HK1066566A - Biologically pure culture of nocardia sp. - Google Patents

Description

Biologically pure culture of Nocardia

The application is a divisional application of Chinese patent application 99808680.0 entitled "nocathiacin antibiotic" filed on 7/13/1999.

Technical Field

The present invention relates to novel peptides of thiazoles antibiotic compounds nocathiacin I, II and III and pharmaceutical compositions thereof and methods of treating bacterial infections using the antibiotic nocathiacin I, II and III.

Background

The emergence of resistant bacteria has become a world health problem for some antibacterial drugs such as lactam antibiotics, beta-macrolides, quinolones and vancomycin. (Cohen, M.L. antimicrobial resistance: facial for public health. trends Microbiol.1994, 2, 422-. The most significant problem in clinical practice is the increased incidence of methicillin-resistant staphylococcus aureus (MRSA) strains. Currently, the most effective drug for treating many MRSA infections is vancomycin. Meanwhile, reports about vancomycin resistance of MRSA isolates also appear. Some other recently emerging clinically relevant multidrug-resistant bacteria are enterococci. Streptococcus pneumoniae, an important public-susceptible pathogen resistant to penicillin and other antibiotics, also poses a worldwide health problem. Several countries, including the united states, have developed resistant bacteria of the streptococcus pneumoniae group. The emergence and spread of this nosocomial and publicly-susceptible drug-resistant pathogen threatens public health worldwide. Therefore, the invention of a novel medicament for treating multi-drug resistant bacterial infection is urgently needed. The present invention is directed to such a study.

Novel thiazole peptide antibiotics are described below (which are referred to herein as nocathiacin (nocathiacin) I, II and III, or nocathiacin antibiotics). It has inhibitory activity against gram-positive bacteria at nanomolar levels. The novel antibiotics described herein were isolated from the liquid medium of Nocardia species ATCC-202099. Among the known thiazolepeptidic antibiotics are the myceliophytin and the norcetin, and glycothiohexide-alpha is reported to have inhibitory activity against gram-positive bacteria in vitro, but no in vivo activity is reported. The antibiotics disclosed herein show in vivo activity against a murine model of systemic staphylococcus aureus infection.

Nocathiacin I has been described previously by J.E.Leet et al (U.S. provisional application 60/093,021, 16.7.1998) and by Sassaki, T.et al, J.of Antibiotics 51, No.8, pp.715-721 (published 25.8.1998). The present invention relates to nocathiacin antibiotics and nosiheptide (Prange T. et al, J.am Chem Soc.99, 6418 (1977); Benazet, F. et al, Experientia 36, 414 (1980); Floss, H.G. et al, J.am Chem Soc.115, 7557 (1993); glycothiohexides (Steinberg, D.A. et al, J.Antibiot.47, 887 (1994); M.D.Lee. et al, J.Antibiot.47, 894(1994) M.D.Lee. et al, J.Antibiot.47,

901 (1994); US 5,451,581, 1995), and antibiotic S-54832A (u.s4,478,831, 1984), but with significant differences.

Disclosure of Invention

The present invention relates to novel peptides of thiazoles antibiotic compounds nocathiacin I, II and III. The antibiotic of the present invention can be obtained by isolating and purifying a fermentation broth of Nocardia species (Nocardiasp.) (WW-12651, ATCC-202099 strain).

The invention also relates to pharmaceutical compositions and methods of treating bacterial infections with the antibiotics nocathiacin I, II and III, and biologically pure media of nocardia species strains for the production of the antibiotics. The invention includes all medicinal derivatives of nocathiacin antibiotics, such as medicinal salts and esters thereof.

It is expected that such gram-positive bacteria inhibiting compounds will be useful in vivo and in vitro as other antibiotics for use in mammals, particularly humans, for which the utility of the title compound in the treatment of bacterial infections is based. The compounds of the present invention have antibacterial activity, particularly against gram-positive bacteria.

