vitro potency was established with determinations of the minimum inhibitory concentration (MIC) of compound PM181104 against bacterial strains, using the Macro-broth dilution method according to the guidelines of the National Committee for Clinical Laboratory Standard (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, United States)] . Unless indicated

15 otherwise, Mueller-Hilton broth was used as a nutritional medium for the trial. Linezolid (prepared by Glenmark Pharma Ltd; Lot No. K2005028) was used as a known standard in all in vitro experiments. For the preparation of the stock solution, compound PM181104 was dissolved in chloroform (5% of the total volume required) and diluted using methanol (95% of the total volume required).

20 Result:

[0070] The results obtained are shown in Table 3 below, and demonstrate that compound PM181104 is useful in the treatment of bacterial infections.

Table 3: MIC of compound PM181104 against bacterial strains

Test body

CIM ( g / ml)  Test body CIM ( g / 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-MRSA1

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 33591

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 C1 3184

0.0625 Streptococcus hirae 55 0.00391

S. aureus (E710)

0.03125 Streptococcus equinus 02 D5 Gr1 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 for A > 1

S. epidermidis 823

0.03125 Pse.aeruginosa (M-35) > 1

S. epidermidis 6098

0.0625 Klebsiella pneumoniae > 1

S. epidermidis 6493II (2) W

0.0625 Citrobacterdiversus 2046E > 1

S. epidermidis 10221II W

0.00781 E. coli SS > 1

The abbreviations used in Table 3 are: S: Staphylococcus E: Enterococci B: Bacillus

In vivo test

[0071] Potency was established in vivo by means of protective dose determinations (PD) of compound 5 PM181104 for its antibiotic activity in three animal models using both Balb / C mice, both male and female.

[0072] The models used were the general purpose efficacy test models and other organ / tissue specific efficacy test models. The general purpose efficacy test models that are

10 used were the systemic infection model (septicemia), the localized infection model (neutropenic thigh model). The specific Organ / Tissue infection models that were used for the efficacy test were kidney model, lung infection, dermal abscess.

Example 9

15 Systemic infection model

[0073] The animals were infected intraperitoneally with ~ 10a to 109 cfu of an overnight culture of methicillin-resistant Staphylococcus aureus E710 (MRSA), suspended in normal saline (0.85% of

20 sodium chloride). The PM181104 solution was prepared in a cremophor-ethanol formulation, as described in example 14. The solution was administered intravenously in doses of 5 mg, 2.5 mg and 1.25 mg / kg, immediately after infection. Each experimental group consisted of ten animals. The PD100 of PM181104 for the septicemia model was determined to be 5 mg / kg compared to the standard Linezolid antibiotic (manufactured by Glenmark Pharma Ltd; Lot No. K2005028) whose PD100 was 25 mg / kg.

25 Example 10

Neutropenic thigh model

[0074] Mice were made neutropenic with cyclophosmamide (150 mg and 100 mg / kg) at 96 and 24 hours, respectively, before infection with S. aureus E-710. Animals were infected in the muscles intramuscularly with ~ 107 cfu of a culture grown overnight of S. aureus E-710, suspended in normal brine (0.85% sodium chloride). Each experimental group consisted of six animals. PM181104 was prepared in

a cremofor-ethanol formulation as described in example 14. The solution was administered intravenously in doses of 5 mg / kg, after two hours of infection. Animals were sacrificed at various points in time and muscle tissue was harvested to determine the count of viable microorganisms. A decrease of approximately 1 log was observed with PM181104 at a dose of 5 mg / kg, at the time point of 6 hours, which was comparable with the standard antibiotic used viz. Linezolid (manufactured by Glenmark Pharma Ltd; Lot No. K2005028) with a dose of 25 mg / kg.

