TW200409816A - Antibiotics GE 81112 factors A,B,B1, pharmaceutically acceptable salts and compositions, and use thereof - Google Patents

Abstract

The invention relates to an antibiotic substances of microbial origin, arbitrarily denominated antibiotic GE 81112 factor A, factor B1 and factor B, which is produced by fermentation of Streptomyces sp. DSMZ 14386, the pharmaceutically acceptable salts and compositions thereof, and their use as an antibacterial agent having inhibitory activity versus susceptible microbes.

Description

200409816 Look at the fiber surface-The present invention relates to an antibiotic substance of microbial origin, arbitrarily named GE81112 factor A, GE81112 factor B, and GE81112 factor B! These factors are mixtures in any ratio. The drug allows the salt and its composition , And its use as an antibacterial agent. Another object of the present invention is a method for preparing GE 81112 factor A, GE 81112 factor B, and GE 81112 factor B !. These factors are in a mixture of any ratio, and the following is to report GE 8 1 1 1 2 compounds or GE 81112 Antibiotic Name Report. Strains and Streptomyces spp. DSMZ 1 43 86 were originally expressed as internal code GE 81112, which were isolated from soil samples and deposited under the name of DSMZ on July 16, 2001 in accordance with the provisions of the Budapest Treaty (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany). This strain carries the accession number DSM Z 1 4 3 8 6. The manufacturing methods of GE 81112 compounds are Streptomyces strains capable of manufacturing these compounds, that is, Streptomyces D SMZ 1 43 8 6 or variants or mutants that maintain the genetic ability to produce these GE 81112 compounds; The obtained antibiotics are separated from the liquid culture medium; the isolated antibiotics are purified; and the three factors A, B, and Bι or the mixture thereof are separated by chromatography. All in all, the method for producing the preferred compound GE 8 1 1 12 is performed under aerobic conditions on an aqueous nutrient medium containing assimilable carbon, nitrogen and inorganic salt sources, and the resulting compound is purified by chromatography. A variety of nutrient media commonly used in fermentation can be used, but certain media are preferred. 6 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 Preferred carbon sources are glucose, xylose, starch, fructose, glycerol and the like. Preferred nitrogen sources are soy flour, protein age, meat extract, yeast extract, trypsin gland, amino acid, hydrolyzed casein, and the like. The inorganic salts that can be blended into the culture medium also include the soluble salts that are conventionally known to produce sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride anions, carbonate, sulfate, phosphate, nitrate and other ions. . Preferably, a strain capable of producing the G E 8 1 1 1 2 compound is cultured in a fermentation tube or a shake flask, and then the culture bacteria are used to inoculate the fermentation bottle to produce a large amount of fermentative substances. The medium used for the pre-cultivation may be the same as that used for the mass fermentation, but other mediums may be used. The germline that produces the GE81112 compound can be grown at a temperature of 20 ° C to 40 ° C, preferably 221 to 37 t. No growth was seen above 4 4 ° C. GE 81 1 12 factor can be monitored during bioassay analysis and / or HPLC analysis of sensitive microorganisms during fermentation. The maximum yield of the GE 81 1 12 compound usually occurs after about 20 hours to 80 hours of fermentation. The compound GE 81 1 12 was produced through the culture of Streptomyces sp. DSMZ 1 43 86 or a mutant or mutant strain thereof capable of producing the compound GE 8 1 1 12 and the compound was produced in a liquid medium. Characteristics of Streptomyces D S M Z 1 4 3 8 6 The characteristics of ursinus streptomyces DSMZ 1 4 3 8 6 grow well in a variety of standard soil media. Microscopic examination and cell size determination using cultured bacteria grown on a tenth-strength humic acid medium (Η. Ν ο η 〇mura, 1 9 8 4-D esig η of a new medium for isolation of soil acti η omycete s. The Actinomy cetes 18 · 2 0 6-209) o In liquid culture (V6 medium, composition reference example 1), no mycelial debris was observed after 2 days of growth at 28 t. 312 / Inventory (Supplement) / 92-02 / 91135061 200409816 Microscopic examination of HV / 2 agar after 15 days incubation at 28 ° C revealed vegetative mycelia that were neither broken nor broadly branched. Aerobic mycelia are also not extensively branched. Aerobic mycelia produce chains containing more than 10 (mostly more than 20) spores; arranged in simple but non-tight 3 to 5 loop spirals. The spore shape includes spheres (average diameter of about 0.9 microns) to oval or cylindrical (0.8 to 0.9 microns x 1.1 to 1.3 microns). _ Yucca DSMZ 143S (culture characteristic of S. streptococcus DSMZ 1 43 86) was grown in V6 liquid medium (medium composition reference example 1) for 2 days. Mycelia were harvested by centrifugation, washed with sterile saline 3 times, and then diluted to obtain Appropriate inoculum. Suspension was cross-hatched and cultured on various media recommended by Shirling and Gottlieb (EB Shirling and D. Gottlieb, 1 966-Method for Characterization of S treptomy ces species-Int. J. Syst. Bacteriol ., 16., 313-340) and Waksen's medium recommended by Waksman (Waksman S.A., 19 61-The Actinomycetes-The Williams and Wilkins Co., Baltimore · Vol 2_, PP 328-334). Use a variety of carbon water The ability of compounds to act as a carbon source and energy source was determined in an IS P8 medium (Shirling and Gottlieb, same article), which contains a carbon source at a final concentration of 1% (w / v). Sodium chloride tolerance and ability to grow at different temperatures It is measured on IS P2 medium. All mediums are inoculated at 28 ° C for 21 days; unless otherwise stated, the explanation here refers to the 14th. Use the color map of Mae ι and Paul under natural light. Evaluating color under natural light (A. Maerz and MR Paul, 1950-A Dictionary of Colour, 2nd edition · McGraw-Hill Book Co. Inc ·, New York). The ability to reduce nitrate to nitrite is at 2 8 ° C After overnight cultivation in aerobic conditions 8 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 Bacto nitrite test strips were used (Dihan followed the manufacturer's method to grade the nitrate liquid culture. The growth of Streptomyces DS Μ Z 1 4 3 8 6 strains, the production of aerobic mycelia, and the production of pigments have recorded that growth of nutrients has occurred, with the exception of G 1 agar, G 1 nitrogen source medium. Any The culture media used are all calm. The physiological characteristics of this line are shown in Table 1 4 3 8 6 shows that it can grow to 7% (w / v) sodium chloride; at the above concentrations, the aerobic mycelium of this strain is not The ability of gelatin liquefaction and milk protein peptidation to grow only weak carbohydrates is shown in Table III. The basal medium was observed, and the vegetative mycelium was limited. Except when all carbon sources used grow white. 312 / Invention Specification (Supplement) / 92-02 / 91135061 I (Difco), culture medium (ISP8 medium), appearance, matrix, and Table I. In all cultures, the agar is the only one that contains inorganic pigments and does not show characteristic pigment II. Streptomyces DSMZ 1% (w / v) sodium chloride and differentiation. Detected after 7 days of growth. Utilizing a variety of aerobic mycelium growth without adding any carbon source Aerobic mycelium, glucose 9 Table I: Growth characteristics of Streptomyces DSMZ 14386