Drawings

FIG. 1 shows the ultraviolet absorption spectrum of nocathiacin I.

FIG. 2 shows the infrared absorption spectrum of nocathiacin I.

FIG. 3 shows the determination of nocathiacin I in deuterated dimethylsulfoxide¹H-NMR spectrum (500 MHz).

FIG. 4 shows the determination of nocathiacin I in deuterated dimethylsulfoxide¹³C-NMR spectrum (125 MHz).

FIG. 5 shows the UV absorption spectrum of nocathiacin II.

FIG. 6 shows the infrared absorption spectrum of nocathiacin II.

FIG. 7 shows the determination of nocathiacin II in deuterated dimethylsulfoxide¹H-NMR spectrum (500 MHz).

FIG. 8 shows the determination of nocathiacin II in deuterated dimethylsulfoxide¹³C-NMR spectrum (125 MHz).

FIG. 9 shows the UV absorption spectrum of nocathiacin III.

FIG. 10 shows an infrared absorption spectrum of nocathiacin III.

FIG. 11 shows the determination of nocathiacin III in deuterated dimethylsulfoxide¹H-NMR spectrum (500 MHz).

FIG. 12 shows the determination of nocathiacin III in deuterated dimethylsulfoxide¹³C-NMR spectrum (125 MHz).

Detailed Description

The antibiotic compounds of nocathiacin I, II and III are found in the fermentation process. The title nocathiacin compound was isolated from the fermentation broth of nocardiasp. (WW-12651, ATCC-202099), purified by extraction and chromatography to obtain an amorphous solid.

Description of the microorganisms

The microorganism used to produce nocathiacin antibiotic is Nocardia sp isolated from soil samples collected in new mexico. Cultures (strain WW-12651) were stored at the American Type Culture Collection (ATCC) of Rockville, MD, deposit number ATCC-202099, 3/4 (1998). The deposit of the ATCC satisfied all of the Budapest treaty requirements. Dormant cultures are also stored at the institute for drug Research culture collection of bristol-meis schnobel, fifth Research park, Wallingford, CT 06492. In addition to the particular microorganisms described herein, it is to be understood that a mutant, such as a mutation caused by a chemical or physical mutagen, including X-rays, and those that have altered genetic composition by molecular biological techniques, can also be cultured to produce nocathiacin antibiotics.

Microscopic studies of strain WW-12651 were carried out in the morphological medium of ISP (ISP2, ISP3, ISP4, ISP5 and ISP7) according to the recommendations of the International Streptomyces Project (ISP) guidelines, and microscopic observations were carried out on days 7, 14 and 21 at 28 ℃ incubation.

The aerial mycelium was white segmented as cream-colored colonies grown on ISP4 medium. Under an optical microscope, spores were visible in the grown mycelia, while few or no spores were visible in the reticulate mycelia. The morphology of this organism is classified as a streptomyces type. A light brown-orange reverse colour was visible in salt-starch agar (ISP 4). There was no production of diffusible pigments in any of the ISP media. Melanoid formation was not observed in tyrosine agar (ISP7) nor was it detected by the modified Arai and Mikami melanin formation assays.

The amino acid components of the cell wall are alanine, L-glutamic acid, aspartic acid and m-diaminopimelic acid isomers, and the sugar components of the cell wall are galactose, arabinose and ribose. Carbon source utilization research showsWhen combined as a monocarbon source with inorganic salt agar (ISP9), glucose, mannitol, sucrose, xylose, and fructose (poorly) can be utilized. Arabinose, inositol, raffinose and rhamnose were not utilized when the ISP9 medium was the sole carbon source (ISP 9). Based on the above properties and analysis of mycolic acid, it can be determined that this microorganism belongs to the genus Nocardia.Fermentation of Nocardia sp. (Strain WW-12651)

The production of nocathiacin antibiotics can be accomplished by culturing nocardibiasp. strain WW-12651 in a suitable nutrient medium, preferably under submerged aerobic conditions, until a certain amount of nocathiacin is detected in the fermentation, by extracting the active ingredient from the growing hyphae using a suitable solvent, concentrating the solution containing the desired ingredient, and then chromatographically separating the concentrated ingredient from other metabolites of the medium.