Example 11

Kidney infection model

[0075] 0.2 ml of 2% λ carrageenan was administered intravenously to Balb / C mice seven days after infection. A growing culture was injected intravenously overnight in the log phase of Enterococcus faecium ATCC 47077 adjusted to approximately 109 cfu / ml, to the mice at a volume of 0.2 ml. The solution of PM181104, prepared in a cremophor-ethanol formulation as described in example 14, was administered to the mice intravenously at 4 hours, 24 hours and 48 hours after infection. At 72 hours after infection, the animals were sacrificed and the kidneys were collected to determine the bacterial load. A decrease in bacterial count of approximately 1 Log was observed with a dose of 5 mg / kg of PM181104, which was comparable with the standard antibiotic used viz. Linezolid (manufactured by Glenmark Pharma Ltd; Lot No. K2005028) in 25 mg / kg and vancomycin hydrochloride (manufactured by HiMedia; Catalog No. RM217-500 mg; Lot No. 06-0350) in doses of 150 mg / kg.

Example 12

Lung Infection Model

[0076] Balb / C mice were neutropenic by intraperitoneal administration of 200 mg / kg of cyclophosmamide four and two days before infection. On the day of infection, the mice were anesthetized and infected with a log phase suspension culture of S. aureus E-710 with bacterial density of approximately 106 to 107 cfu / ml. At the 24 and 36 hours after infection, the respective first and second doses of the drug were administered intravenously. Euthanasia was performed in mice 48 hours after infection and their lungs were collected aseptically to determine the count of viable bacteria. In this model, PM181104 was tested at a dose of 5 mg / kg prepared in a cremophor-ethanol formulation as described in example 14. Two standard viz antibiotics were used as positive controls. Linezolid (a single dose of 80 mg / kg at 24 hours after infection) and Vancomycin (two doses of 110 mg / kg at 24 and 48 hours after infection). PM181104 was shown to have bacteriostatic activity, which was comparable to that of the Linezolid standard (manufactured by Glenmark Pharma Ltd; Lot No. K2005028). Standard vancomycin hydrochloride (manufactured by HiMedia; catalog No. RM217-500 mg; Lot No. 060350) showed a bacterial profile. There was approximately 2 Log difference in bacterial count in the lungs of animals treated with PM181104 compared to those of untreated control animals.

Example 13

Skin abscess model

[0077] Balb / C mice were infected subcutaneously with approximately 108 cfu of a growing overnight culture of methicillin-resistant S. aureus E710 (MRSA). The bacteria were suspended in a 1: 1 mixture of 2% cytodex beads in normal saline (0.85% sodium chloride). PM181104 was prepared in a cremofor-ethanol formulation as described in example 14. The solution was administered intravenously in doses of 2.5 mg, 5 mg and 10 mg / kg, two hours after infection. Each experimental group contained six animals. After abscess formation, animals were sacrificed and abscesses were harvested to determine viable counts. A decrease in bacterial count of approximately 1 Log was observed with the 5 mg / kg dose of PM181104, which was comparable with the standard antibiotic used viz. Linezolid (manufactured by Glenmark Pharma Ltd; Lot No. K2005028) in doses of 50 mg / kg.

FORMULATION OF COMPOUND PM181104

Example 14

[0078] Injectable formulations were prepared by the following general procedure:

Ethanol and cremofor EL were mixed in a 1: 1 ratio (by weight). To this was added PM181104 and vortexed. This mixture was sonic at 25 ° C. This mixture (considering it as a 10% constituent) was diluted by adding water (90%) and vortexed to obtain the injectable formulation.

COMPOUND DERIVATIVES PM181104 5 Example 15

PM181104 butyric acid ester derivative

[0079] To a solution of PM181104 (0.13 g, 0.085 mmol) in dichloromethane (2 ml) was added butyric acid (0.008 µl, 0.085 mmol), DCC (0.018 g, 0.085 mmol) and a catalytic amount of DMAP (0.002 g, 0.016 mmol) and the reaction mixture was stirred for 18 h under a nitrogen atmosphere. To the reaction mixture was added cold water and the organic phase was separated; The aqueous phase was extracted with dichloromethane (3 x 50 ml), the organic extracts were combined and washed with water (2 x 30 ml). The organic phase was dried over anhydrous sodium sulfate and concentrated.

The crude product was purified using 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)

[0080] The derivative of the butyric acid ester of PM181104 showed a MIC value of 2.5 μg / ml against E. 20 faecium R-2, bacterial strain (VRE).

