PT1855705E - Antibiotic 107891, its factors, pharmaceutically acceptable salts and compositions, and use thereof - Google Patents

Description

1

DESCRIPTION

" ANTIBIOTIC 107891, ITS FACTORS, ITS ACCEPTABLE SATISFACTS THROUGH PHARMACEUTICAL VISION AND ITS COMPOSITIONS, AND ITS USE " WO2005 / 014628 discloses antibiotic 107891, its components Factors AI and A2, their preparation and use. The present invention relates to an antibiotic substance of microbial origin, arbitrarily referred to as antibiotic 107891, which is a complex. The present invention is further characterized by the compounds of claim 1. The present invention further relates to the pharmaceutically acceptable salts thereof, to the pharmaceutical compositions thereof and to their use as an antibacterial agent. Also described is a process for the preparation of antibiotic 107891 which includes the culture of Microbispora sp. 107891 (hereinafter referred to as Microbispora sp. ATCC PTA-5024) or a variant or mutant thereof which retains the ability to produce said antibiotic, recovering the antibiotic of the present invention from the mycelium and / or from the fermentation broth , separation of the pure substance by chromatographic processes. Antibiotic 107891 is a novel antimicrobial agent with a peptide structure containing lanthionine and methyl-lanthionine as constituents. These are characteristic attributes of lantibiotics and, in particular, of the subgroup that acts primarily on cell wall biosynthesis. 2

Lantibiotics are peptides which contain the amino acid lanthionine with a thioether group as well as some other modified amino acids (HG Sahl and G. Bierbaum, (1998) " Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria " Ann. Rev. Microbiol., 52: 41-79). Most of the known lantibiotics have antibacterial activity, although some have been presented as active in different pharmacological targets. Antibacterial lantibiotics can be broadly divided into two groups based on their structures: type A lipobiotics are usually elongated (" elongated "), amphiphilic peptides, while type B lantibiotics are compact and globular (0. McAuliffe, RP Ross and C. Hill, (2001): " Lantibiotics: structure, biosynthesis and mode of action ", FEMS Microb Rev. 25: 285-308). Nisin is the typical representative of type A lithium antibiotics, while actagardine (gardimicin) and mersacidine belong to the subtype of type B lantibiotics. Both lysine antibiotics of the nisin and mersacidine type interact with lipids II precursors of membrane bound peptidoglycans , although the two classes differ in the effects they produce in the process of bacterial proliferation. Nisin-type lantibiotics essentially kill bacteria by permeabilization of the cytoplasmic membrane (H. Brotz, M. Josten, I. Wiedemann, U. Schneider, F. Gotz, G. Bierbaum and HG Sahl, (1998): "Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics ", Microbiol 30: 317-27), while mersacidine-type lantibiotics kill the bacterial cell first by inhibiting cell wall biosynthesis (H. Brotz, G. Bierbaum, K. Leopold, PE Reynolds and HG Sahl, (1998): " The lantibiotic mersacidin inhibits peptidoglycan synthesis by targeting lipid II ", Antimicrob Agents Chemother, 42: 154-60). 3

In U.S. Patent No. 6,551,591, two antibiotics produced by Microbispora corallina strain NRRLL 30420, identified as antibiotics MF-BA-1768cxl and MF-BA-17 68βΐ, respectively, are described. The physicochemical data described in the previously identified patent (e.g. mass spectral data, molecular weight, amino acid content) and comparison of retention times in experimental analyzes of LC-MS clearly show that antibiotic complexes 107891 are chemical entities different from the antibiotics MF-BA 1768o and MF-BA-1768β. European Patent EP 0592835A2 describes the antitumor antibiotics BU-4803TAi, A2, B, Ci, C2 and D. The antibiotics BU-4803TAi A2 and B are recovered from the fermentation broth of Microbispora ATCC 55327 (AA 9966) while antibiotics BU4803TCi, C2 and D are products of transformation of antibiotics BU 4803ΤΑχ, A2 and B, respectively, when these products are stored in dimethylsulfoxide. The physico-chemical data described in EP 0592 835 A for prior antibiotics (e.g., appearance, U.V., absorption, molecular weight, antitumor activity, clearly show that they are chemical substances other than the antibiotic complex 107891.

STRAIN AND FERMENTATION

From the environment Microbispora sp. 107891 and filed on February 27, 2003 at the American Type Culture Collection (ATCC), 10801 University Blvd, Manassas VA, 20110-2209 U.S.A., under the provisions of the Budapest Treaty. The strain was granted accession number PTA-5024. The production of antibiotic 107891 is carried out by cultivating a strain of Microbispora sp. its producer, namely Microbispora sp. ATCC PTA-5024 or a variant or mutant thereof which maintains the ability to produce said antibiotic; isolating the resulting antibiotic from the whole culture broth and / or from the separated mycelium and / or from the filtered fermentation broth; and purifying the isolated antibiotic by chromatographic procedures. In any case, it is preferred to produce the antibiotic 107891 under aerobic conditions in an aqueous nutrient medium containing readily assimilable sources of carbon, nitrogen, and inorganic salts. Many of the nutrient media commonly used in the field of fermentation may be used, however, certain media are preferred.

Preferred carbon sources are sucrose, fructose, glucose, xylose, and the like. Preferred nitrogen sources are ground soybeans ("soybean meai"), peptone, meat extract, yeast extract, tryptone, amino acids, hydrolyzed casein, and the like. Among the inorganic salts which may be incorporated into the culture medium are the usual soluble salts which can provide sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, sulfate, phosphate, nitrate, and the ions similar.

Preferably, the antibiotic producing strain 107891 is pre-cultured in a fermentation tube or in a shake flask, after which the fermenter culture ("fermenters") is used for the production of large quantities of substances. The medium used for the pre-culture may be the same as that used for large-scale fermentations, but other means may also be used. The antibiotic producing strain 107891 may be grown at a temperature between 17 ° C and 37 ° C, with optimum temperatures being as low as 28-30 ° C.

During fermentation, the production of antibiotic 107891 can be monitored by bioassays regarding susceptible microorganisms and / or HPLC analyzes. The maximum production of antibiotic 107891 generally occurs after about 90 hours and before 200 hours of fermentation. Antibiotic 107891 is produced by culturing Microbispora sp. ATCC PTA-5024 or a variant or mutant thereof capable of producing the antibiotic 107891, being found in the culture broths and / or in the mycelium.

In the present description and claims the expression " antibiotic 107891 ", unless otherwise specified, identifies the antibiotic complex 107891. MORPHOLOGICAL CHARACTERISTICS OF Microbispora sp. ATCC PTA-5024 Microbispora sp. ATCC PTA-5024 grows well in a number of conventional solid media. Microscopic dimensions were measured using culture growth in Salic Acid Humic Acid (composition in g / 1: humic acid 0.5, FeS04 * 7H20.001, MnCl2 * 4H20.001, ZnS04 * 7H20.001, NiS04 * A solution of vitamins (thiamine hydrochloride 25 mg / 1, calcium pantothenate 250 mg / 1, nicotinic acid 250 mg / 1, biotin 0, 620 μg 0,001, MOPS 2, agar 20) was added. , 5 mg / l, riboflavin 1.25 g / l, cyanocobalamin 6.25 mg / l, paraminobenzoic acid 25 mg / l, folic acid 500 mg / l, pyridoxal hydrochloride 500 mg / l).

In liquid culture (V6 medium, composition in g / 1: dextrose 22, meat extract 5, yeast extract 5, casein 3, 6

NaCl 1.5) no fragmentation of the mycelium is observed after 6 days growth at 28 ° C. Microscopic examination on Humic Acid - Salt Trace Agar (after 21 days of incubation at 28 ° C) reveals on the substrate a non-fragmented branched mycelium and a monopodial branched aerial mycelium; are also visible many long, linear and slightly branched hyphae. Pairs of characteristic longitudinal spores sprout through short sporophore arising laterally from branches or directly from major air hyphae. The spores are globular and not mobile. No sporangium-like bodies or other particular structures are observed. CULTURAL CHARACTERISTICS OF Microbispora sp. ATCC PTA-5024

Microbispora sp. ATCC PTA-5024 for six days in AF / MS liquid medium (see Example 1) at 28øC and 200 rpm, then transferred (5% inoculum) to a new AF / MS liquid medium and cultured over 6 days and finally inoculated (7% inoculum) into 100 ml V6 liquid medium (see Example 1). After 6 days of growth at 28 ° C and 200 rpm, the mycelium was collected by centrifugation and washed three times using sterile saline solution ("saline solution"), after which it was diluted to give inoculum. Suspension aliquots were seeded in a cross-culturing mode on various media recommended by Shirling and Gottlieb (EB Shirling and D. Gottlieb, (1966): " Method for Characterization of Streptomyces species ", Int. J. Syst Bacteriol 16: 313-340), and media recommended by SA Waksman (1961): " The Actinomycetes ", The Williams and Wilkins Co., Baltimore. Vol.2: 328-334). The ability to utilize various carbohydrates as a source of carbon and energy was determined using ISP4 starch-free medium, to which 1 ml / 1 of the above-described vitamin solution 7 was added as the basic medium; each carbon source was added at the final concentration of 1% (w / v). Tolerance to NaCl, growth pH limits as well as the ability to grow at different temperatures were determined on ISP2 medium. All media were incubated at 28 ° C for three weeks; the descriptions refer to 21 days unless specified. The color was evaluated in the natural light of the day, using the Color Atlas of Maerz and Paul (A. Maerz and M.R. Paul, 1950 - A Dictionary of Color 2nd Edition, McGraw-Hill Book Co. Inc., New York). The ability to reduce nitrates to nitrites in inconsistent nitrate medium (" sloppy ") was evaluated according to the procedure described by Williams et al. (S.T. Williams, M.Goodfellow, G. Allen, E. M. Wellington, P.H.A. Sneath & M.J. Sackin, 1983 Numerical classification of Streptomyces and related generates J. Gen. Microbiol., 129, 1743-1813). Growth, colony appearance, color of the substrate and aerial mycelium and the production of pigments of the strain Microbispora sp. ATCC PTA-5024 were recorded in Table I. Vegetative growth is present in most media used, unlike aerial mycelium which is only present in some of them. No evident pigmentation is observed in any of the media used. The physiological characteristics of the strain are shown in Table II. Growth and production of aerial mycelium are present at 17 ° C but not at 43 ° C. The production of the aerial mycelium in ISP2 is present at a pH higher than 6, while it is absent in the presence of 1% NaCl. The ability to utilize various carbohydrates for growth is shown in Table III. 8