Medium Growth and Morphology Inverse Color Code ISP 2 Yeast Extract-Malt Extract Agar Growing densely, circling, becoming crusty near the end of the streak culture; orange / brown; discoloration of growth edges. White aerobic mycelium clusters. No soluble pigments were produced. Thick earthy flavor. 12L9 ISP3 oatmeal agar thick and smooth with smooth surface; cream yellow. A thin layer of white aerobic mycelia appears only near the growth edge. No soluble pigments were produced. 11H3 ISP 4 inorganic salt-starch agar grows densely, spirals, and becomes crusty near the end of the streak culture; yellow. White aerobic mycelia appear, fluffy. No soluble pigments were made. Thick earthy flavor. 10F3 ISP5 glycerol-asparagine agar Separately grown to good growth; colorless to pale yellow; feathery and faded at the edges. Fine white mycelia appeared and turned pinkish (10A2) with aging. No soluble pigment was produced. 10G2 ISP 6 protein difficulty-yeast extract-iron agar Separate growth, wrinkling near the end of the streak culture; small colony diameter; less expansion from the streak area; sticky; light brown. No aerobic mycelium was produced. Mycelium is slightly darker. 11 J3 ISP7 tyrosine agar grows densely, spirals, and becomes crusty near the end of the culture line; brown; feathery and faded at the edges. The production of white aerobic mycelia is good and powdery. A brown soluble pigment was produced. Light earthy taste. 14L9 SYE starch lion extract agar grows densely circling, becomes hard crust near the end of the streak culture; yellow-brown; broad-edged, feathery and faded at the edges. Limited growth of white sparse aerobic mycelia. No soluble pigments were produced. Light earthy taste. 10L7 CZ-GLU Czapeck-glucose agar Separately grown to good growth, producing crusts near the end of the streak culture; small colony diameter; opalescent-light brown: feathered edges. Growth of white sparse aerobic mycelia is restricted. No soluble pigments were produced. Light earthy taste. 12H6 GAUZE 1 agar does not grow • GAUZE 2 agar grows well, the end of the streak culture is sparse and wrinkled and crusty, the intersection of the streak culture is flat; yellow / brown. Sparse white aerobic mycelia rarely occur in flat areas. Mycelium is slightly darker. 11L6 NA Nutritional Agar Separately grows well to grow 'smooth; creamy yellow. No aerobic mycelium was produced. No soluble pigments were produced. 11L2 SE Soil Extract Agar There are almost no vegetative mycelia visible by sight; colorless; rarely spread by streaking. No aerobic mycelium was produced "• No soluble pigment was produced. ND HV / 2W straight acid · vitamin agar The growth of vegetative mycelia is good, penetrating deep into the agar; dark brown: feathery edges. Presence of white aerobic mycelia, powdery. ND 312 / Invention Specification (Supplement) / 92-02 / 91135061 10 200409816 Physiological characteristics of DSMZ 1 4 3 8 6 ------- Test reaction starch hydrolysis ~ -------- Positive casein hydrolysis negative malate Calcium digestion positive litmus milk protein positive (weak) litmus milk coagulation negative gelatin liquefaction positive (weak) tyrosine reaction positive than ISP6 hydrogen sulfide production negative (4th): + acetic acid test paper weak positive nitrate Reduction-negative sodium chloride tolerance ^ 7% Table III: Streptomyces DS M Z 1 4 3 8 6 Use of carbon source Carbon source nutrition Mycelium growth Aerobic mycelium growth Arabinose + + /-Fructose + + + + Inositol + + /-mannitol + + + raffinose + + + rhamnose + + + + sucrose + /-xylose + + + + glucose + + + + + good growth; + growing Degree; -Deficiency / No growth 312 / Invention specification (Supplement) / 92-02 / 91135061 200409816 § Chemical classification characteristics of Trichoderma DSMZ 1 4 3 8 6 Streptomyces DS MH Z 1 4 3 8 6 grows in sand (S aut ο η) medium for 4 weeks, then mycelia were harvested, the mycelia were washed three times with sterile distilled water and then Lyophilized. The stereoisomeric form of diaminopimelic acid (DAP) is determined according to the methods of Staneck and Roberts (JL Staneck and GD Roberts, Simplified approach to identification of aerobic actinomy cetes by thin-layer chromatography, Appl. Microbiol. Plex, 226 -23 1, 1, 9 7 4). In order to extract fatty acids, the wet biomass block uses the Miller method (LT Miller, A single deri vatization method for bacterial fatty acid methyl esters including hydroxy acids, J. Clin. Microbiol., 5 84 -5 8 6, 1 98 2). Extraction with slight modification (LD · Kuykendall, MA Roy, JJ〇'Neill and TE Devine, Fatty acid, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium j aponicium, Int. J. System. Bact. 38_, 3 5 1 -3 6 1, 1 9 8 8). Analytical systems such as R.M.

Performed as described by Kroppenstedt (RM Kroppenstedt, E. Stackebrandt and M. Goodfellow, Taxonomic revision of the actinomycete genera Actinomadura and Microtetraspora, System. Appl. Microbiol. J_i, 1 48- 1 60, 1 990), using a microbiological identification system Inspection (LT. Miller, same text). The Streptomyces DSMZ 1 43 8 6 strain contains LL-diaminopimelate and peptidoglycan, and does not contain mycolic acid. Iso- and anti-iso-branched fatty acids were present, and no hydroxy fatty acid or 10-methyl-branched fatty acid was detected, that is, fatty acid type 2c streptomyces. 12 312 / Instruction of the Invention (Supplement) / 92-02 / 91135061 200409816 Identification of the Streptomyces DS M Z 1 4 3 8 6 Strain that can produce the compound GE 8 1 1 1 2 is named on the chain because it has the following chemical characteristics Mycelia:-Vegetative mycelia that form branches but are not segmented. 10 aerobic hyphae (mostly > 20) arthrospore chains;-According to Kroppenstedt, there are LL · diamine and 2c fatty acid side pictures on the cell wall (RM Kroppenstedt, Fatty menaquinone analysis of actinomy cetes and organisms, pp .1 7 3-199, in: Chemical Methods in Systematics, M. Goodfellow and DE Minnikin eds Accademic Press, 1985). Like other microorganisms, the bacteria that make GE81112 compounds have changed. For example, artificial mutants and mutants of this strain can be obtained by treating various mutants such as ultraviolet light and treatments such as nitrous acid, N-methyl-N-nitrosoguanidine, and various other chemicals. All natural and artificial variants and mutants of the strand 1 4 3 8 6 which contain the genetic ability of the ΐ 8 111 2 compound are considered to be equivalent to the present invention and thus fall within the scope of the present invention. Antibiotics can be recovered from the surface portion of the fermentation broth. Extraction of GE 8 1 1 1 2 compounds and pure GE 8 1 1 1 2 compounds are performed by known techniques for the recovery of fermentation broths that can produce microorganisms. Known recovery techniques such as extraction in the presence of adjuvants, by adding non-solvents, PH precipitation of salt or changing solution, operation, reversed-phase separation chromatography, ion exchange chromatography, molecular row, etc. 312 / Description of the Invention (Supplement) / 92-02 / 91135061 Morphology and production of acid and related bacterial greater than pimelic acid; London, Strain characteristics are also made by the use of -N'nitro bacteria DSMZ Ding for the purpose of making GE The procedure for removing chromatography according to the used solvent extraction sorting chromatography 13 200409816 to recover the antibiotic