Example 16

Stearic acid derivative of PM181104

[0081] To a solution of PM181104 (0.12 g, 0.079 mmol) in dichloromethane (2 ml) were added stearic acid (0.022 µl, 0.079 mmol), DCC (0.016 g, 0.079 mmol) and a catalytic amount of DMA (0.002 g, 0.016 mmol) and the reaction mixture was stirred for 6 h under a nitrogen atmosphere. To the reaction mixture was added cold water and the organic phase was separated; The aqueous phase was extracted with dichloromethane, (3 x 50 ml), the organic extracts were

30 combined and washed with water (2 x 3 0 ml). The organic phase was dried with anhydrous sodium sulfate and concentrated. The crude product was purified using 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).

[0082] The stearic acid ester derivative of PM181104 showed a MIC value of 1.25 μg / ml against the bacterial strain (VRE) E. faecium R-2.

Example 17

40 Nicotinic acid ester derivative of PM181104

[0083] To a solution of PM181104 (0.02 g, 0.013 mmol) in N, N-dimethylformamide (1 ml) was 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 mol) and the reaction mixture was stirred for 18 h under a nitrogen atmosphere. The solvent was removed and at

The residue was added with 10 ml of dichloromethane. Undissolved urea was filtered and the filtrate was washed with water (2 x 10 ml). The organic phase was dried with anhydrous 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).

[0084] The nicotinic acid ester derivative of PM181104 was tested against bacterial strains. The results obtained are shown in table 4 below, and demonstrate that the derivative of the nicotinic acid ester of PM181104 is useful in the treatment of bacterial infections.

55 Table 4. MIC of the nicotinic acid ester derivative of PM181104 against bacterial strains.

Test body

CIM ( g / ml)  Test body CIM ( g / ml)

E. faecalis ATCC 51299

0.312 E. faecium R-2 (VRE) 0.312

E. faecalis ATCC 51575

0.312 S. aureus MRSA ATCC 33591 > 10

E. faecalis ATCC BAA472

0.312 S. aureus MRSA E710 > 10

E. faecium ATCC 51559

0.156

wherein R = H, alkyl, alkylcarbonyl, (HO) 2PO-, alkyl-OPO (OH) -, (alkyl-O) 2PO-, cycloalkyl, cycloalkylcarbonyl,

Claims (12)

1. Compounds of the following formula I; aryl, arylcarbonyl, heterocyclyl and heterocyclyl carbonyl; or stereoisomers or tautomers or pharmaceutically acceptable salts thereof.

2.

 The compound as indicated in claim 1, wherein in the compounds of formula I, R is H; said compound is designated as PM181104; or a stereoisomer or a tautomer or a pharmaceutically acceptable salt thereof.

3.

 The compound designated as PM181104 as indicated in claim 2, or a stereoisomer or tautomer thereof, whose compound is isolated from the fermented broth of the microorganism belonging to the Kocuria species (ZMA B-1 / MTCC 5269) and is characterized by :

(to)

 molecular weight of 1514,

(b)

 molecular formula C69H66N18O13S5,

(C)

 UV spectrum absorbances (MeOH) at 205.2, 220.6 and 306.2 nm,

(d)

 IR spectrum absorbances (KBr) at 3368, 2981, 1654, 1516, 1429, 1314, 1199, 1269, 1074, 1034, 805, 580 cm -1,

(and)

 1H NMR spectrum (500 MHz, DMSO-d6): δ 10.03 (s, 1 H), 9.48 (s, 1 H), 9.16 (s, 1 H), 9.07 (s, 1 H), 8.81 (t, 2H), 8.65 (s, 1 H), 8.57-8.58 (d, 1 H), 8.49-8.50 (d, 1 H), 8.27-8.29 (d, 2H), 7.95 (s, 1 H), 7.90 (s, 1 H), 7.69 (s, 1 H), 7.53 (s, 1 H), 7.33-7.35 (d, 1H), 7.30 (s, 1 H), 7.26 (d, 4H), 7.17-7.18 (m, 1 H), 7 , 04-7.06 (d, 2H), 6.86 (s, 1 H), 6.59-6.61 (d, 2H), 6.50 (s, 1H), 6.07 (s, 1 H), 5.92 (s, 1 H), 5.83 (s, 1 H), 5.63 (s, 1 H), 5.52 (s, 1 H), 5.33 (m, 1 H), 5.23 (t, 1 H), 4.93-4.97 (t, 1 H), 4.89 (m, 1 H), 4.80 (m, 1 H), 4.69 -4.71 (t, 1 H), 4.49-4.50 (d, 1 H), 3.77 (s, 2H), 3.69 (m, 1 H), 3.62 (t, 2H), 3.24-3.26 (m, 1 H), 3.17-3.21 (m, 1 H), 2.97-2.99 (d, 1 H), 2.76-2 , 82 (m, 2H), 2.68 (s, 3H), 2.40-2.42 (ma, 1 H), 2.25-2.28 (da, 1 H), 2.12 (ma , 1 H), 2,052.06 (ma, 1 H), 1.94 (m, 4H) and 1.33-1.34 (d, 3H),