Any temperature beneficial to the growth of the culture, such as 16 ℃ and 40 ℃, will affect the production of Nocathiacin, but is preferably carried out at 22-32 ℃. The liquid medium is necessarily incubated for a period of time necessary to ensure that production of Nocathiacin is sufficiently performed, typically monitored by HPLC for 1-6 days, with a rotational shaker speed of 50-300rpm, preferably 150-200rpm, during production.

Growth of the microorganism can be accomplished by a common process using an appropriate medium. Briefly, carbon sources include glucose, fructose, mannose, maltose, galactose, mannitol and glycerol, other sugars and sugar alcohols, starch and other carbohydrates, or carbohydrate derivatives such as dextran, cerelose and complex nutrients such as oat flour, corn flour, millet, cereals and the like. The precise amount of carbon source used in the medium will depend to some extent on the other components of the medium, and it has also been found that a medium containing 0.5-10% carbohydrate is satisfactory. These carbon sources can be used alone or in combination in the same medium. Some preferred carbon sources are set forth below.

The N source comprises amino acids such as glycerol, arginine, threonine, methionine, ammonium salt, yeast extract with complex source, corn extract, soluble distillate, soybean powder, cotton seed powder, fish powder, etc., and different N sources can be used in culture medium with 0.05-5 wt% alone.

The nutrient inorganic salts which can be incorporated into the culture medium are the usual salts which produce Na, K, Mg, Ca, P, S, Cl, carbonic acid, etc., and also trace metals including cobalt, manganese, iron, molybdenum, zinc, cadmium, etc.

Typically, Nocardiasp. (strain WW-12651) is grown in 500ml flasks containing 100ml nutrient medium: wherein each liter of deionized water contains 20g of starch; 5g of glucose; 3g N-Z case; 2g yeast extract; 5g of fish meat extract; 5g of calcium carbonate. The cultures were incubated for 3 days at 32 ℃ in a rotary shaker at 250 rpm. The nutrient medium is mixed with a quantity of cryoprotectant solution containing 100g of sucrose and 200g of glycerol per litre of deionised water. Every 4ml portion of the mixture was transferred to a sterile cryovial (5ml capacity), frozen in a dry ice-acetone bath, and the resulting frozen nutrient medium was stored at-80 ℃.

Seed cultures for the production of nocathiacin antibiotic products were prepared by the following procedure. 4ml of the medium containing the cryoprotectant was transferred to a 500ml flask containing 100ml of sterile nutrient culture (same composition as before). The seed medium was incubated for 3 days at 28 ℃ to 32 ℃ in a rotary shaker at 250 rpm. This seed medium was cultured in flasks containing 100ml of each 4ml of the following medium: HY Yest 421, 10g, 20g glucose, 10g Nutrisoy per liter deionized water, this is the preferred culture. The culture was incubated at 24-32 ℃ in a rotary shaker at 180-250 rpm. The best nocathiacin antibiotics are usually obtained after 4-5 days of fermentation.

When nocathiacin I, II or III compounds are used as pharmaceutical ingredients for the treatment of bacterial infections, they may be combined with one or more pharmaceutically acceptable carriers, such as solvents, diluents, and the like. It can also be administered parenterally in the form of oral administration, such as tablets, capsules, dispersible powders, granules or suspensions containing, for example, about 0.05-5% of a suspension, syrups containing, for example, about 10-50% of a syrup, elixirs containing, for example, about 20-50% ethanol, etc., or in the form of suspensions or sterile injectable solutions containing 0.05-5% of a suspension in an isotonic medium. The pharmaceutical formulations herein may be about 0.05 to about 90% of the active ingredient combined with the carrier, but are more typically about 5% to about 60% by weight.