substance of the present invention from the fermentation broth includes the extraction of GE 8 1 11 2 by ion exchange chromatography. According to this procedure, the fermentation broth after the furnace can be contacted with the ion exchange resin. Elution can be performed by changing the pH or ion intensity. Ion exchange resins that can be conveniently used to recover the compounds of the present invention are, for example, anionic and cationic ion exchange resins, that is, synthetic polymers (such as polystyrene, acrylic polymers) or natural polymers (such as cellulose , Sephar ose polymer, dioxide, etc. The matrix is introduced by acid (such as COOH, -SChH) or test (such as _n (CH3) 2, -n + (ch3) 3) Functional group derived resin. The aforementioned resins are, for example, Dowex 50W resin (Dow Chemical Co.), Amberlite IR-200 and IR-120 (Rohm & Haas), PL-SCX 1000 and APL-SCX 4000 A (Polymer Laboratories), Isolute SCX IST (International Sorbent Technology), SP Westfaros Fast Flow and SP Sephadex G25 (Pharmacia Biotech). Another procedure for the recovery of antibiotic substances involves adsorption onto a suitable substrate, followed by elution with a polar water-miscible solvent or a mixture thereof, concentration under reduced pressure to form a water residue, and precipitation using the aforementioned type of precipitant. The pH can be adjusted as appropriate. Adsorption matrices that can be conveniently used to recover the compounds of the present invention are, for example, activated carbon (such as Darco G60, Norit CG1), polystyrene, or mixed polystyrene-divinylbenzene resins (such as Dyne ( Diaion) HP20, Mitsui Chemicals Corporation; M112 or S112 The Dow Chemical Company; Amberlite XAD2 or XAD4, Rohm and Haas Corporation), acrylic resins (such as XAD7 or XAD8, Rohm and Haas Corporation), phenolic adsorbents ( Example 14 312 / Explanation of the Invention (Supplement) / 92-02 / 91135061 200409816 Such as DuolUe X AD 761 (Rohm and Haas Company), polyamide resins such as polycaprolactam, nylon and cross-linked polyethylene Pyridoxone (eg Polyamide-CC 6, Polyamide-SC 6'Polyamine-CC 6.6, Polyamide-CC 6AC and Polyamide-SC 6AC, Macherey-Nagel ) Company, West Germany; PA400, M. Woelm AG AG, West Germany; PVP-CL, Aldrich Chemical Company, West Germany, cross-linked glucosan (such as West France Days LH-20, Famacia Fine Chemicals). Activated carbon and / or polystyrene resins are commonly used, particularly as Dane HP 20 resin (Mitsui Chemical Co., Ltd.). Adsorption on the aforementioned types of adsorption matrices can also use ion (e.g. cation) exchange chromatography as a procedure to purify the crude product recovered from the fermentation broth. It is preferred that the solvent for the elution of the GE 81 1 12 compound by the adsorption matrix is determined based on the specific stationary phase. When activated carbon is used, the elution is achieved by changing to an acidic pH elution buffer, which typically contains a water-miscible solvent such as a lower ketone such as acetone or a lower alcohol such as methanol. In this way, the aqueous mixture is adjusted to an appropriate pH. When polystyrene resin, polystyrene-divinylbenzene resin, acrylic resin or polyamide resin is used, the eluent is preferably a water-miscible solvent or an aqueous mixture thereof such as water and (Cl-C3) alkanol. Of a mixture. The term "water-miscible solvent" used in this case is intended to indicate the current definition of the word in the industry when not specifically stated, meaning that a solvent can be miscible with water in a reasonably wide concentration range under the conditions of use and is suitable for anticipation use. Water-miscible effective solvents that can be used to elute the compounds of the present invention are, for example, lower alkanols such as (ChCd alcohols such as methanol, ethanol, and propanol; phenyl (Ci-Cd alkanols such as benzyl alcohol; lower ketones such as ( C3-C4) ketones such as acetone and ethyl methyl ketone; cyclic ethers such as di # alkane and tetrahydrofuran; glycols and partial etherification products thereof such as 15 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 Ethylene glycol, propylene glycol, and ethylene glycol monomethyl ether; low-carbon ammonium amines such as dimethylformamide and diethylformamide; acetic acid, dimethylarsox, and acetonitrile. The method for recovering the GE 81 1 12 compound is to extract the GE 8 1 1 1 2 compound or a salt thereof using a water-miscible organic solvent, and adjust the pH to an appropriate pH value during the extraction, and / or via salting, and then add the The antibiotic dissolved in the extraction solvent forms an appropriate organic salt of the ion pair. The antibiotic is then precipitated from the concentrated extract by, for example, adding a precipitant. The term "water-immiscible solvent" used in this case is intended to have the definition currently used in the industry, indicating ~ Solvents are slightly miscible or practical under the conditions of use Miscible in water. Water-immiscible organic solvents that can be used to extract the compounds of the present invention from fermentation broth. For example, alkanols containing at least 4 carbon atoms can be linear, branched or cyclic such as n-butanol, 1-pentanol, 2- Amyl alcohol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, 3,3-dimethyl-1-butanol, 4-methyl-1 · pentanol, 3 · methyl 1-pentyl alcohol, 2,2-dimethyl-3-pentanol, 2, cardiac dimethyl-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-cyclopentylethanol, 3-cyclopentyl-1-propanol, cyclohexanol, cycloheptanol, 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 containing at least 5 carbon atoms, such as methyl isopropyl ketone, methyl isobutyl ketone, methyl n-pentyl ketone, methyl isoamyl ketone and mixtures thereof. With GE 8 1 1 1 2 Convenient ion pairs formed by compounds are obtained, for example, by the addition of an acid at the appropriate pH. Thus, for example, sulfonic acid is represented by p-toluenesulfonic acid, pentanesulfonic acid, hexanesulfonic acid, heptanesulfonic acid, or the like; or carboxylic acid is represented by hexanoic acid, heptanoic acid, benzoic acid, etc. In addition, the ion pair may be passed Example 3 of adding cations / Instruction of the invention (Supplement) / 92-〇2 / 91135061 200409816 It is produced at the appropriate pH such as triethylcopper, tetrabutylcopper, etc. The purification of crude GE 8 1 1 1 2 compound can be borrowed. Known technology is achieved, but it is preferably performed using chromatography. Conveniently, so-called stereo exclusion chromatography can also be used to obtain good results. In particular, this technique usually uses cross-linked glucosan with controlled porosity, most of which have been alkylated, such as Sifa Days LH-2C (Famacia Fine Chemicals). For example, a medium pressure liquid chromatography separation system can be used to perform reverse-phase chromatography on R P · 8 or R P -1 8 functionalized silica gel and elute with ammonium formate solution. The crude product and purified product separated from the fermentation broth depends on the fermentation and purification conditions, and usually contains unequal proportions of a single GE 81112 factor human, 31 and B mixture. The separation and purification of the individual factors A, B, and B! Can be conveniently performed by semi-preparative HPLC using a formulation containing the GE 8 1 1 12 compound mixture. The preparative HPLC technique, which is preferably used for the separation of pure GE 8 1 1 1 2 factors A, B! And