Table I: Growth characteristics of Microbispora sp. ATCC PTA-5024 MEAN GROWTH AND MORPHOLOGY Reverse Color Code ISP 2 Yeast extract agar Malt extract Abundant growth, rough surface; good production of aerial mycelium (2A8) pink. Small production of orange / light brown soluble pigment. 5 E 12 orange / red ISP 3 Oat flour auger Abundant growth / good production of rosy aerial mycelium (2A8), especially on the branches of crossed crop lines. Small production of orange soluble pigment. 11 H 10 orange / pink ISP 4 Agar Starch Inorganic salts Good growth; absence of aerial mycelium. Absence of soluble pigments. Hydrolyzed starch. 11 I 9 Orange Glu / Asp Agar Asparagine Glucose Fragile, discontinuous growth / production of fragile beige / rosy (9B4) aerial mycelium on the branches of cross-culture lines. Absence of soluble pigments. 12 K 12 orange / light brown ISP 6 Agar Iron Peptone Yeast extract Scarce growth, with individual pink colonies growing in height, convoluted, with a smooth surface; absence of aerial mycelium. No darkening of the medium. Nd ISP 7 Agar Tyrosine Significant growth on the substrate of a fragile orange / light brown mycelium. No darkening of the medium. Nd ISP 3 + EL Agar Oatmeal / 1% Yeast Extract Abundant growth, rough surface; very scarce production of pink fragile aerial mycelium. Absence of soluble pigments. 4 B 12 orange / red (ISP4 and Agar Asparagine Glucose added with 1ml / 1 of vitamins solution)

Quadry II t Physiological characteristics of Microbispora sp. ATCC PTA-5024 REACTION REACTION Assay hydrolysis Positive Casein hydrolysis Negattivation Calcium malate digestion Negat iva Peptide of milk Litmus Negat Coagulation of milk Litmus Negative Gelatine liquefaction Negative to slightly positive Tyrosine reaction Negation Nitrate reduction Positive Limits of growth pH (14 days) Absence of growth at 4.2, good at 5.5 to 8.8; Not tested outside of this range. Absence of aerial mycelium at pH = 6.5 CINa tolerance in% <2; absence of aerial mycelium at a 1. Growth temperature limits 17 ° C to 37 ° C. Presence of aerial mycelium throughout the range; absence of growth at 43 ° C.

Table III: Use of carbon sources by Microbispora sp. ATCC PTA-5024 9

Carbon source Growth (14 days) Arabinose + + Cellulose - Fructose + + Inositol +/- Mannitol +++ Rafinose - Ramnose - Sucrose +++ Xylose +++ Glucose ++ Glycerol ++ No sugar - +++ abundant; ++ good growth; +/- scarce growth; - lack of growth; aerial mycelium is always absent. CHARACTERISTICS OF Microbispora sp. ATCC PTA-5024

Microbispora sp. ATCC PTA-5024 in GYM medium (4g glucose / 1; yeast extract 4g / 1; 10g / 1 malt extract) was added at 28 ° C on a rotary shaker and the mycelium was collected, twice with sterile distilled water and subsequently lyophilized. Amino acid analyzes were performed according to the method of Staneck and Roberts, (JL Staneck and GD Roberts, (1974): " Simplified approach to Identification of aerobic actinomycetes by thin layer chromatography ", Appl. Microbiol. : 226-231). Menaquinones and polar lipids were extracted following the procedure of Minnikin et al. (DE Minnikin, AG O'Donnell, M. Goodfellow, G. Alderson, M. Athalye, A. Schaal and JH Parlett, (1984): " An integrated procedure of isoprenoid quinones and polar lipids ", J. Microbiol. Meth 2: 233-241). By thin layer chromatography polar lipids were analyzed (DE Minnikin, V. Patel, L. Alshamaony, and M. Goodfellow, (1977): "Polar lipid composition in the classification of Nocardia and related bacteria" Int. J. Syst Bacteriol 27: 104-117), menaquinones by HPLC

Kroppenstedt, (1982): " Separation of bacterial menaquinones by HPLC using reverse phase RP18 and silver loaded ion exchanger as stationary phase ", J. Liquid Chromat 5: 2359-2367; R.M. Kroppenstedt, (1985): " Fatty acid and menaquinone analysis of actinomycetes and related organisms ", in: Chemical Methods in Bacterial Systematics. No 20 SAB Technical Series pp. 173-199, M. Goodfellow and D.E. Minnikin eds, Academic Press, London) and fatty acid methyl esters by gas-liquid chromatography, respectively (LT Miller, (1982): " A single derivatization method for bacterial fatty acid methyl esters including hydroxy acids ", J. Clin. Microbiol 16: 584-586; M. Sasser, (1990): " Identification of bacteria by gas chromatography of cellular fatty acids ", USFCC News Letters 20: 1-6). The presence of mycolic acids was verified by the method of Minnikin et al. (DE Minnikin, L. Alshamaony, and M. Goodfellow, (1975): " Differentiation of Mycobacterium, Nocardia and related taxa by thin layer chromatographic analysis of whole organism methanolyzates ", J. Gen. Microbiol 88: 200-204) .

The complete cell hydrolysates of the strain Microbispora sp. ATCC PTA-5024 contain a meso-diamino-pimelic acid such as diamino acid present in the peptidoglycan. The predominant menaquinones are MK-9 (III, VIII-H4), MK-9 (H2) and MK-9 (H0). The polar lipid model is characterized by the presence of phosphatidylethanolamine, methylphosphatidylethanolamine, phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl inositol, phosphatidyl inositol mannosides and N-acetylglucosamine containing phospholipids, i.e. type IV phospholipids according to Lechevalier et al. al. (H.A. Lechevalier, C. De Briève and M.P. Lechevalier, (1977): " Chemotaxonomy of Aerobic Actinomycetes: Phospholipid Composition ", Biochem Syst.Ecol., 5: 246-260). The major components of the fatty acid model are antioxidant 15: 0, iso 16: 0, n-16: 0, antiethyl 17: 0, and 10-methylhep-octadanoic acid (10-Me-17: 0), i.e. , 3c sensu Kroppenstedt (RM Kroppenstedt, (1985): " Fatty acid and menaquinone analysis of 11 actinomycetes and related organisms ", in: Chemical Methods in Bacterial Systematics No20 SAB Technical Series p.173-199 M. Goodfellow and DE Minnikin eds, Academic Press, London). Mycolic acids were not detected. Sequence of Microbispora sp. ATCC PTA-5024 The partial gene sequence encoding the 16 rRNA (16S rDNA), i.e. 1443 nucleotides, corresponding to 95% of the entire rRNA of the strain Microbispora sp. ATCC PTA-5024, was obtained following the published procedures (P. Mazza, P. Monciardini, L. Cavaletti, M. Sosio and S. Donadio, (2003): " Diversity of Actinoplanes and related genera isolated from an italian soil " Microbial Ecol. 5: 362-372). It is presented in the form of SEQ ID NO 1.

This sequence was compared to that of the Microbispora coralline strain NRRL 30420 (MF-BA-1768), as described in U.S. Patent No. 6,551,591 B1. The two sequences were aligned and differences were found in 31 of 1456 aligned positions, representing a global sequence divergence of 2.13%. Any two strains that share less than 97.5% sequence identity generally belong to different species (Stackebrandt, E. and Embley, M. T. (2000) " Diversity of Uncoated Microorganisms in the Environment ". In: Non-culturable Microorganisms in the

Environment, R. R. Colwell and D.J. Grimes (eds). ASM, Press, Washington DC, p. 57-75). Therefore, a 2% level of divergence in the sequence is quite high (Rosselló-Mora, R., and Amann, R. (2001). &Quot; The Species Concept for Prokaryotes ". FEMS Microbiol Rev. 25:39 -67) and indicates that Microbispora sp. ATCC PTA-5024 and Microbispora coralline NRRL 30420 (MF-BA-1768) are different strains. 12 IDENTITY OF STIRPE Microblspora sp. ATCC PTA-5024 The antibiotic producing strain 107891 is attributed to the genus Microbispora, to the family Streptosporangiaceae because of the following chemotaxonomic and morphological characteristics - Presence of meso-diaminopimelic acid in the wall of the cell;

Principal amount of MK-9 (III, VIII-H4) and type IV phospholipid according to Lechevalier et al. (THERE IS.

Lechevalier, C. De. Briève and M.P. Lechevalier, (1977): " Chemotaxonomy of aerobic actinomycetes: phospholipid composition ", Biochem. Syst. Ecol. 5: 246-260); - Fatty acid profile of 3c sensu Kroppenstedt (R.M.

Kroppenstedt, (1992): " The genus Nocardiopsis ", in: The

Prokaryotes, Vol. II, p. 1139-1156, A. Balows, H. Truper, M. Dworkin, W. Harder and K.H. Schleifer eds, New York, Springer-Verlag); - Absence of mycolic acids;

Formation of characteristic longitudinal spore pairs at the ends of short sporophores branching from aerial hyphae. Non-mobile spores. - Partial sequence of the gene encoding the 16 rRNA (16S rDNA), i.e. 1443 nucleotides, corresponding to 95% of the entire rRNA described in SEQ ID NO: 1, exhibiting identity > 97% relative to the 16S rDNA sequences from the described Microblspora species.