(F)

 13C NMR spectrum (125 MHz, DMSO-d6): δ 174.90, 174.14, 173.51, 171.96, 171.77, 171.03, 170.39, 169.68, 167.95, 165 , 58, 163.35, 162.79, 161.54 (2C), 161.27, 159.06, 156.45, 156.27, 154.20, 153.43, 152.08, 151.04, 150.83, 149.73, 147.97, 140.92, 137.67, 136.87, 135.17, 133.99, 130.99 (2C), 130.11, 129.76 (2C), 129.18, 128.71 (2C), 127.36, 127.08, 124.33, 123.39, 122.84, 118.77, 116.92, 115.46 (2C), 107.52, 104.94, 103.89, 77.80, 60.56, 59.92, 54.43, 52.69, 48.88, 47.88, 47.76, 47.22, 38.73, 38, 62, 37.00, 36.67, 33.09, 29.42, 25.16 (2C), 16.88 and 12.04.

Four.

 A process for the production of the compound designated as PM181104 as indicated in claim 2 or claim 3, comprising the steps of:

(a) culturing the microorganism of the Kocuria species (ZMA B-1 / MTCC 5269) or one of its variants or mutants under aerobic conditions submerged in a nutritive medium containing carbon and nitrogen sources to produce the compound PM181104,

(b)

 isolate compound PM181104 from fermented broth, and 5 (c) purify compound PM 181104.

5. A process as set forth in claim 4, further comprising the step of converting the compound designated as PM181104 into its pharmaceutically acceptable salt.

The method as indicated in claim 4, further comprising the step of reacting the compound designated as PM181104 with an acid having the formula RCOOH; wherein R is alkyl, cycloalkyl, aryl or heterocyclyl, to obtain the compound of formula I as indicated in claim 1, wherein R = alkyl, cycloalkyl, aryl and heterocyclyl.

The compound as indicated in claim 1, wherein R = CH3CH2CH2CO, CH3 (CH2) 15CH2CO or

8. A pharmaceutical composition, which comprises an effective amount of a compound as indicated in the claim 1, with at least one pharmaceutically acceptable excipient or additive or auxiliary. twenty 9. The pharmaceutical composition as indicated in claim 8, wherein the pharmaceutical composition is in the form of a tablet, coated tablet, capsule, granule, powder, cream, ointment, gel, emulsion, suspension or solution for injection. The pharmaceutical composition as indicated in claim 8, wherein said composition is adapted for the treatment of a bacterial infection. 11. The pharmaceutical composition as indicated in claim 10, wherein said bacterial infection It is caused by bacteria belonging to the species Staphylococcus, Streptococcus, Enterococcus or Bacillus. 30 12. The pharmaceutical composition as indicated in claim 11, wherein said bacteria belong to the species Staphylococcus or Enterococcus. 13. The pharmaceutical composition as indicated in claim 12, wherein said bacterium belonging to the Staphylococcus species is resistant to methicillin. 14. The pharmaceutical composition as indicated in claim 12, wherein said bacterium belonging to the Staphylococcus species is resistant to vancomycin. The pharmaceutical composition as indicated in claim 12, wherein said bacterium belonging to the Enterococcus species is resistant to vancomycin. 16. Use of the compounds as indicated in claim 1, for the manufacture of a medicament for The treatment of a bacterial infection. Four. Five