B, is performed on a semi-preparative HPLC instrument (Shimadzu-LC8A) equipped with Hibar Lichrosorb (Merck) 25 x 250 mm column RP 8 (7 micron particle size), using 40 mM ammonium formate and adjusting to pH 4.5 with formic acid, eluting at a flow rate of 35 ml / min. The repeated chromatography elution products containing the isolated GE 81112 factor were pooled according to their contents, and concentrated under reduced pressure to form an aqueous solution. The aqueous solution was lyophilized to obtain purified GE 81112 factors A,: ^, and B. As is common in the industry, manufacturing, recovery, and purification steps can be monitored by a variety of analytical procedures, including bioassay analysis and / or HPLC procedures using sensitive microorganisms. A better analytical HPLC technique was performed on a Shimadzu LC10 instrument equipped with a 250 x 4.6 mm column mounted symmetrical 17 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 (Symmetry) (Watt Waters) or Altima (Alltech) 5 micron particle size C18 stationary phase, adjusted to p Η 4 · 5 with 40 mM ammonium formate and formic acid, and washed at a flow rate of 1 ml / min mention. Detected at 230 nm. In this case, the retention times of factors A, B !, and B are typically 13, 18, and 21 minutes, respectively. Because the antibiotic substance of the present invention has acid and base functional groups, it can also exist in the form of internal salts or zwitterions at appropriate pH. In addition, salts can be formed according to conventional procedures. Representative acid addition salts of the compounds of the present invention include salts formed by standard reactions with organic and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid. , Succinic acid, citric acid, ascorbic acid, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, pamolic acid, mucoic acid, glutamic acid, camphoric acid, pentyl acid Diacid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid, etc. Representative examples of bases that can be used to form addition salts with GE 81 1 12 compounds are: alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, and calcium hydroxide; ammonia and organic aliphatic amines, cycloaliphatic Amines or aromatic amines such as methylamine, dimethylamine, trimethylamine and methylpyridine. The conversion of the free-state compound or zwitterionic compound of the present invention into the corresponding salt, and conversely, the conversion of the salt of the compound of the present invention to the zwitterionic or free-state compound form is within the skill of those skilled in the art and is within the scope of the present invention. For example, the compound of the present invention can be converted to the corresponding acid or base by dissolving the non-addition salt form in an aqueous solvent and adding a slight excess of the selected acid or base. 200409816 into salt. The resulting solution or suspension is then lyophilized to recover the desired salt. If the salt form of the final product is insoluble in the solvent and the non-salt form is soluble in the solvent, it can be recovered from the solution by filtration. Non-salt forms can be prepared by dissolving the corresponding acid or base salt in an aqueous solvent and then neutralizing to release the non-salt form. When desalination is required after neutralization, common desalination procedures can be used. For example, it can be conveniently used for column chromatography on silylated silicone, non-functional polystyrene, acrylic and polydextrose resins with controlled pore size (such as Sifa Days LH 20) or activated carbon. After the undesired salt is eluted with an aqueous solution, the desired product is eluted with a linear gradient or a step gradient of a mixture of water and a polar or non-polar organic solvent, such as acetonitrile / water 50:50 to about 100% acetonitrile Elution. As known in the industry, addition salts with pharmaceutically acceptable acids (or bases) or non-pharmacologically acceptable acids (or bases) can be used as convenient purification techniques. After formation and separation, the acid addition salt form of the GE 8 1 1 12 compound can be converted to the corresponding free state, zwitterion, or into a different drug tolerable addition salt. Physicochemical properties of GE 8 111 2 factor A A) Mass spectrometry MS experiment of factor 81 GE 81112 on a Thermofinnigan LCQ deca instrument equipped with an electrospray source, using a thermofenigan calibration mixture One protonation was performed at m / z = 644. Electrospray conditions were: spray voltage: 4.7 kV; capillary temperature: 250 ° C; capillary voltage: 9 volts; infusion mode 5 μl / min. The spectra were recorded from a solution of 0.01 mg / ml in acetonitrile: water 50:50 (v / v). Figure 1 shows that the collision energy of GE 81 1 12 factor A after using regularization is 25%. 19 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 After sorting with one of the protonated ions at m / z = 644 Typical MS-MS spectrum. GE 81112 Factor A was obtained in an experiment with Bruker's Daltonics APEXII, 4 · 7 Tesla spectrometer equipped with an electrospray source at m / z = 644.2 1 8 6 The exact mass of one of the protonated ions. FTMS conditions are: electrospray: analysis source, off-axis spray, 60 μl / h; dry gas at 200 ° C; capillary voltage: 70 volts; degreaser voltage: 10 volts; progressive: 40 scans; broadband mode: analysis Degree 20'000. B) Infrared spectroscopy The infrared spectrum of GE 8 1 1 1 2 factor A is shown in Figure 2. The spectrum was measured with a lujol mull using an IFS-48 Fourier transform spectrophotometer. The following main absorption bands (cm ^) were observed: 3356: 2956; 2855; 2256; 2128; 1663; 1596; 1455; 1378; 1345; 1123; 1050; 1026; L002; 825; 764 °.

C ^ H-NMR and 13C-NMR with trifluoroacetic acid (TFA) were recorded at 25 ° C at DMS 0-d6. The spectrum of GE 81112 factor A was recorded at 100 ΜΗζΑ-ΝΜΙΙ (Figure 3) and 150 MHz 13C-NMR spectrum (Figure 4). The two-spectrum detection comes from the formate signal (1)] of the purification process (chemical shifts 8.13 and 7.07-7.25ppm; 13C chemical shift 163.0ppm). Tables IV and V report the 1Η and 13C NMR spectra observed for factor A. 20 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 Table IV: 1H-NMR resonance chemical shift diversity 屮 chemical position diversity (ppm) shift (ppm) of GE81112 (factor A) in DMSO-ch + TFA ) 1.52 br d 4.70 m 1.57-1.67 m 5.06 d 1.8 1 br d 6.46 br 1.88 m 6.94 s 2.87 dd 7.29 s 2.90 m 7.99 d 3.12 dd 8.17 d 3.15 m 8, .3 0 m 3.65 m 8.57 br d 3.83 m 8.95 d 3.94 br d 9.06 br d 4.40 brs 14.09 br 4.46 m 14.27 br 4.50 dd--21 312 / Description of the Invention (Supplement) / 92-02 / 91135061 200409816 Table V: GE 81112 Factor A in DMSO-ch + TFA 13C-NMR resonance 13c Chemical shift diversity 13c Chemical shift diversity (ppm) (ppm) 15.9 t 67.76 t 27.19 t 116.83 d 28.41 t 118.49 d 35.3 1 t 128.4 1 s 42.86 t 129.03 s 50.13 d 133.65 d 51.30 d 139.66 s 56.86 d 156.73 s * 59.7 1 d 167.39 s 64.07 d 170.16 s 65.11 d 17 1.29 s 66.88 d 171.65 s GE 8 1 1 1 2 Physical and chemical properties of factor B A) Mass spectrometry GE 81112 factor B is equipped with an electrospray source MS experiment with Thermal Fernigan LCQ Dicastal instrument, calibrated with Thermal Fernigan A composition for protonated at m / z = 65 9. Electrospray conditions were spray voltage: 4.7 kV; capillary temperature: 25 0 ° C; capillary voltage: 9 volts; infusion mode 5 μl / min. The optical spectrum was recorded from a solution of 0.01 mg / ml in acetonitrile: water 50:50 (Wv).