As with other microorganisms, the characteristics of the strains producing the antibiotic 107891 are subject to variation. For example, variants and artificial mutants of the strain can be obtained by treatment with various known mutagenic agents, such as U.V. rays, and chemicals such as nitrous acid, N-methyl-N'-nitro-N-nitrosoguanidine, and many others. All variants and the natural and artificial mutants of the strain Microbispora sp. ATCC PTA-5024 capable of producing the antibiotic 107891 are considered equivalent to that antibiotic for the purposes of the present invention and therefore fall within the scope of the same invention. EXTRACTION AND PURIFICATION OF ANTIBIOTIC 107891

As mentioned above, the antibiotic 107891 is distributed almost equally in both the mycelium and the filtered fraction of the fermentation broth. The collected broth can be treated to separate the mycelium from the broth supernatant from the fermentation and the mycelium can be extracted with a water miscible solvent to give a solution containing the antibiotic 107891, after the removal of the depleted mycelium (" spent mycelium "). This mycelial extract can then be treated separately or together (" in pool ") with the supernatant according to the procedures reported hereinafter for the supernatant fraction. When the water-miscible solvent can interfere with the steps of recovering the antibiotic from the mycelial extract, such a water-miscible solvent can be removed by distillation or diluted with water to a concentration that does not interfere. The term " water miscible solvent " as used in the present application is intended to have the meaning currently given in the art to that term and refers to solvents which, under the conditions of use, are miscible with water over a reasonably broad concentration range . Examples of water miscible organic solvents which may be used in the extraction of the compounds of the present invention are: lower alkanols, for example (C 1 -C 3) alkanols such as methanol, ethanol and propanol; phenyl (C 1 -C 3) alkanols such as benzyl alcohol; lower ketones, for example (C 3 -C 4) ketones such as acetone and ethyl methyl ketone; cyclic ethers such as dioxane and tetrahydrofuran; glycols and their partial etherification products such as ethylene glycol, propylene glycol, and ethylene glycol monomethyl ether, lower amides such as dimethylformamide and diethylformamide; acetic acid, dimethylsulfoxide and acetonitrile. The recovery of the compound from the supernatant of the fermentation broth of the producing microorganism is carried out according to techniques known per se, which include solvent extraction, precipitation by addition of nonsolvent or by alteration of the pH of the solution, by means of partition chromatography , reverse phase partition chromatography, ion exchange chromatography, molecular exclusion chromatography, and the like, or an association of two or more such techniques. A procedure for recovering the compounds of the present invention from the filtered fermentation broth includes extracting the antibiotic 107891 with non-water miscible organic solvents, followed by precipitation from the concentrated extracts, possibly by the addition of an agent of precipitation

Also in this case, the term " solvent not miscible with water " when used in the present application is intended to have the meaning commonly indicated in the art for said term and refers to solvents which, under the conditions of use, are slightly miscible or practically immiscible with water in a suitable solvent. range of concentrations reasonably broad, suitable for the intended use.

Examples of non-water miscible organic solvents which may be used in the extraction of the compounds of the present invention from the fermentation broth are: alkanols of at least four carbon atoms which may be linear, branched or cyclic such as n-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, 3,3-dimethyl-1-butanol, 4-methyl-1-pentanol, 3-methyl-1-pentanol 3-pentanol, 4,4-dimethyl-2-pentanol, 5-methyl-2-hexanol, 1-heptanol, 2-heptanol, 5- methyl-1-hexanol, 2-ethyl-1-hexanol, 2-methyl-3-hexanol, 1-octanol, 2-octanol, cyclopentanol, 2-cyclopentethanol, 3-cyclopentyl-1-propanol, cyclohexanol, cyclohexanol, cyclooctanol, 2,3-dimethylcyclohexanol, 4-ethylcyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol, and 3-decanol; ketones of at least five carbon atoms such as methyl isopropyl ketone, methyl isobutyl ketone, methyl n-amyl ketone, methyl isoamyl ketone and mixtures thereof.

As is known in the art, extraction of the product from the filtered fermentation broth can be improved by adjusting the pH to an appropriate value, and / or by adding an appropriate organic salt which forms an ionic pair with the antibiotic, which is soluble in the extraction solvent.

As is known in the art, phase separation can be improved by the addition of an electrolyte (" salting ") to the aqueous phase. 16

When, after extraction, an organic phase containing a large amount of water is recovered, it may be convenient to separate the water from the organic phase by azeotropic distillation. Generally, this requires the addition of a solvent capable of forming minimal azeotropic mixtures with water, followed by the addition of a precipitating agent to precipitate the desired product, if necessary. Representative examples of organic solvents capable of forming minimal azeotropic mixtures with water are: n-butanol, benzene, toluene, butyl ether, carbon tetrachloride, chloroform, cyclohexane, 2,5-dimethylfuran, hexane, and m-xylene ; with n-butanol being the preferred solvent.

Examples of precipitating agents are petroleum ether, lower alkyl ethers, such as ethyl ether, propyl ether, and butyl ether, and lower alkyl ketones such as acetone.

According to a preferred method for the recovery of the antibiotic 107891, the filtered fermentation broth is contacted with an adsorption matrix followed by elution with a polar solvent miscible with water or a mixture thereof, a concentration until obtaining of an oily residue under reduced pressure, and a precipitation with a precipitating agent of the aforementioned type.

Examples of suitable adsorption matrices for the recovery of the compounds of the present invention are polystyrene or mixed polystyrene-divinylbenzene resins (for example M112 or S112, Dow Chemical Co., Amberlite® XAD2 or XAD4, Rohm & Haas (Eg XAD7 or XAD8, Rohm & Haas), polyamides such as polycaprolactams, nylons and crosslinked polyvinylpyrrolidones (for example, Polyamide CC-176, Polyamide-SC 6, Polyamide-CC 6 , 6 Polyamide-CC 6AC and Polyamide-SC 6AC, Macherey-Nagel & Co., Germany, PA 400, M. Woelm AG, Germany), and PVP-CL polyvinylpyrrolidone resin (Aldrich Chemie GmbH and Co., KG, Germany) and controlled pore cross-linked dextrans (e.g. Sephadex® LH-20, Pharmacia Fine Chemicals, AB). Preferably, polystyrene resins are used, with Diaion HP 20 resin being especially preferred.

In the case of polystyrene resins, polystyrene-divinylbenzene resins, polyamide resins or acrylic resins, the preferred eluent is a solvent miscible with water or aqueous mixtures thereof. The aqueous mixtures may contain buffers at an appropriate pH value.

Also in this case, the term " water miscible solvent ", when used in that description and in those claims, has the meaning commonly given in the art to said expression as described above.

Successive procedures for the isolation and purification of the antibiotic may be carried out in the pooled extracts from broth and mycelial supernatant. For example, when recovering the portion of the antibiotic product contained in the filtered fermentation broth or in the supernatant by absorption on an absorption resin and extracting the portion of the antibiotic product contained in the mycelium with a water miscible solvent followed by adsorption on an absorption resin, the fractions eluted from each of the two sets of absorption resins may optionally be combined after concentration and then further treated as a unit culture. Alternatively, when the two sets of absorption resins in the separate extraction steps are of the same type 18 and exhibit the same functional characteristics, they may be brought together together and the blend may undergo a unit elution step, with a water miscible solvent or a mixture thereof with water.

In any event, whatever procedure is adopted for the recovery of the crude antibiotic 107981, the successive purification step is generally carried out in the blending of the raw materials resulting from the association of the products from the separate extraction phases. Purification of the crude antibiotic 107891 can be carried out by any of the techniques known per se but is preferably carried out by means of chromatographic techniques. Examples of such chromatographic techniques are those described with respect to the recovery step and also include chromatography on stationary phases such as silica gel, alumina, activated magnesium silicate and others or reverse phase chromatography on silanized silica gel having various functional derivatizations, and eluting with water miscible solvents or an aqueous mixture of water miscible solvents of the type mentioned above.

For example, preparative HPLC chromatography may be applied using RP-8 or RP-18 as the stationary phase and a mixture of HCOONH 4: CH 3 CN buffer as the elution system.

The active fractions recovered from the purification step are pooled, concentrated in vacuo, precipitated by addition of a precipitating agent of the aforementioned type, and dried or lyophilized in single or iterative cycles. In the case where the product contains residual amounts of ammonium formate or other buffering salts, these may be removed by absorption of antibiotic 107891 on a solid phase extraction column, for example a reverse phase resin column such as SPE Superclean LCP18 Supelco (Bellefonte PA, USA), followed by washing with distilled water and elution with an appropriate aqueous solvent mixture, for example ethanol: water. The antibiotic is then recovered by elimination of the elution solvents.

Accordingly, a purified dry preparation of the antibiotic complex 107891 is obtained as a white powder.

As is customary in this art, the production steps as well as the recovery and purification steps can be monitored by a variety of analytical procedures including inhibitory assay against sensitive microorganisms and analytical control using HPLC or HPLC coupled to mass spectrometry.

In a Waters apparatus (Waters Chromathography, Milford, MA) equipped with a Waters Simmetry-shield RP8, 5μ (250 x 4.6 mm) column eluted at a flow rate of 1 ml / min and at 50 ° C A preferred analytical HPLC technique is provided. Elution occurred using a multi-phase program: Time = 0 (B phase 30%); Time = 8 min (Phase B 30%); Time = 28 min (40% of Phase B). Phase A was acetonitrile: 100 mM ammonium formate buffer (pH: 5.0) 5:95 (v / v) and to Step B was acetonitrile. The UV detector ran at 282 nm.

The effluent from the column was divided by a ratio of 5:95 and most (ca 950 μΐ / min) was diverted to a detector with a photodiode array. The remaining 50 μΐ / min were diverted to " interface " ESI from a Finnigan LCQ mass spectrometer with an " ion-trap " (Thermoquest, Finnigan MAT, San Josè CA). Mass spectrometric analysis was performed under the following conditions:

Sample entry conditions: Separator gas (N2) 60 psi; Auxiliary gas (N2) 5 psi;

Capillary heater 250 ° C;

Sample input potential settings

Positive and negative polarity;

Ion atomization potential +/- 5 kV;

Capillary potential +/- 19 V;

Scan conditions (" scan "): maximum ion time 200 ms;

5 ms ion time;

Complete micro-verification 3;

Section: duration 30 minutes, positive (150-2000 m / z) and negative (150-2000 m / z) for occurrences per holding.