Figure 5 shows a typical MS-MS spectrum of GE 8 1 1 1 2 factor B after using a regularized collision energy of 28% at m / z = 65 9 to sort protonated ions. GE 22 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 8Π12 Factor B was obtained in an experiment with a Baker Datonic APEX II, 4.7 Tesla spectrometer equipped with an electrospray source, obtained at m / z = 6 5 9.22 The exact mass of one of the protonated ions. FTMS conditions are: electrospray: analysis source, off-axis spray, 60 μl / h; dry gas at 200 ° C; capillary voltage: 70 volts ^ 'eye loss voltage: 10 volts; progressive: 4 0 scan; Broadband mode: Resolution 20,000. B) Infrared spectroscopy

The infrared spectrum of GE 8 1 1 1 2 factor B is shown in Figure 6. The spectrum was measured with potassium bromide using an IFS-48 Fourier transform spectrophotometer. The following main absorption bands were observed (cm 1: 3359: 2958; 2852; 2258; 2129; 1681; 1591; 1450; 1385; 1347; 1122; 1072; 1025; 998; 765. C ^ H-NMR and 13C-NMR addition Trifluoroacetic acid (TFA) was recorded at 25 ° C in DMS 0-d6. SoOMHzj-NMR spectrum of GE 81112 factor B (Figure 7) and 150MHz13C-NMR spectrum (Figure 8). Two-spectrum detection of formic acid from the purification process Salt signal (1 η chemical shift 8.13 and 7.07-7.25 ppm; 13C chemical shift 163.0 ppm). Table VI and Table VII report the 1Η and 13C NMR spectra observed for factor B. 23 312 / Description of the Invention (Supplement) / 92 -〇2 / 91135061 200409816 Table VI: 1H-NMR resonance of GE 81112 factor B at DMS〇-cU + TFA 1 Η Chemical shift diversity 屮 Chemical shift diversity (ppm) (ppm) 1.52 br d 4.60 m 1.57-1.72 m 5.05 d 1.83 br d 6.46 br 1.90 m 6.48 s 2.63 dd 6.93 s 2.88 dd 7.40 brs 2.92 m 7.84 d 3.13 br d 8.05 d 3.64 m 8.33 m 3.83 m 8.54 d 3.95 br d 9.00 br d 4.35 br s 11.72 brs 4.46 m 11.75 brs 4.50 dd--24 312 / Description of the Invention (Supplement) / 92-02 / 91135061 200409816 Table VII: GE 81112 13C-NMR resonance of DMS 0-d6 + TFA 13c Chemical shift diversity 13C Chemical shift diversity (ppm) (ppm) 15.9 t 67.77 t 27.36 t 110.3 d 28.40 t 117.85 d 35.47 t 122.5 s 42.87 t 128.4 s 50.1 d 139.66 s 5 1.20 d 146.7 s 56.78 d 156.7 s 59.73 d 167.4 s 64.09 d 17 0.5 s 65.14 d 17 1.3 s 66.90 d 17 1.38 s GE 8 1 1 1 2 Physical and chemical properties of factor B! A) Mass spectrometry GE Factor 81112! In a MS experiment with a thermal Fernigan LCQ Dicastal instrument equipped with an electrospray source, a thermal Fernigan calibration mixture was used to perform a protonation at m / z = 65 8. Electrospray conditions were spray voltage: 4.7 kV; capillary temperature: 250 ° C; capillary voltage: 9 volts; 5 microliters / minute in infusion mode. The optical spectrum was recorded from a solution of 0.01 mg / ml in acetonitrile: water 50:50 (v / v). Figure 9 shows a typical MS-MS spectrum of a GE 81 1 12 factor using a regularized collision energy of 25% at m / z = 658 after protonated ion sorting. 25 312 / Explanation of the invention (Supplement) / 92-02 / 91135061 200409816 GE 81112 factor dagger was used in the experiment of APEX II, 4.7 Tesla spectrometer equipped with electrospray source, obtained at m / z = 6 5 8.2 4 5 9 The exact mass of one of the protonated ions. FTMS conditions are: electrospray: analysis source, off-axis spray, 60 μl / h; dry gas 200 ° C; capillary voltage: 70 volts; degreaser voltage: 10 volts; progressive: 40 scans; broadband mode: resolution 20,000.

B) W-NMR and 13C-NMR were recorded at 25 ° C in DMS 0-d6 at 600 MHz j-NMR spectrum (Figure 10) and 150 MHz 13C-NMR spectrum (Figure 11). 2 Spectral detection comes from the formate signal of the purification process (the chemical shift ΓΗ is 8.32; the 13C chemical shift is 165.2 8ppm). Tables VIII and IX report the observed 4 and 13C NMR spectra for Factor B !. 26 312 / Description of the Invention (Supplement) / 92-02 / 91135061 200409816 Table VIII: GE 81112 Factor DMS 1-D6 + TFA 1H-NMR Resonance Chemical Shift Diversity 4 Chemical Shift Diversity (ppm) (ppm) 1.38 brd 4.40 brm 1.56-1.64 m 4.46 m 1.76 m 5.05 d 2.70 m 5.60 br s 2.82 brs 6.21 br 2.92 m 6.52 br 3.01-3.06 m 6.78 brs 3.49 brm 7.13 br 3.57 brs 7.80 br d 4.20 brdd 7.90 br d 4.26 br 8.44 br 27 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 Table IX: Factor of σβ81112 focusing on DMS 0-d6 + 13C-NMR of TFA + 13C chemical shift diversity 13C chemical shift diversity (ppm) (ppm) 17.95 t 66.88 d 27.97 t 111.07 d 29.8 t 118.97 d-36.54 t 122.47 s 43.75 t 127.88 s 45.65 d 148.08 s 50.52 d 159.18 s 51.9 d 169.44 s 57.75 d 171.53 s 61.0 d 171.81 d 031.81 -— Bovine living parts GE 8 1 1 1 2 The antimicrobial activity of factors A, B and B i was determined using the micro-dilution method using a standard U-shaped bottom 96-well plate according to the method of the National Committee for Clinical Laboratory Standards (Methods for Dilutio n Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically a Third edition; Approved Standard. NCCLS document M 7-A 3 V o 1. 1 3 N o. 2 5). The results are reported in Table 10. The strains used were clinically isolated or obtained from the American Type Culture Collection (ATCC). GE 81 1 factor 12 is dissolved in DMS0; this solution is further diluted with distilled water. 28 312 / Invention (Supplement) / 92-02 / 91135061 200409816 The culture medium used is cation-adjusted Mueller Hinton liquid medium (CAMHB) for Escherichia coli, Staphylococcus aureus ( Staphylococcus aureus) 'Moraxella catarrhalis, Enterococcus faecalis; Todd Hewitt liquid medium (THB) for Streptococcus pyogenes, pneumonia chain Streptococcus pneumoniae; antibiotic medium 3 (AM3) and basal medium Davis Mingioli liquid medium + 2% (w / v) glucose + 100 μg / ml asparagine (MM) for Bacillus subtilis (Bacillus sub till s); Davis Siming Geely liquid medium + 2% (w / v) glucose (MM) for E. coli; RPMI 1 640 (RPMI) and minimum required medium 40 + 0.1% vitamin (MM) for white Candida (Candida albicans). Unless otherwise indicated, the inoculum is 104 CFU / ml. All strains were cultured in air at 35 ° C. Incubation time is 18-24 hours. After visual inspection, the MIC is defined as the lower limit radon concentration that can completely inhibit the growth of the microorganisms under test. 29 312 / Instruction of the invention (Supplement) / 92-02 / 91135061 200409816 Table XGE 8 1 1 1 2 Antimicrobial activity of IC A (micrograms / ml) Strain (medium) 81112 A 81112 B 81112 B1 819 S. aureus SMITH ATCC 1 9636 (CAMHB) > 5 12 > 5 12 > 5 12 49 Streptococcus pyogenes cl.is. (ΤΗΒ) > 5 12 5 12 5 12 44 Streptococcus pneumoniae cl. is. (THB) 64 64 64 560 Enterococcus faecalis Van A (CAMHB) 32 32 5 12 102 Bacillus subtilis ATCC6633 (MM) 0.13 0.008 0.06 102 ATCC 6 6 3 3 (AM3) > 5 12 256 5 12 3 2 9 2 Moraxella catarrhalis cl.is. (CAMHB) 2 1 2 47 E. coli (MM) 0.06 0.03 0.13 47 E. coli (CAMHB) > 5 12 5 12 5 12 145 Candida albicans (MM) > 5 12 > 5 12 > 5 12 145 Candida albicans (RPMI) > 5 12 > 5 12 > 5 12 GE81112 factor VI ^ and Bi can inhibit the growth of Moraxella catarrhalis, MIC is at 1- 2 μg / ml range. The strain used was a clinical single isolate. GE81112 Factors A, B, and B! Showed marginal activity against clinical isolates of Streptococcus pneumoniae (MIC 64 μg / ml). In addition, factors a and B are resistant to Vancomycin (VanA). Enterococcus faecalis has MI032 µg / ml, and the factor is not active until the concentration is 512 µg / ml. Moraxella catarrhalis and Streptococcus pneumoniae are known to be the main human pathogens. It is a common cause of otitis media in children with respiratory infections and lower respiratory tract infections in the elderly. Moraxella catarrhalis and Streptococcus pneumoniae were recently known as the most common pathogens of the respiratory tract (MC · Enright and H. McKenzy, Moraxella 30 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 (Branhamella) catarrha 1 is-Clinical and molecular aspect of a rediscovered pathogen, J. Med. Microbiol. 16., 3 6 0-7 1, 1997). Vancomycin-resistant enterococci (VRE) become major hospital infections that cause severe human infections (such as endocarditis, meningitis, and sepsis), and gradually pose clinical challenges (Y. Cetinkaya, P. Falk, CG Mayhall, Vancomycin -resistant enterococci, Clin. Microbiol. Rev. 13, 686-707, 2000; LB Rice Emergence of vancomycin-resistant enterococci, Emerg. Infec · Dis.l, 183-7, 2001) o GE 81 1 12 factors A, B And B! Can inhibit the growth of E. coli and Bacillus subtilis at very low concentrations (exactly defined as less than 1 μg / ml), and these bacteria are cultured in the minimum necessary medium (MM). When these bacterial lines are grown in rich media, the MICs of factors A, B, and ^ are in the range of 256-512 μg / ml or more. GE 81112 Factors A and B will not inhibit the growth of Candida albicans in the minimum necessary medium or in rich culture medium. GE 8 Π 1 2 factors A, B, and B! Differential expression of bacteria such as E. coli and B. subtilis compared to eukaryotes such as Candida albicans, suggesting that GE 8 1 1 1 2 compounds have specific effects on prokaryotes turn. According to the recent trend of antimicrobial treatment, it is advantageous to use agents with a narrow antimicrobial range that have selective activity against isolated pathogens instead of agents with a wide range of activity, which can reduce the selectivity of resistance Pressure (JC Gyssens: "Quality Measures of Antimicrobial Drug Use" in Int. J. Antimicrobial Agents, 17, 2001, 9-19; J. Chopra "Research and Development of 31 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816

Antibacteiial Agents "in Current Opinion in Microbiology, 1998, 1,49 5-50 1). Therefore, the GE 81112 compound of the present invention is particularly suitable as a selective therapeutic agent for respiratory infections caused by Moraxella catarrhalis. The compounds of the present invention can be It may be administered as a pharmaceutically acceptable composition, or it may be administered in admixture with a pharmaceutically acceptable carrier. It may also be administered in combination with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides. Combination therapy also includes sequential, simultaneous and separate administration of the active compound, so that the efficacy of the first-administered drug does not completely disappear, and subsequent administration. The compounds of the present invention can be used as drugs; single factors A, B and L can be used alone or in two or Two or more mixtures are used in any ratio. The mixture can be obtained by mixing a predetermined amount of two or more factors. In addition, the factor mixture can be directly obtained from the fermentation product of Streptomyces DSMZ 14386 according to the aforementioned method. Preferred compounds of the present invention are formulated for parenteral administration according to procedures known in the industry and reported in references. And / or oral and / or topical formulations. For parenteral administration for the treatment of any infection involving microorganisms that are sensitive to antibiotics, the formulations are, for example, water with a suitable solubilizer such as polypropylene glycol or Dimethylacetamide and a surfactant such as polyoxyethylene sorbitol monooleate or polyethoxylated castor oil in sterile water for injection. The pH of the formulation for injection is preferably 7 ± 0.5. If If necessary, it is recommended to adjust the pH of the preparation with a suitable buffer. Conveniently, phosphate can also be used as a buffer. In addition, the active ingredient can be prepared as a lyophilized powder for reconstitution. If necessary, if necessary Common freeze-drying auxiliaries can be added to obtain lyophilized materials in powder form 32 312 / Invention Specification (Supplement) / 92_〇2 / 91135061 200409816. Antibiotics can also be used in appropriate pharmaceutical dosage forms such as capsules, lozenges or aqueous solutions Or suspensions (such as syrups) for oral administration; or blended into conventional creams or gels for topical application; or formulated as a liquid formulation suitable for inhalation therapy. Dosage is determined based on a number of factors including patient type, age and condition, specific active ingredients and formulations selected for administration, dosing schedule, etc. Effective antimicrobial doses are usually used for each unit dosage form. Usually repeated administrations / Administration is preferably, for example, 2 to 6 times a day. The effective dose is usually in the range of 0.5-50 mg / kg / body weight / day. The preferred topical formulation is an ointment containing 1% to 10% of the compound of the present invention. The doctor in g will determine the optimal dosage for a specific patient in a specific situation. The following examples further illustrate the invention without limitation. Example 1: Fermentation of Streptomyces DSMZ 1 4 3 8 6 Recovery of GE 8 1 1 1 2 Compound seed culture bacteria was stored in a vial of -8 for inoculation of a 500 ml Erlenmeyer flask containing 100 ml of V6 medium (V6). The composition of V6 is as follows (g / l "Glucose 20, meat extract 5, yeast extract 5, peptone 5, hydrolyzed casein 3, and sodium chloride 1.5. The culture medium was prepared using distilled water, the pH was adjusted to 7.5 with sodium hydroxide, and then sterilized at 12 CTC for 20 minutes. Inoculation flasks were incubated at 28 ° C on a rotary shaker at 200 rpm for 40-4 8 hours, and then used to inoculate a 4 liter bioreactor containing 3 liters of V6 medium. The cultured bacteria were grown at 27 ° C for the first 30 hours, and then grown at 24 ° C for 18 hours until the time of inoculation. Stir at 700 rpm and allow a flow of 0.5 v / v / min. This culture 33 312 / Invention specification (Supplement) / 92-02 / 91] 3 5061 200409816 Inoculate bacteria (1.5 liters) in a fermentation tank containing 200 liters of production medium to 300 liters. The composition of the medium is as follows (g / liter): Glycerin 30, soy flour 15, sodium carbonate 5 and sodium chloride 2. The culture medium was prepared using deionized water, the pH was adjusted to 7.3 with sodium hydroxide, and 30 ml of Hodag AFM-5 was added as an antifoaming agent. This culture was fermented at 28 ° C for 62 hours. The air flow was 60 liters / minute for the first 18 hours, and then increased to 100 liters / minute (0.5 v / W minutes) to harvest time. The agitation used was 180 rpm. The harvested culture solution was then diluted to 200 liters with distilled water, and a Hyflo filter aid was added, followed by filtration. Streptomyces was discharged, and the filtrate was adjusted to pH 3.5 by adding about 600 ml of 3.25 M hydrochloric acid. Then the GE81112 antibiotic mixture was adsorbed in acid form to 2.5 liters of Dowitz 50W x2 cation exchange resin (The Dow Chemical Company), and after stirring overnight in batches, the resin was recovered by filtration and the culture solution was discharged. The two fermentation tanks were processed in parallel as described above, and then Dowes resins from the respective operation rounds were pooled. The resin (5 liters) was loaded on a column (12.5 cm diameter; bed height 45 cm) and washed with 6 liters of 20 mM sodium phosphate pH 3.5 buffer at a flow rate of 90 ml / min; then 10 liters of 20 mM sodium phosphate pH 3.5 Buffer: methanol 8: 2 (Wv) mixture was washed. Elution was then performed with 257 mM ammonia in phosphate buffered pH 10. 