Under these analytical HPLC conditions, Factors AI and A2 of antibiotic 107891 showed retention times of 13.2 min and 13.9 min, respectively. In the same HPLC system the Ramoplanin Factor A2 (L. Gastaldo, R.Ciabatti, F.Assi, E. Restelli, JK Kettenring, LF Zerilli, G. Romano, M. Denaro and B. Cavalleri, (1992): "; Isolation, structure determination and biological activity of A-16686 factors A'l, 21A'2 and A'3 glycolipodepsipeptid antibiotics ", J. Ind. Microbiol 11: 13-18) eluted with a retention time of 7.5 minutes. Since antibiotics 107891 as demonstrated by acid / base titration in 2-methoxyethanol (MCS): H2 O 12:03 (v / v) contain a basic function, they are capable of forming salts with suitable acids according to standard procedures and may exist also in the form of free base. Antibiotic 107891, when obtained as the free base, may be converted with acids into the corresponding salts, which include non-toxic pharmaceutically acceptable salts. Suitable salts include salts formed by the normal reaction with organic and inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, trichloroacetic, succinic, citric, ascorbic, lactic, maleic, fumaric, palmitic, , pamoic, mucic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and the like acids. Addition salts of antibiotic 107891, acid derivatives may be prepared according to customary procedures commonly used. As an example, the antibiotic 107891 in the free base form dissolves in the minimum amount of a suitable solvent, typically a lower alkanol, or a lower alkanol / water mixture, the stoichiometric amount of a chosen acid being gradually added to the solution obtained and the salt obtained precipitated by the addition of a non-solvent. The addition salt which is formed is then recovered by filtration or evaporation of the solvents. 22

Alternatively, such salts may be prepared in an extremely anhydrous form by lyophilization; in which case a salt of antibiotic 107891 derived from a volatile acid is dissolved in a suitable amount of a non-volatile acid. The solution is then filtered to remove any insoluble materials and is lyophilized in a single cycle or iterative cycles.

A specific addition salt may also be obtained from a solution of another antibiotic salt 107891 when the desired salt precipitates upon addition of the appropriate anion. The conversion of the compound of the present invention not in the form of salts into the corresponding addition salts, and the reverse, i.e., the transformation of an addition salt of a compound of the present invention to form a form which is not a salt are encompassed by common technical experience and are included in the present invention. The formation of salts of antibiotic 107891 may serve a variety of purposes, including separation, purification of said antibiotic 107891 and its use as a therapeutic agent or animal growth promoter. For therapeutic purposes, pharmaceutically acceptable salts are generally used. The term " pharmaceutically acceptable salts " identifies the non-toxic salts that can be used in the therapy of warm-blooded animals. Antibiotic complex 107981 may be administered as such or in the form of mixtures with pharmaceutically acceptable carriers and may also be administered in combination with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides.

A combined therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutic effects of the first compound administered do not entirely disappear when the following compound is administered.

The compounds of the present invention, or pharmaceutically acceptable addition salts thereof, may be formulated in forms suitable for parenteral, oral or topical administration. For iv administration in the treatment of any infection involving an antibiotic-sensitive microorganism, a parenteral formulation is, for example, in water with a suitable solubilizing agent such as polypropylene glycol or dimethylacetamide and a surfactant (for example, polyoxyethylene sorbitan monooleate or polyethoxylated castor oil) or formulations based on cyclodextrins or phospholipids in sterile water for injections. An injectable formulation may also be obtained with an appropriate cyclodextrin. The antibiotic complex 107981 may also be used in a suitable pharmaceutical form as a capsule, tablet or aqueous suspension for oral administration or as conventional creams or ointments (" jellies ") for topical applications. In addition to their use in the form of medicaments in human and veterinary therapy, the compounds of the present invention may also be used as animal growth promoters. To this end, a compound of the present invention is administered orally in a suitable ration. The exact concentration used is that which is required to provide the active agent in an effective growth promoting amount when normal amounts of the feed are consumed. The addition of the active compound of the present invention to animal feed is preferably achieved by preparing a feed premix containing the active compound in an effective amount and incorporating that premix into the complete feed. Alternatively, an intermediate concentrate or a food supplement containing the active component may be mixed into the feed. The ways in which such premixes and complete feedingstuffs can be prepared and administered are described in reference books ("Applied Animal Nutrition", WH Freedman and Co., S. Francisco, USA, 1969 or "Livestock Feeds and Feeding " 0 and B books, Corvallis, Oregon, USA, 1977). PHYSICAL-CHEMICAL CHARACTERISTICS OF THE ANTIBIOTIC 107891 A) Mass spectrometry [(MS) Mass spectrometry]:

In MS experiments with a Thermo Finnigan LCQ Deca apparatus equipped with an electrospray source using a calibration mixture of the Thermo Finnigan apparatus, the antibiotic 107891 discloses two doubly protonated ions at m / z = 1124 and m / z 1116 corresponding to the isotope composition lower than the AI and A2 Factors of the complex. The electrospray conditions were: Spray Potential: 4.7 kV; Capillary temperature: 220 ° C; Capillary tension: 3 V; Perfusion Mode 10 μΐ / min. The spectra were recorded from a 0.2 mg / ml solution in 80/20 (v / v) methanol / water with 0.1% trifluoroacetic acid and are depicted in Fig. 1A (low resolution spectrum per holding " scan ") and 1B [high resolution scanning spectrum (" scan ") by zooming]. 25 Β) no infrared spectrum in KBr of antibiotic 107891 with a Bruker FT-IR spectrophotometer model IFS 48, exhibits absorption maxima at (cm-1): 3263; 2929; 1661, 1533; 1402; 1114; 1026. Infrared spectrum is described in

Fig.2. The absorption bands at 1631, 1596 and 1346 are assigned the residual amount of ammonium formate. C) The spectrum in U.V. of antibiotic 107891, performed in methanol / H 2 O (80:20 ratio) with a Perkin-Elmer Lambda 16 spectrophotometer, exhibits two inflections (" shoulders ") at 226 and 267 nm. The UV spectrum is depicted in Fig. D) 1 H-NMR spectrum was recorded in the 40:10 (v / v) methanol-d 4: H 2 O (40: 3 HCl) mixture at 40øC on a Bruker AMX 600 spectrometer using a suppression sequence of water. As the internal standard, the residual signal of methanol-d4 at 3.31 ppm was considered. The 1 H-NMR spectrum of antibiotic 107891 is depicted in Fig. The 1 H-NMR spectrum of antibiotic 107891 dissolved in methanol-d 4: H 2 O (0.01 N HCl) 40:10 (v / v) exhibits the following signal groups (in ppm) at 600 MHz using MeOH-d4 as the standard internal (3.31 ppm), [Î'= ppm, multiplicity; (assignment)]: 0.93 d (CH3), 0.98 d (CH3), 1.07 t (overlapping CH3), 1.18 t (CH3 overlapping), 1.26 s (CH3), 1.30 t (overlapping CH3), 1.62-1.74 m (CH2), 1.78 d (CH3), 1.80 d

(CH 3), 2.03 m (CH 2), 2.24 m (CH), 2.36 m (CH 2), 2.72-3.8 m (peptidic CH 3), 3.8-5.2 m (CH alpha peptide), 5.53-6.08 s (CH 2), 5.62 d (CH double bond), 6.42 m (CH), 6.92 d (double bond CH), 7.0- 7.55 m (aromatic CH), 7.62-10.4 dem (aromatic NH and peptidic). E) 13 C-NMR spectrum was recorded in the 40:10 (v / v) methanol-d 4: H 2 O (40: 3 HCl) mixture at 40øC on a Bruker AMX 600 spectrometer using as internal standard 26 Residual signal of methanol-d4 at 49.15 ppm. The decoded 13 C-NMR spectrum of bb of antibiotic 107891 is depicted in Fig. The13 C-NMR spectrum of antibiotic 107891 dissolved in methanol-d4: H2 0 (0.01 N HCl) 40:10 (v / v) exhibits the following signal groups (in ppm) at 600 MHz using MeOH-d4 as the standard internal (49.15 ppm), [Î'= ppm; (attribute)): 13.6-23.2 (aliphatic CH3), 26, 16-73 (0α aliphatic and CH alpha peptide), 105-136 (aromatic and double bonded CH and quaternary carbons), 164,3- 176.3 (peptidic carbonyls). F) Antibiotic complex 107891 was dissolved in 2-methoxyethanol (MCS): H2 O 12:03 (v / v) containing a molar excess of 0.01 M hydrochloric acid. The solution was then titrated again with a solution of 0.01 N potassium hydroxide. The resulting titration curve presented a basic ionizable function. COMPOSITION OF ANTIBIOTIC AMINO ACIDS 107891 A) Determination of " acid-resistant amino acids " in the Antibiotic complex 107891

Antibiotic 107891 was subjected to complete acid hydrolysis (6N HCl, 105 ° C, 24h) and the acid treatment resistant amino acid components of the antibiotic were identified. The labile amino acids are not detectable with this technique. The hydrolysis was studied by HPLC-MS and GC-MS analysis, following suitable derivatization, as compared to a similarly derived mixture of standard amino acids. For the HPLC analysis the hydrolyzed sample was treated with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag ™ Fluor reagent kit) for GC analysis with a mixture of 3N HCl in anhydrous methanol and trifluoroacetic anhydride. Qualitative HPLC analysis was performed on a liquid chromatography system with simultaneous DAD and MS detection. The HPLC method had the following conditions:

Column: AccQ-Tag ™ (Waters C18 NovoPak 4pm 3.9 x 150mm) Column temperature: 37 ° C Flow: 1 ml / min.

Step A: 140 mM ammonium acetate pH 5 (acetic acid)

Step B: water: acetonitrile 60:40 (v / v)

Elution program

Time (min) 0 5 30 35 40 41% B 5 5 80 95 95 5

UV detection: 254 nm

MS conditions were as follows:

Spectrometer: Finnigan LCQ Deca equipped with conventional electrospray source.

Capillary temperature: 250 ° C Voltage source: 4.70 KV Supply current: 80 μΑ Capillary potential: -15V The qualitative GC analysis was performed on a gas chromatograph equipped with MS-EI detection. The GC method had the following conditions:

Column: J & W Scientific DB-5, 30m x 0.254mm ID x 0.25pm FT Carrier gas: Helium

Injection mode: without fractionation

Injector temperature: 200 ° C

Transfer line temperature: 300 ° C

Temperature range: 50 ° C to 100 ° C at 2.5 ° C / min (10

min), from 100 ° C to 250 ° C at 10 ° C / min (15 min), 15 min at 250 ° C 28

Injection volume: 1 μΐ

The MS conditions were as follows:

Used Spectrometer: Finnigan TSQ700

Ionization Mode: Impact of Electrons

Potential adjustment:

Filament Current: 400 mA Electron Multiplier: 1400 V Electron Energy: 70 eV Positive ion mode

Scan condition (" scan "):

Scan time (" scan "): 40-650 amu Scan time (" scan "): 1 sec

In the LC / MS and GC / MS chromatograms obtained on the hydrolyzate of antibiotic 107891, the following amino acids were identified along with other unidentified peaks: lanthionine, methyl-lanthionine, glycine, proline, valine, aspartic acid (NMR indicate that this is a transformation product of asparagine, which generates aspartic acid by hydrolysis), phenylalanine and leucine.