1 liter of eluate was collected, which was immediately adjusted to about pH 6 by adding phosphoric acid. The antibiotic content of each elution fraction was graded according to the method described in Example 2 by bioassay analysis, that is, inhibition of growth of E. coli and Bacillus subtilis cultured in the minimum necessary medium (MM), and by analytical HPLC. The eluted fractions 5.10 from Elvis resin were pooled to obtain 5.8 liters of a solution rich in a mixture of GE 8 11 12 compounds. A sample of the solution (50 ml) was concentrated in vacuo and then lyophilized to obtain 1.48 g of a crude product of a GE 8 1 1 12 compound preparation. 34 312 / Invention (Supplement) / 92-02 / 91135061 200409816 Example 2 GE 8 1 1 1 2 Separation and purification of factors A, B and B! The solution obtained in Example 1 and 1.2 liters of polybenzene Vinyl resin HP 20 (Mitsui Chemical Co., Ltd.) was stirred overnight in batches. The resin was then recovered by filtration and loaded on a string of 7.5 cm long and 27 cm bed height. The column was then eluted with 6 liters of 1: 9 (v / v) methanol: distilled water and then 4 liters of 3: 7 (v / x0 mixture) at a flow rate of 40 ml / min. 1 liter of eluate was collected and analyzed by HP LC The initial three-elution fractions showed a factor 8-rich GE 8 1 1 1 2 factor mixture and were pooled. The subsequent three-elution fractions were enriched with factors Bι and B and also pooled. The second pool was concentrated in vacuo to a small amount. The volume was then lyophilized to obtain 3.8 grams and 1.7 grams of solids enriched with factor A and factor 1 plus B, respectively. Then preparative HPLC (Ninjin-LC8A) was used in Sibelico (Mock) 25 x 2 5 0 mm column RP8 (7 micron particle size), using 40 mM ammonium formate, adjusted to pH 4.5 with formic acid, and eluted at a flow rate of 3.5 ml / min to obtain individual pure elution fractions. About mg of the aforementioned antibiotic preparation was dissolved in 3 50 micron eluent phase, and subjected to chromatography. Factors A, B !, and B are typically eluted after about 9.5, 12.4, and 13.4 minutes. Pooled fractions of repeated chromatography rounds showing homogeneous antibiotic content , And concentrated in vacuo to a water residue. The solution was then lyophilized, dissolved again in 40 ml of distilled water, and lyophilized again to obtain 116 13. 10 mg antibiotics GE81112 factor A, B! * B. HP LC analysis was performed on a Shimadzu LC 10-AS instrument equipped with an SPD-M 1 0 A diode array detector and a Si 1 · 9 A autoinjector. Chromatography column was "Adima" RP 18.5, 5 micrometers' 2 5 0 X 4 · 6 mm inner diameter, purchased from All-Technology Company. Use 40 mM ammonium formate buffer to adjust pH to 4.5 by adding formic acid. Isocratic elution was performed at a flow rate of 1 ml / min. UV detection was performed at 230 nm. 35 312 / Instruction Manual (Supplement) / 92-02 / 91135061 200409816 The three factors are under the aforementioned analytical HPLC conditions The following test shows the following retention times: factor A, 14.1 minutes, factor B !, 18.4 minutes, and factor B, 2 1.6 minutes. [Simplified illustration of the figure] Figure 1 shows a regularized collision energy of 25% in m / z = MSMS spectrum of antibiotic GE 81112 factor A after protonation of one of the 644 protonated ions. Figure 2 shows the IR absorption spectrum of antibiotic GE 81112 factor A in lubricant paste. · Figure 3 shows antibiotic GE81112 factor A at 600 MHz. UMR spectra measured on DMSO-d6 and TFA (several drops). Figure 4 shows the resistance Proton decoupling 13C-NMR spectrum of biotin GE81112 factor A measured at 150 MHz in DMS 0-d6 and TFA (several drops). Figure 5 shows one with a regularized collision energy of 28% at m / z = 65 9 MS MS spectrum of protonated ion GE 81112 factor B after segmentation. Figure 6 shows the I.R. absorption spectrum of antibiotic GE81112 factor B on potassium bromide. Figure 7 shows the 'H-NMR spectrum of the antibiotic GE81112 factor B measured at 600 MHz on DMS 0-d6 and TFA (several drops). Fig. 8 shows the 13C-NMR spectrum of proton decoupling measured for antibiotic GE81112 factor B at 150 MHz in DMSO-d6 and TFA (several drops). Figure 9 shows the MSMS spectrum of the antibiotic GE 81112 factor heart with a regularized collision energy of 25% at m / z = 65 8 and a protonated ion. FIG. 10 shows a 1 Η -N M R spectrum of the antibiotic GE 81112 factor B1 at 600 MHz and DMS 0-d6. Figure 11 shows the antibiotic GE81112 factor Bι at 150 MHz at DMS 0-d6 36 312 / Invention specification (Supplement) / 92-02 / 91135061 200409816 Detected proton measurement] 3C-NMR spectrum 312 / Invention Specification (Supplement) / 92-02 / 91135061 37

Claims (1)

200409816 1. An antibiotic GE 81112 factor A, which has the following characteristics: A) The mass spectrum is recorded from a solution of 0.01 mg / ml in acetonitrile: water 50:50 (v / v), and it is recorded in Thermofinnigan LCQ ( Deca) Yiyi obtained one of the protonated ions at 644 m / z under the following electrospray conditions: spray voltage: 4.7 kV; capillary temperature: 2 50 ° C; capillary voltage: 9 volts; 5 μl infusion mode / Minute; B) Infrared spectroscopy (Figure 2), using the lubricating oil paste (nujol mull) using IFS-48 Fourier transform spectrophotometer to measure and display the following main absorption band (cm- 丨) · 3356; 2956; 2855; 2256; 2128; 1 663; 1 5 96; 1 45 5; 1378; 1345; 1123; 1050; 1026; 1002; 825; 764; CVH-NMR spectrum (Figure 3) at 600 MHz at 25 ° C at 25 ° C DMS〇-d6 trifluoroacetic acid (several drops) records, showing the following 4 signals: chemical shift (ppm) diversity chemical shift (ppm) diversity 1.52 brd 4.70 m 1.57-1.67 m 5.06 d 1.8 1 br d 6.46 br 1.88 m 6.94 s 2.87 dd 7.29 s 2.90 m 7.99 d 3.12 dd 8.17 d 3.15 m 8.30 m 3.65 m 8.57 b rd 3.83 m 8.95 d 312 / Invention Specification (Supplement) / 92-02 / 91135061 38 200409816 3.94 brd 9.06 brd 4.40 brs 14.09 br 4.46 m 14.27 br 4.50 dd-— D) 13C-NMR spectrum (Figure 4), in Recorded at 150 ° C at 5 ° C in DMS 0-d6 and trifluoroacetic acid (several drops), showing the following 13C signal chemical shift diversity chemical