Factors AI and A2 of antibiotic 107891 (outside the scope of the present invention) were subjected to complete acid hydrolysis under the same conditions (derivatization and HPLC-MS) described for the complex. GC-MS analysis was performed on a Thermo Finnigan Trace GC-MS instrument equipped with a PTV injector. The GC method had the following conditions:

Column: Restek RTX-5MS, 15m x 0.25mm ID x 0.25 pm FT Carrier gas: Helium

Contact face temperature: 250 ° C

Temperature program: 1.5 min at 50 ° C, 50 ° C to 100 ° C at 20 ° C / min, 1 min at 100 ° C, 100 ° C to 135 ° C at 20 ° C / min, 1 min at 135 ° C, 135 ° C to 250 ° at 20 ° C / min, 1 min at 250 ° C

Injection volume: 1 μΐ

Injector: mode without fractionation, base temperature 50 ° C, transfer temperature 280 ° C, transfer rate 14.5 ° C / min

MS conditions were as follows:

Ionization mode: electron impact

Potential adjustment:

Filament current: 149 μΑ Electron multiplier: 200 V Electron energy: 70 eV

Positive ion mode:

Scan condition (" scan "):

Scan interval: 33-500 amu Scan time: 0.6 sec B) Determination of 5-chlorotryptophan in the antibiotic complex 107891. Complete hydrolysis of the purified 107891 complex was carried out in the presence of 5% was determined according to the method described by Simpson RJ, Neuberger MR, Liu TY, " Complete Aminoacid Analysis of Proteins from a Single Hydrolyze ". Journal Biol. Chem (United States), April 10, 1976, 251 (7), 1936-1940.

This hydrolysis process avoids the degradation of normally unstable amino acids during digestion with mineral acids, thus allowing the determination of such amino acids, including tryptophan, from a hydrolyzate of a peptide. A standard sample of 5-chloro-DL-tryptophan was purchased from Biosynt AG, Staad, Switzerland, and its structure confirmed by NMR analysis; DL-tryptophan was purchased from Merck KGaA, Darmstadt, Germany.

The tryptophan and 5-chlorotryptophan standards elute at retention times of 8.1 minutes and 11.5 minutes corresponding to M + H + at m / z 205 and 239, respectively.

With the chromatographic system used with a detection limit of 0.3 pg / ml, tryptophan Standard was detected. This value is lower than the value that would have been indicative of the presence of said amino acid in the sample of the antibiotic under test. COMPARISON OF THE FACTOR AI AND THE FACTOR A2 OF ANTIBIOTIC 107891 (outside the scope of the present invention) Amino Acid Microblspora NNRL 30420 (MF-BA-1768), described in U.S. Pat. U.S. Patent No. 6,551,591, was purchased from the NNRL collection. In one exploratory experiment, M. corallina strain NNRL 30420 (MF-BA-1768) was subjected to fermentation in an Erlenmeyer under the conditions described in U.S. Patent 6,551,591 B1 referred to above. The culture broth collected was extracted by dilution with methanol. After centrifugation of the mycelium, the supernatant was placed on an HP20 polystyrene absorption resin, eluted with a 70:30 methanol: water mixture, reduced to a small volume and then lyophilized. In the chromatogram two peaks showed signals of 1091 and 1108 [M + 2H] 2+, corresponding to the [M + 2H] 2+ described in U.S. Patent No. 6,551,581 Bl for ΜΓ-ΒΑ-1768β and MF -BA-1768, respectively. B) In a further experiment, the fermentation of Microbispora sp. strain NRRL 30420 (MF-BA-1768) and the collected juice was treated following the disclosure of U.S. Patent No. 6,551,591. After the sequential purification steps on HP20 polystyrene resin and polyamide CC resin 60.1-0.3 mm (Macherey-Nagel), two individual substances in the pure form were obtained by preparative HPLC on a Phenomenex C18 column with μ10 particles (Torrance CA, USA) Luna (250x12.2 mm) eluted at a flow rate of 27 ml / min with the following multi-phase program: Time = 0 min (32% phase B); Time = 8 min (32% phase B); Time = 20 min (36% phase B); Time = 32 min (90% Phase B). Phase A was 0.05% (v / v) formic acid in water, Phase B was CH 3 CN.

These substances showed antibacterial activity against staphylococci and enterococci as shown in Table IV. In LC-MS experiments the two substances will show signals of doubly protonated ions [M + 2H] ++ corresponding to the antibiotics MF-BA-1768al and MF-BA-1768pi, as described in U.S. Patent 6,551,591 Bl. 32

Table IV STRAIN MIC (μg / ml) MF-BA-1768al MF-BA-1768pl 107891 AI 107891 A2 Complex 107891 1400 Staphylococcus aureus cl.isol.Met r 0.13 0.5 0.13 0.13 0.13 568 Enterococcus faecium cl. isol. 4 16 1 2 569 Enterococcus faecium cl. isol. Van A 4 8 1 2 2 559 Enterococcus faecalis cl. isol. 4 8 1 2 1 560 Enterococcus faecalis cl. isol. Van A 4 8 0.5 1 0.5

The experimental conditions of the antimicrobial assays were the same as those used in the assays described in Table VI below.

LC-MS analyzes of the isolated antibiotics MF-BA-1768 oi and ΜΕ-ΒΑ-17 68β were carried out on a Symmetry Ci8 (5: m) 4, 6 x 250 mm column (Waters; Milford MA, USA), equipped with a Symmetry Cis (5: m) 3.9 x 20 mm pre-column (both kept in a 50 ° C oven). Elution was carried out at a flow rate of 1 ml / min with the following multi-phase elution schedule: time = 0 min (30% Phase B); Time = 8 min (30% Phase B); Time = 20 min (45% Phase B); Time = 24 min (90% Phase B), and time = 28 min (90% Phase B). Phase A was 25 mM HCOONH 4 buffer pH 4.5: CH 3 CN 95: 5 (v / v) and phase B was CH 3 CN. The HPLC equipment was coupled to a Finnigan LCQ Mass Spectrometer with an " ion-trap " (Thermoquest, Finnigan MAT, San Josè CA, USA). The column effluents were diverted 100 μΐ / min to the ESI contact face of the LCQ Mass Spectrometer. The MS analysis was performed under the following conditions: sample inlet: gas flow separator (N2) 25 psi, auxiliary gas flow 5 psi; capillary heater: 210 ° C, positive and negative sample input potential polarity; atomization potential of 33 ions: +/- 4.75 KV, capillary potential: +/- 12 V, scan conditions: maximum ion time 50 ms; complete micro: scan (" scan ") 3.

The antibiotic Factors MF-BA-688 and γ-γ-1768β and the Factors AI and A2 of antibiotic 107891 (outside the scope of the present invention) were analyzed individually and in admixture. The results are summarized in the following Table V.

Table V

Ret. (min) [M + 2H] MF-BA-17 68βι 12.86 1091 Antibiotic 107891 AI 16.323 Antibiotic 107891 A2 16.81 1116 MF-BA-1768 (¾ 18.1 1108

In the same chromatographic system, the factor A2 of the plantarin was eluted (L. Gastaldo, R. Ciabatti, F. Assi, E. Restelli, JK Kettenring, LF Zerilli, G. Romano, M. Denaro and B. Cavalleri, (1992 ): " Isolation, structure determination and biological activity of A-16686 factors A'l, A'2 and A'3 glycolipodepsipeptide antibiotics ", J. Ind. Microbiol 11: 13-18) with the retention time of 11, 00 minutes. NMR SPECTROSCOPY OF FACTOR AI AND FACTOR A2 OF ANTIBIOTIC 107891 (outside the scope of the present invention)

1H-NMR spectra of Factor AI and Factor A2, outside the scope of the present invention, of antibiotic 107891 were recorded in the CD3CN: D20 (1: 1) mixture at 298 K on a Bruker AMX 600 spectrometer using a suppression sequence of water. As the internal standard the residual signal of acetonitrile-d3 at 1.94 ppm was considered. A) 1 H-NMR spectrum of the Factor AI (outside the scope of the present invention) of antibiotic 107891 is depicted in Fig. B) The unbound 1H-NMR spectrum of bb of Factor A2 of antibiotic 107891 (outside the scope of the present invention) is depicted in Fig.

13 C-NMR spectra of Factor A1 and Factor A2 of antibiotic 107891 (outside the scope of the present invention) were recorded in the CD3 CN: D20 (1: 1) mixture at 298 K on a Bruker AMX 600 spectrometer using as an internal standard the residual signal of acetonitrile-d3 at 1.39 ppm. C) 13C-NMR spectra of Factor A1 (outside the scope of the present invention) is shown in Fig. 10. D) The decoded 13 C-NMR spectrum of bb of Factor A2 of antibiotic 107891 (outside the scope of the present invention ) is depicted in Fig. UV and IV FACTOR SPECTROS Al and FACTOR A2 OF ANTIBIOTIC 107891. (outside the scope of the present invention) A) Infrared spectrum of Factor Al (outside the scope of the present invention) of the antibiotic 107891 recorded in KBr with a spectrophotometer FT- IR Bruker model IFS 48, is depicted in Fig. 12. B) The UV spectrum of Factor Al of antibiotic 107891, (outside the scope of the present invention) recorded in methanol: H 2 O 80:20 (v / v) with a spectrophotometer Perkin-Elmer Lambda 16, is described in Fig. 13. C) Infrared spectrum of Factor A2 of antibiotic 107891 (outside the scope of the present invention) recorded in KBr with a Bruker FT-IR spectrophotometer model IPS 48, Fig. 14. D) The UV spectrum of Factor A2 of antibiotic 107891 (outside the scope of the present invention) recorded in methanol: H 2 O 80:20 (v / v) with a Perkin-Elmer Lambda spectrophotometer 16, is depicted in Fig.

Based on the physicochemical data described above, the following structure formula can be attributed to the antibiotic 107891: the compounds according to claim 1:

in which X is halogen (F, Cl, Br, I), and wherein R1, R2, R3, and R4 are independently selected from the group consisting of H, OHR5, R6, R7 and R8 are independently selected from the group consisting of H, OH, (branched or unbranched, substituted or unsubstituted), and (substituted or unsubstituted) aryl, with the proviso that when R 2 is OH or R 1 and R 2 is OH, at least one R3 to R8 is not H or X is not Cl or Cl. In an alternative embodiment R1, R2, R3 and R4 may represent H or OH.

Thus, the possible combinations of R 1, R 2, R 3, and R 4 include the following.

R 2, R 3, R 4, R 4, R 5, R 4, R 5, R 4, OH OH

Likewise, represent H or OH. R 5, R 6, R 7, and R 5 may

Thus, the possible combinations of R5, R6, R7, and R8 include the following. 37

H, OH, OH, OH, OH, OH, OH, OH, OH, OH, OH, The antimicrobial activity of antibiotic 107891 was determined by the microdilution method of the broth according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, document M7-A5).

The strains used were clinical isolates or strains from the American Type Culture Collection (ATCC). The results of the tests are given in Table VI and Table VII. Antibiotic 107891 was dissolved in DMSO to give a stock solution of 1000 pg / ml, and subsequently diluted with water to give the working solution. The media used were cation-adjusted Mueller Hinton broth (CAMHB) for Staphylococci, M. catarrhalis, Enterococci and L. monocytogenes; Todd Hewitt broth (THB) for Streptococci; GC medium + 1% IsoVitaleX + 1% hemin 38 for Neisseria spp; Brain and Heart Perfusion + 1% C supplement for H. influenzae; Lactobacillus broth for Lactobacilli culture; Middlebrook 7H9 enriched with Middlebrook OADC for M. smegmatis; RPMI 1640 Medium for C.albicans. Wilkins Chalgren + oxirase broth (1:25 v / v) for Clostridia; Broth for Brucella containing cistern (0.5 g / 1) for Propionibacteria. The inoculums for the bacteria were 105 CFU / ml. The inoculum of C. albicans was 1 x 10 4 CFU / ml. All assays were performed in the presence of 0.02% bovine serum albumin (BSA). Cultures were incubated in the air at 35 ° C, except the Clostridia and Propioniobacteria strains which required an anaerobic atmosphere. After 18-24 hours visual readings were performed and MICs were determined. The MIC was defined as the lowest concentration of the antibiotic in which there is no visible growth. TABLE VI: Antimicrobial activity of antibiotic 107891 MIC (pg / ml) Microorganism 107891 819 Staph. aureus Smith ATCC 19636 &lt; 0.11 4061 Staph. Aureus LIM1 <0, 13 3798 Staph. Aureus clin. Isolate VISA 2 1400 Staph. Aureus clin. Isolate Met-R &lt; 0, 13 613 Staph. Aureus clin. Isolate Met-R &lt; 0, 13 3797 Staph. Aureus clin. Isolate VISA ME 2 4064 Staph. Aureus LIM2 GISA Met-R 0.5 1729 Staph. Haemolyticus Met-R 8 1730 Met-S 2 147 Staph. Epidermidis ATCC 12228 <0, 13 1139 4 44 Strept. Pneumoniae Pen-S &lt; 0, 13 2868 Pen-I &lt; 0, 13 49 Strept. pyogenes &lt; 0, 13 559 Ent. Faecalis Van-S 1 560 Ent. Faecalis Van-A 0.5 A333 Ent. Faecalis Van-A 1 568 Ent. Faecium Van-S 2 569 Ent. Faecium Van-A 1 B518 Ent. Faecium Van-A 2 A6345 Ent. Faecium Van-A Lnz-R 4 3754 Mycobacterium smegmatis 32 884 Listeria garviae &lt; 0, 13 148 Listeria delbrueckii ATCC 4797 4 1450 Listeria monocytogenes 0, 125 833 Haemophilus influenzae 32 970 Haemophilus influenzae ATCC 19418 32 3924 Moraxella catharralis 1 Moraxella catharralis ATCC 8176 0.25 1613 Neisseria meningitidis ATCC 13090 0.5 997 Neisseria gonorrhaee 0.25 47 Escherichia coli &gt; 128 145 Candida albicans &gt; 128 40 TABLE VII: Antimicrobial activity of antibiotic 107891 against anaerobic bacteria 40 Microorganism ATCC 27520 Propionibacterium ammphophilum ATCC 25564 Propionibacterium acnes ATCC 6922 Propionibacterium acnes ATCC 6922 Propionibacterium acnes ATCC 1322 Propionibacterium acnes ATCC Propionibacterium acnes ATCC Propionibacterium acnes ATCC Propionibacterium acnes ATCC Propionibacterium acnes ATCC Propionibacterium acnes 0.125 &lt; 0.0039 0.25 4018 Clostridium difficile &lt; 0.125 4025 Clostridium difficile &lt; 0.125 4022 Clostridium difficile &lt; 0.125 4032 Clostridium perfringens &lt; 0.125 4043 Clostridium butyricum ^ 0,125 4009 Clostridium beijerinckii &lt; 0.125 4052 Clostridium septicum &lt; 0.125 60601 Peptostreptococcus anaerobius &gt; 128 Antibiotic 107891 shows good antibacterial activity against Gram bacteria -positive. The MIC range (&quot; minimum inhibitory concentration &quot;) against Staphylococcus spp., Including methicillin resistant strains (MRSA) and Glycopeptide Intermediates (GISA), is = 0.13-4 pg / ml and against recent clinical isolates of Enterococcus spp., Including vancomycin resistant (VRE), is 0.5-4 pg / ml. Against Streptococcus spp. the MICs are 0.13 pg / ml. Antibiotic 107891 is also active against anaerobic Gram-positive strains; the MICs are &lt; 0.13 pg / ml against Clostridia and ≤ 0.004-4pg / ml against Propionibacteria. 41

Antimicrobial activities against strains of L. monocytogenes (MIC 0.125 pg / ml) and Lactobacilli strains (MIC limits 0.13-4 pg / ml) were reported.

Some Gram-negative bacteria are susceptible to antibiotic 107891; the MICs are between 1 and 0.25 pg / ml versus M. catharralis, 0.5 and 0.25 pg / ml against Neisseria spp. and 32 pg / ml against H. influenzae. Antibiotic 107891 is not active against the E. coli and Candida albicans strains tested. Antibiotic 107891 in experiments to determine the rate of bacterial killing (&quot; time-kill &quot;) exhibits bactericidal activity against S. aureus GISA and strain E. faecalis Van A; at 24 hours the bactericidal concentration is the MIC value in Mueller Hinton broth. S. aureus can cause life-threatening infections and MRSA is of particular clinical importance because it is resistant to all penicillins and cephalosporins and also to several other antibiotics; in addition, it easily spreads from patient to patient causing outbreaks of infection with important implications for health facilities (W. Witte, (1999): &quot; Antibiotic resistance in Gram-positive bacteria: epidemiological aspects &quot;, Journal of Antimicrobial Chemotherapy 44: 1-9). The National Nosocomial Infection Surveillance System (NNIS) reported that methicillin resistance among S. aureus in US hospitals increased from 2.4% in 1975 to 29% in 1991, with a higher degree of resistance in intensive care units (L. Archibald, L. Philips, D. Monnet, JEJr McGowan, F. Tenover, R.Gaynes, (1997): "Antimicrobial resistance in isolates from inpatients and outpatients in the United States: increasing importance of the intensive care unit &quot;, Clinic Infect Dis 24: 211-5). Nosocomial staphylococcal infections are associated with considerable morbidity and mortality, extending length of stay and increasing hospitalization costs. Most MRSA strains are resistant to several of the most commonly used antimicrobial agents, including macrolides, aminoglycosides, and β-lactam antibiotics currently in use, including the latest generation of cephalosporins.

Pathogens acquired in hospitals that are resistant to vancomycin responsible for infections (such as endocarditis, meningitis, and septicemia) are placing a growing therapeutic challenge (Y. Cetinkaya, P. Falk and CG Mayhall, (2000): "Vancomycin-resistant enterococci" , Clin Microbiol Rev. 13: 686-707, LB Rice, (2001): &quot; Emergence of vancomycin-resistant enterococci &quot;. Infection Dis. 7: 183-7). S. pneumoniae and M. catarrhalis are recognized as important pathogens of humans. They are a common cause of respiratory tract infections, especially otitis media in children and lower respiratory tract infections in the elderly. M. catarrhalis and S. pneumoniae have recently been recognized as the most common pathogens of the respiratory tract (MC Enright and H. McKenzy, (1997): "Moraxella (Branhamella) catarrhalis.") Clinical and molecular aspect of a rediscovered pathogen Med Microbiol 46: 360-71).

Clostridia are responsible for different diseases: gas gangrene and related wound infections, tetanus, botulism, antibiotic-associated diarrhea (CDAD) and pseudomembranous colitis. Most of these microorganisms produce exotoxins that play an important role in the pathogenesis of diseases. C. difficile is the causative agent responsible for 25% of cases of CDAD and for almost all cases of pseudomembranous colitis. In recent years the occurrence of C. difficile co-infection has been observed in patients with vancomycin-resistant enterococci infection or colonization (JG Bartlett, (1992): &quot; Antibiotic associated diarrhea &quot;, Clinic Infect Dis 15: 573-581). IN VITRO BIOLOGICAL ACTIVITY OF THE AI AND A2 FACTORS (OUTSIDE THE FIELD OF THE INVENTION) OF ANTIBIOTIC 107891. Table VIII describes the antimicrobial activities of individual Factors AI and A2 (outside the scope of the present invention), which are outside the scope of the present invention of antibiotic 107891. MICs were determined by microcatch dilution method as described above.