shift diversity (ppm) (ppm) 15.9 t 67.76 t 27.19 t 116.83 d 28.41 t 118.49 d 35.31 t 128.41 s 42.86 t 129.03 s 50.13 d 133.65 d 5 1.30 d 139.66 s 56.86 d 156.73 s 59.7 1 d 167.39 s 64.07 d 170.16 s 65.11 d 171.29 s 66.88 d 171.65 s E 4) 1) Analysis η PLC Shimadzu LC 1 0-AS measurement, the instrument is equipped with "Adima" RP 18.5, 5 micron, 250 X 4.6 mm inner diameter column; use 40 m mM ammonium formate buffer to adjust by adding formic acid Isocratic elution was carried out to pH 4.5 at a flow rate of 1 ml / min; UV detection was performed at 230 nm. 39 Description of the Invention (Supplement) / 92-02 / 91135061 200409816 2. An antibiotic GE 81112 factor B, which has the following characteristics: A) Mass spectrometry is from 0.01 mg / ml to acetonitrile: water 5 0:50 0 (v / v ) Solution records, protonated ions at 65 9 m / z were obtained under the conditions of electrospray on a thermal Fernigan LC Q Dicastal instrument: spray voltage: 4.7 kV; capillary temperature: 25 (TC; capillary Voltage: 9 volts; 5 μl / min in infusion mode; B) Infrared spectroscopy (Figure 6), using potassium bromide and IFS-48 Fourier transform spectrophotometer, shows the following main absorption band (cm · 1): 3 3 5 9; 2 9 5 8; 2 8 5 2; 22 5 8; 2 129; 1681; 1591; 1 45 0; 1 3 8 5; 1 3 4 7; 1122; 1072; 1025; 998; 765 〇 ; CVH-NMR spectrum (Figure 7), recorded at 600 MHz at 25 ° C in DMS 0-d6 and trifluoroacetic acid (several drops), showing the following 1H signals: Chemical shift diversity Chemical shift diversity (ppm) (ppm) 1.52 br d 4.60 m 1.57-1.72 m 5.05 d 1.83 brd 6.46 br 1.90 m 6.48 s 2.63 dd 6.93 s 2.88 dd 7.40 brs 2.92 m 7.84 d 3.13 br d 8.05 d 3.64 m 8.33 m 3.83 m 8.54 d 3.95 br d 9.00 br d 4.35 br s 11.72 br s 312 / Invention Specification (Supplement) / 92 · 02/91135061 40 200409816 4.46 m 11.75 brs 4.50 dd Dguang C-NMR spectrum (Figure 8), recorded at 150 MHz at 2 50 ° C in DMS 0- and trifluoroacetic acid (several drops), showing the following 13 c signal] 3c chemical shift diversity I3C chemical shift diversity (ppm) (ppm) 15.9 t 67.77 t 27.36 t 1 10.3 d 28.40 t 117.85 d 35.47 t 122.5 s 42.8 7 t 128.4 s 50.1 d 139.66 s 5 1.20 d 146.7 s 56.78 d 156.7 s 59.73 d 167.4 s 64.09 d 170.5 s 65.14 d 17 66.90 d 17 1.38 s E) Retention time 21.6 minutes, measured by analytical HPLC Shimadzu LC10-AS, equipped with "Adima" RP18, 5 micron, 25 0 X 4.6 mm id column; 40 mM ammonium formate The buffer was adjusted to pH 4.5 by adding formic acid, and the isocratic elution was performed at a flow rate of 1 ml / min; UV detection was performed at 230 nm. 3.—An antibiotic GE 81112 factor, which has the following characteristics: A) Mass spectrometry is from 0.01 mg / ml in acetonitrile: water 50:50 (v / v) solution 41 312 / Invention specification (Supplement) / 92-〇 2 / 91135〇61 200409816 record, one of the protonated ions obtained at 6 5 8 m / z under the conditions of electrospray on a thermal Fernigan LCQ Dicastal instrument: spray voltage: 4.7 kV; capillary temperature: 2 5 0 ° C; capillary voltage: 9 volts; infusion mode 5 μl / min; B ^ H-NMR spectrum (Figure 10), recorded at 600 MHz at 25 ° C in DMS 0-d6, showing the following 1 Η signal: Chemistry Displacement diversity Chemical displacement diversity (ppm) (ppm) 1.38 br d 4.40 br m 1.56-1.64 m 4.46 m 1.76 m 5.05 d 2.70 m 5.60 brs 2.82 br s 6.21 br 2.92 m 6.52 br 3.01-3.06 m 6.78 br s 3.49 br m 7.13 br 3.57 brs 7.80 br d 4.20 brdd 7.90 br d 4.26 br 8.44 br C) 13C-NMR spectrum (Figure 11), recorded at 150 MHz at 2 5 ° C in DMS 0-, showing the following 13C signal chemical shifts are various Chemical shift diversity (ppm) (ppm) 17.95 t 6 6.86 d 27.97 t 111.07 d 29.8 t 118.97 d 312 / Invention (Supplement) / 92-02 / 91135061 42 200409816 36.54 t 122.47 s 43.7 5 t 127.88 s 45.65 d 148.08 s 50.52 d 159.18 s 5 1.9 d 169.44 s 57.75 d 171.53 s 61.0 d 171.81 s 64.03 d s 66.7 d-〇) Retention time 18.4 minutes, measured by analysis ^ 1 ^ Shimadzu 1 ^ 10-Aug. 3, the instrument is equipped with "Adima" RP 1 8, 5 microns, 2 50 x 4.6 mm inner diameter Column; 40 mM ammonium formate buffer was used to adjust the pH to 4.5 with the addition of formic acid, and the eSQCratic elution was performed at a flow rate of 1 ml / min; UV detection was performed at 230 nm. 4 · A method for manufacturing a GE 81112 antibiotic such as any one of claims 1 to 3 in the scope of patent application, which method comprises:-under aerobic conditions, an assimilable carbon source, a nitrogen source, and an inorganic salt source Aqueous nutrient medium 'culture of Streptomyces sp. DSMZ 1 43 8 6 or its mutant strain or mutant strain which maintains hereditary ability to produce GE 81112 antibiotics; • antibiotic mixture obtained from fermentation broth;-purification Recovered antibiotic mixture, and separation of three antibiotics, GE 81112 Factor A, B, and B !. 5 · —A mixture 'which contains two or more antibiotics such as any one of claims 1 to 3 of the scope of patent application GE 8 1 1 1 2 factor at any ratio 43 312 / Invention Specification (Supplement) / 92-02 / 91135061 200409816 mixed with its drug tolerable salt. 6. The mixture of item 5 in the scope of the patent application, which is a mixture of the antibiotic ge 8 1 1 1 2 factors in any ratio, and the antibiotic GE 8 111 2 factor mixture is made of Streptomyces DSMZ 1 43 86 or maintained The GE 81 丨 12 antibiotic producing mutant strain or mutant strain which can produce the genetic ability of the GE 81 1 12 antibiotic is obtained by separation. 7. A method for manufacturing an antibiotic GE 8 1 Π 2 factor mixture as described in claim 6 of the scope of patent application, the method comprises: '-culture of Streptomyces DSMZ 1 4 3 8 6 or its maintenance can produce GE 81 1 12 antibiotics Mutants or mutants of heritability;-an antibiotic mixture of the factor GE 8 1 1 12 obtained from the liquid culture medium; and-a purified antibiotic GE 81112 factor mixture. 8. The method of claim 4 or 7, wherein the Streptomyces DSMZ 14386 or a mutant strain or mutant strain that maintains hereditary ability to produce the GE 81112 antibiotic is subjected to preculture. 9. The method according to item 4 or 7 of the scope of patent application, wherein the recovery is performed on an ion exchange resin, and purification is performed on an adsorption matrix, preferably on polystyrene or mixed polystyrene · divinylbenzene resin. 10 · The method according to item 4 of the scope of patent application, wherein the antibiotic GE 8 1 1 12 factor A, B and B! Are separated by chromatography. 11. The method of claim 10 in the scope of patent application, wherein the chromatography is a preparative HPLC technique. 1 2 · A pharmaceutical composition comprising an antibiotic such as any one of claims 1 to 3 and 5. 44 312 / Description of the Invention (Supplement) / 92-02 / 91135061 200409816 1 3. If the pharmaceutical composition in the scope of the patent application No. 12 contains a drug tolerable carrier. 1 4. A use of antibiotics according to any one of claims 1 to 3 and 5 of the scope of patent application, which is used for the manufacture of drugs for the treatment of bacterial infections. 45 312 / Invention Specification (Supplement) / 92-02 / 91135061