Table VIII: Antimicrobial Activity of Factors AI and A2 (outside the scope of the present invention) of antibiotic 107891

Micro-organism MIC (pg / ml) Factor AI Factor A2 819 Staph. Aureus Met-S &lt; 0.03 &lt; 0.03 1524 Staph. Aureus Met-R &lt; 0.03 &lt; 0.03 2235 Staph. Aureus Met-R0,060,096 3894 Staph. Epidermidis Met-R &lt; 0.03 0.06 3881 Staph. Epidermidis Met-R0.06 &lt; 0.03602 Staph. Haemolyticus Met-R 0.25 0.25 3919 Strept. Pneumoniae Pen-R? 0.015 &lt; 0.015 3915 Strept. Pneumoniae Pen-S &lt; 0.015 &lt; 0.015 4323 Ent. Faecalis Van-A &lt; 0.03 &lt; 0.03 J1 Ent. Faecalis Van-A 1 1 4341 Ent. Faecalis Van-B 0.5 0, 5 4397 Ent. Faecalis Van-B 1 1 4341 Ent. Faecalis Van-B 2 2 6349 Ent. Faecium Van-A Lnz-R 2 2 44 4 Ent. Faecium Van-A 1 1 3 Ent. Faecium Van-A 0.55, 5 D561 Ent. Faecium Van-A 2 2 A8 Ent. Faecium Van-A 0.5 0.5439 Ent. Faecium Van-D 0.25 0.25 4174 Ent.gallinarum 1 1 997 Neisseria gonorrhaee 0.5 0.25 1613 Neisseria meningitidis 0.25 0.25 1016 Propionibacterium acnes &lt; 0.03-0.06 IN VIVO BIOLOGICAL ACTIVITY ANTIBIOTIC 107891

Female ICR mice (Harlan Italia SpA, Pietro al Natisone, Italy) weighing between 23 and 25 g were used in acute lethal infection experiments in immunocompetent or neutropenic mice. Neutropenia was induced by two intraperitoneal administrations of cyclophosphamide, 200 and 100 mg / kg, on four days and one day, respectively, before the mice were infected. Infection was induced by intraperitoneal inoculation in immunocompetent mice (8 animals / dose / treatment group) of a bacterial suspension of any clinical isolate of methicillin resistant staphylococci (Staph. Aureus SA3817) or of a standard methicillin susceptible strain (Staph aureus Smith ATCC19636), or by inoculation in neutropenic mice from a clinical isolate of glycopeptide-resistant enterococci (Ent faecalis A533). Bacterial (&quot; bacterial challenges &quot;) stimulation (about 106 cells / mouse) was administered suspended in 0.5 ml of bacterial mucin 5% (Difco). Untreated animals died within 24 to 72 hours after infection. Antibiotic treatment began within 10-15 min after challenge (&quot; challenge &quot;). Antibiotic 107891 was once administered intravenously or subcutaneously in different aqueous formulations. The 50% effective dose (ED 50) and 95% confidence limits were calculated by the Spearman-Karber method (DJ Finney, (1952): &quot; The Spearman-Karber method &quot;, in: Statistical methods in biological assay, pp. 524-530,

Charles Griffin &amp; Co., Ltd., London) from the percentage of surviving animals on the seventh day. The results are shown in Table IX below. Antibiotic 107891 is non-toxic up to the maximum test dose of 200 mg / kg.

Table IX DE5o of antibiotic 107891 in acute lethal infections in rats

Formulation | Strain | Route I 50 mg / kg | Confidence limits 95% A MSSA iv 2.1 1.7-2.7 SC 2.1 1.7-2.7 A VanA iv 3.2 2.7-3.9 SC 11.1 9.2- 13.5 B MRSA SC 4.2 3.5-5.1 C VanA iv 3.7 2.8-4.9 SC 12.7 10.7-15.0

Formulations: A: 10% (v / v) DMSO, 10% (w / v) beta-hydroxypropyl cyclodextrin (Sigma), 80% (v / v) 5% (w / v) glucose in H2O B: 10% (v / v) DMSO, 40% (v / v) PEG 400 in 0.1 M aqueous CH 3 COOH C: 50% (v / v) PEG 400 in H 2 O

Strains: I MSSA: Staph. aureus ATCC19636 Smith 819 II MRSA Staph. aureus 3817, isolated clinic III. VanA: Ent. faecalis A533, clinical isolate, in neutropenic mice 46

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A (low resolution spectrum developed in the &quot; full scan &quot; mode) and Fig. 1B (high resolution spectrum developed in &quot; zoom-scan &quot; mode) represent mass spectra of antibiotic 107891 which exhibits a double-protonated ion in m / z 1124 at / z 1116. FIG. 2 depicts the absorption spectrum at IR of antibiotic 107891 dispersed in KBr. FIG. 3 depicts the UV spectrum of antibiotic 107891 dissolved in methanol: H2 0. FIG. 4 represents the 1 H-NMR spectrum recorded in the 40:10 (v / v) methanol-d 4: H 2 O (40: 3 HCl) mixture at 40øC on a Bruker AMX 600 spectrometer using a Water.

FIG. 5 represents the13 C-NMR spectrum recorded in the 40:10 (v / v) methanol-d4: H20 (pH 4.3 HCl) mixture at 40øC on a Bruker AMX 600 spectrometer. FIG.6A (low resolution spectrum developed in &quot; full scan &quot;) and 6B (high resolution spectrum developed in &quot; zoom-scan &quot; mode) represent mass spectra of antibiotic 107891 Factor AI (outside the scope of present invention) having doubly protonated ions [M + 2H] 2+ am / z 1124. FIG. 7A (low resolution spectrum developed in the &quot; full scan &quot; mode) and 7B (high resolution spectrum developed in &quot; zoom-scan &quot; mode) represent mass spectrums of Factor A2 of antibiotic 107891 (outside the scope of the present invention ) showing double-protonated ions [M + 2H] 2+ am / z 1116. FIG. 8 depicts the 1 H-NMR spectrum of the Antibody I-Factor 107891 (outside the scope of the present invention) recorded in the CD3 CN: : 1) at 288 K on a Bruker AMX 600 spectrometer using a water suppression sequence. 9 shows the 1 H-NMR spectrum of Factor A2 of antibiotic 107891 (outside the scope of the present invention) recorded in the CD3CN: D20 (1: 1) mixture at 298 K on a Bruker AMX 600 spectrometer using a sequence of water suppression. FIG. 10 represents the13 C-NMR spectrum of Factor A1 of antibiotic 107891 (outside the scope of the present invention) recorded in the CD3 CN: D2 O (1: 1) mixture at 298 K on a Bruker AMX 600 spectrometer. FIG. 11 represents the13 C-NMR spectrum of Factor A2 of antibiotic 107891 (outside the scope of the present invention) recorded in the CD3 CN: D20 (1: 1) mixture at 298 K on a Bruker AMX 600 spectrometer. FIG. 12 represents the absorption spectrum in the Factor A1 of the antibiotic 107891 (outside the scope of the present invention) dispersed in KBr. FIG. 13 represents the UV spectrum of Factor A1 of antibiotic 107891 (outside the scope of the present invention) dissolved in methanol: H2 O. Fig. 14 depicts the absorption spectrum in Factor A2 of Antibiotic 107891 (outside the scope of the present invention) dispersed in KBr. 15 shows the UV spectrum of Factor A2 of antibiotic 101891 (outside the scope of the present invention) dissolved in methanol: H2 0.

EXAMPLES

Example 1: Method of fermentation of Microbispora sp. ATCC PTA-5024

The strain Microbispora sp. ATCC PTA-5024 in tubes (&quot; slants &quot;) containing oatmeal agar medium for 2-3 weeks at 28 ° C. The microbial content of a tube was scraped with 5 ml of sterile water and inoculated into 500 ml Erlenmeyers containing 100 ml of sowing medium (AF / MS) which is composed of (g / l): dextrose 20 , yeast extract 2, soybean meal 8, NaCl 1 and calcium carbonate 4. The medium was prepared with distilled water and the pH was adjusted to 7.3 before sterilization at 121øC for 20 minutes. The inoculated containers were grown at 28øC on a rotary shaker operating at 200 rpm. After 4 to 6 days, 5% of this culture was inoculated into a second series of containers containing the same fermentation medium. Seventy-two hours after incubation, 200 ml was transferred to a 4 1 bioreactor containing 3 l of the same vegetative medium. The fermentation was carried out at 30øC with stirring at 700 rpm and ventilation at 0.5 vvm (&quot; Gas volume per reactor volume per minute &quot;). After 72 hours the culture (1.5 L) was transferred to a 20 1 49 bioreactor containing 15 l of the same vegetative medium. The fermentation was carried out for 48 hours at 30 ° C, under stirring at 500 rpm and ventilation of 0.5 vvm and then transferred to the production tank. Production of antibiotic 107891 was carried out in a 300 L fermentor containing 200 l of M8 production medium composed of (g / l): starch 20, glucose 10, yeast extract 2, hydrolyzed casein 4, meat extract 2 and calcium carbonate 3. The medium was prepared in deionized water and the pH was adjusted to 7.2 before sterilization at 121 ° C for 25 min. After cooling the fermenter was inoculated with approximately 141 (7%) preculture. The fermentation was carried out at 29 ° C at 180 rpm of stirring and ventilation at 0.5 vvm with a head pressure of 0.36 bar. After 98 hours of fermentation, the fermenter was harvested. Production of antibiotic 107891 was monitored by HPLC as described above, after extraction of the whole culture broth with the same volume of methanol. Extraction was carried out at room temperature under stirring for one hour.

Example 2: Alternative Fermentation Method of Microbispora sp. ATCC PTA-5024

In 500 ml Erlenmeyers containing 100 ml of growth medium (Gl) consisting of (g / l): glucose 10, maltose 10, soybean oil 10, soybean meal 8, yeast extract 2 and calcium carbonate 4, if Microbispora sp. ATCC PTA-5024. The medium was prepared in deionized water and sterilized at 120 ° C x 20 minutes without adjustment of pH. The inoculated containers were incubated for 120-168 hours at 28øC under agitation at 50-200 rpm until a good growth was observed. The containers were then used to inoculate (3%) a 4 1 bioreactor containing 3 L of AF / MS sowing medium, which is composed as described in Example 1. After 120 hours of fermentation at 30øC, stirring at 700 rpm and 0.5 vvm ventilation, 1.5 l of culture was transferred to a 20 l bioreactor containing 15 l of the same vegetative medium. The fermentation was conducted for 96 hours at 30øC, shaking at 600 rpm and ventilation at 0.5 vvm, and then transferred to the production tank. The production of the antibiotic was obtained in a 300 l fermenter containing 200 l of the production medium (v6) consisting of (g / l): dextrose 20, yeast extract 5, meat extract 5, blockhouse hydrolyzate 3, peptone 5 and NaCl 1.5. The medium was prepared in deionized water at a pH adjusted to 7.5 with NaOH, and sterilized at 121 ° C for 20 min.

The fermentor was inoculated with 14 L of seed culture (7%) and the fermentation was carried out at 29øC, stirred at 180 rpm and vented with 100 l of conventional air per minute (0, 5 vvm). Production of antibiotic 107891 was monitored by HPLC as described above. The fermentation was collected after about 160 hours.

Example 3 Recovery of antibiotic 107891

The fermentation broth described in Example 1 was filtered through a tangential filtration system (pore membrane 0.1 μm in size, Koch Carbo-COR, Koch Wilmington, USA) to obtain 17β of supernatant and 30 1 of concentrated mycelium. Antibiotic complex 107891 was found in both the filtrate (A) and the mycelium (B). (A) The filtered broth was stirred overnight at room temperature in the presence of Diaion HP-20 polystyrene resin (4 1). The resin was then recovered, washed with 10 l of 4: 6 (v / v) methanol: water and eluted initially with 10 l of methanol: water 9: 1 (v / v) and then with 10 l of methanol: butanol: water: 9: 1: 1 (v / v). The pooled eluate fractions containing antibiotic 107891 were concentrated to a small volume on a rotary evaporator and then lyophilized to give 32 g of crude material. This crude material was dissolved in n-butanol (11) and extracted three times sequentially with 800 ml of water. The organic layer was concentrated under reduced pressure to an oily residue, which was dissolved in methanol. After the addition of petroleum ether, 5 g of a crude antibiotic preparation was obtained by precipitation. (B) After the addition of 25 l of methanol, the portion of retained material containing the mycelium was stirred for 1 hour and filtered to give 45 l of the extract of the mycelium. This solution was then diluted with water (20 L) and stirred overnight at room temperature with Diaion HP-20 (11) polystyrene resin. The resin was then recovered, washed with 2 l of 40:60 (v / v) methanol: water and eluted discontinuously with 3 l of 85:15 (v / v) methanol: water and then with 2 l of methanol: water 90:10 (v / v). Fractions eluted for the presence of antibiotic 107891 were monitored by agar diffusion assay with Staphylococcus aureus and using an analytical HPLC method as previously described. 52

The eluted fractions containing antibiotic 107891 pooled, concentrated under reduced pressure and lyophilized, yielding 8.1 grams of the crude antibiotic 107891.

Example 4: Alternative Recovery of Antibiotic 107891 The broth collected from the fermentation of the 200 L tank described in Example 2 was brought to pH 6.8 and the broth was filtered by tangential filtration (membrane with pore size 0, 1 μ, Koch Carbo-Co). The permeate (180 L) was stirred overnight at room temperature with 21 of Diaion HP20 resin (Mitsubishi Chemical) and then the resin was collected.

Methanol (25 L) was added to the portion retained in the tangential filtration apparatus (about 20 L) containing the concentrated mycelium. This suspension was stirred for 1 hour and then filtered with the microfiltration system to a residual volume retained of about 20 l. Further methanol (25 l) was then added and the above procedure sequentially repeated for a total of 5 cycles. The combined extracts of methanol (about 125 L) were diluted with 160 l of demineralized water and discontinuously stirred overnight at room temperature with 3 l of Diaion HP 20 resin. The resin was then collected, and added to the Diaion HP 20 resin used to extract the permeate from the broth according to the process described above. The pooled resin was washed on a chromatographic column with 20 L of 6: 4 (v / v) water: methanol. Antibiotic 107891 was eluted with 23 l of methanol: 50 mM ammonium formate buffer pH 3.5: n-butanol 9: 1: 1 (v / v). This eluate was then concentrated in vacuo to a final volume of 34 ° C. The concentrated solution was then compressed to pH 4.5 on a 53.5 1 column of polyamide CC 60.1-0.3 mm (Macherey -Nagel) packed with water: 7: 3 (v / v) methanol. The column was washed with 7: 3 (v / v) water: methanol and then with 25 mM ammonium formate buffer pH 3.5: methanol 7: 3 (v / v). The antibiotic was eluted with a 3: 7 (v / v) water: methanol mixture and then with 1: 9 (v / v). Elution was accomplished with 25 mM ammonium formate buffer pH 2.8: methanol in a ratio of 1: 9 (v / v). The eluates containing the antibiotic 107891 were pooled and concentrated in vacuo to give a final volume of 11. The pH of the concentrated solution was adjusted from 4 to 5.7 with 7M ammonium hydroxide and then centrifuged the mixture to collect the precipitate. This solid was suspended in water and freeze-dried to give 6.96 g of the preparation containing antibiotic 107891.

Example 5: Purification of antibiotic 107891 The crude antibiotic 107891 (3.6 g), prepared as described in Example 3, was purified by medium pressure chromatography on 100 g of C8 (EC) reverse phase with particles of dimensions between 40 and 70 pm, pore size 60A, IST (International Sorbent Technology, Mid-Glamorgan, UK) using a Buchi B-680 medium pressure chromatography system (Buchi laboratoriums technik AG, Flawil Switzerland) equipped with B-687 gradient, fractions B-684 manifold, B-685 70 X 460 mm glass column. The resin was preconditioned with a phase A: phase B 8: 2 (v / v) mixture and then eluted at 25 ml / min with linear gradient over 60 minutes from 20% to 60% of phase B over 60 minutes .

As phase A was used acetonitrile: 20 mM ammonium formate buffer (pH 6.6) 10:90 (v / v); and acetonitrile: 20 mM ammonium formate buffer (pH: 6, 6) 90:10 (v / v) was used as phase B 54.

Fractions containing antibiotic 107891 were pooled, concentrated in vacuo, and lyophilized twice from water to give 430 mg of the purified antibiotic 107891.

Antibiotic 107891 was further purified by preparative HPLC on a LiChrosorb RP8 column previously containing Hibar (particle size 7: m) RT 250-25 mm, Merck, using a linear gradient elution of 25 minutes from 30% to 45% of Step B at a flow rate of 30 ml / min. As phase A was used 25 mM ammonium formate buffer pH 4.5: 95: 5 (v / v) acetonitrile and acetonitrile was used as phase B.

A sample of antibiotic 107891 from Example 5 (300 mg) was dissolved in 1.5 ml 350: 1 of DMSO: formic acid 95: 5 (v / v) and 300 μ tratar was treated by chromatographic assay. Antibiotic 107891 was eluted characteristically in 15-16 minutes. The fractions eluted from 5 chromatographic assays containing the antibiotic 107891 were pooled, and concentrated in vacuo. The residual solution was lyophilized three times sequentially from the water, yielding 31 mg of antibiotic 107891 as a white powder.

Example 7: Separation and Purification of Individual Factors Al and A2 from Antibiotic 107891

Factors A1 and A2 (outside the scope of the present invention) were separated and purified from the antibiotic 107891 complex of Example 5 by preparative HPLC on a Symmetry Prep C18 (particle size 7 pm) column 7.8x300 mm Waters (Mildfold USA) using two different elution programs. A) Factor AI (not included within the scope of the present invention) was purified by a 25 minute linear gradient elution from 30% to 45% Phase B at a flow rate of 3.5 ml. Phase A was a 25 mM ammonium formate buffer pH 4.5: 95: 5 (v / v) acetonitrile and the B phase was acetonitrile. The purified antibiotic 107891 complex (15 mg) was dissolved in 350 μΐ of DMSO: formic acid 95: 5 (v / v) and subjected to a chromatographic assay. The factors AI and A2 were eluted normally within a period of 11-13 minutes. The fractions eluted by HPLC were then analyzed under the analytical conditions described above. Fractions from 14 chromatographic assays containing the AI factor (outside the scope of the present invention) of the pure antibiotic 107891 were pooled and concentrated in vacuo. The residual solution was lyophilized sequentially in water three times to give 15 mg pure factor AI (outside the scope of the present invention) as a white powder. B) Factor A2 (outside the scope of the present invention) was purified by isocratic elution to a 7 ml flow with 100 mM ammonium formate buffer pH 4: acetonitrile 82.5: 17.5 (v / v) . The purified antibiotic 107891 complex (5 mg) was dissolved in a 250 μΐ mixture of acetic acid: acetonitrile: 100 mM ammonium formate buffer pH 4 50: 120: 80 (v / v) and subjected to assay chromatographic. Factors AI and A2 (outside the scope of the present invention) were eluted normally over a period of 9-10 minutes. The eluted fractions were then analyzed by HPLC under the analytical conditions described above. The fractions from 20 chromatographic assays containing Factor A2 (outside the scope of the present invention) of the pure antibiotic 107891 were pooled, and concentrated in vacuo. The residual solution was lyophilized twice in water to give 8 mg pure factor A2 (outside the scope of the present invention) as a white powder.

Lisbon, October 10, 2012.

Claims (12)

A compound of formula in which X is selected from the group consisting of F, Cl atoms. Br, and I; and R 2, R 3, and R 4 are independently selected from the group consisting of H, OH and R 5, R 6, R 7 and R 8 are independently selected from the group consisting of H, OH, alkyl and aryl; the proviso that at least one of R3 to R8 is not H or X is not Cl when R2 is OH or R1 and R2 are OH. A compound according to claim 1, wherein R1 is H, R2 is H, and R3 is H, R4, R5, R6, R7, and R8 are H. A compound according to claim 1, wherein R1 is H, R2 is H, and R3 is OH, R4, R5, R6, R7, and R8 are H. A compound according to claim 1, wherein R1 is H, R2 is OH, and R3 is OH, R4, R5, R6, R7 and R8 are H. A compound according to claim 1, wherein R1 is OH, R2 is OH, and R3 is OH, R4, R4, R6, R7, and R8 are H. A compound according to claim 1, wherein R1 is OH, R2 is H, and R3 is H, R4, R5, R6, R7, and R8 are H. A compound according to claim 1, wherein R1 is OH, R2 is H, and R3 is OH, R4, R8, R6, R7, and R8 are H. A pharmaceutical composition comprising an antibiotic selected from the compounds of claim 1 or a pharmaceutically acceptable salt thereof derived from an acid. A pharmaceutical composition according to claim 8, further comprising a pharmaceutically acceptable carrier. A compound according to claim 1 or a pharmaceutically acceptable salt thereof derived from an acid for use as a medicament. The use of compounds according to claim 1 or pharmaceutically acceptable salts thereof derived from an acid for the preparation of a medicament for the treatment or prevention of bacterial infections. Use of compounds according to claim 1, and of the non-toxic salts thereof derived from an acid as animal growth promoters. Lisbon, October 10, 2012.