The effective dosage of the active ingredient may vary depending upon the particular compound employed, the method of administration and the severity of the condition being treated, and thus in general satisfactory efficacy is obtained when the compound of the invention is administered in a daily dosage of about 0.5 to 500mg per kg of animal body weight, preferably in two to four different doses a day, or in a sustained release form. For most large mammals, the total daily dose is from 1 to 100mg, preferably from 2 to 80 mg. Dosage forms suitable for internal administration contain 0.5-500mg of the active ingredient in admixture with a solid or liquid pharmaceutically acceptable carrier, which mode of administration may be selected to provide optimum therapeutic effect. For example, the dosage may be administered daily in several different doses or reduced proportionally to the particular needs of the treatment situation.

These active ingredients can be administered orally and by intravenous, intramuscular or subcutaneous routes. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, liquid carriers include sterile water, polyethylene glycols, nonionic surfactants and edible oils such as corn, peanut and sesame oils, as appropriate to the nature of the active ingredient and the particular form of administration desired. Adjuvants customarily employed in the preparation of pharmaceutical compounds are advantageous, for example flavoring agents, coloring agents, preserving agents and antioxidants, such as VE, ascorbic acid, BHT and BHA.

These active compounds can also be administered parenterally or intraperitoneally. Like the free base and pharmacologically acceptable salts. Solutions or suspensions of such active ingredients can be prepared by mixing in water with a surfactant such as hydroxypropylcellulose. Colloidal solutions (Dispersions) can be prepared in glycerol, liquid polyethylene glycols and mixed oils of these. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the proliferation of bacteria.

Pharmaceutical dosage forms for injection include sterile aqueous solutions or colloidal dispersions. In all cases, such dosage forms must be sterile and must be fluid to the extent that they are needle-through. It must be stable in production and storage and have the ability to resist bacterial, fungal contamination. The carrier can be a solvent or dispersion medium containing water, ethanol, polyol (such as glycerol, propylene glycol and liquid polyethylene glycol), and suitable mixtures and vegetable oils.

The term "pharmaceutically acceptable salts" includes solvates, hydrates, acid addition salts and quaternary salts. Acid addition salts can be prepared from nocathiacin I, II or III compounds containing basic nitrogen, and pharmaceutically acceptable organic or inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methane sulfate, acetic acid, citric acid, malonic acid, succinic acid, fumaric acid, maleic acid, sulfonic acid, or tartaric acid. The quaternary salts are obtained from basic nocathiacin I, II or III compounds and alkyl or aralkyl halides, preferably methyl or benzyl bromide.

The known nocathiacin compounds of the present invention may include different stereoisomers.

Description of the MREF analysis method

The biological activity of nocathiacin antibiotic compounds was found in the HPLC assay in crude extracts prepared from cultures of Nocardia sp (ATCC 202099). This screen was based on the growth inhibition of enterococcus faecalis a28152 incubated in agar growth medium. Enterococcus faecalis is resistant to many antibiotics such as penicillin G, vancomycin, ciprofloxacin, teicoplanin, tetracycline, streptomycin, gentamycin, erythromycin, clindamycin and ramification, but is sensitive to chloramphenicol, followed by imipenem. The name MREF therefore refers to multidrug resistant enterococcus faecalis.

Enterococcus faecalis strain A28152 was inoculated into Brain Heart Infusion Broth and grown to log phase with agitation at 37 ℃. Periodically, 0.2ml of the culture was removed and transferred to a plastic flat pan containing 96 wells and the optical density was measured at 595 nm. When the optical density is 0.2-0.4, the separation is 1000 XgBacteria were obtained in 10 minutes. The cell pellet was suspended in Mueller-Hinton II growth medium and the suspension was inoculated into molten Mueller-Hinton II growth medium containing 1% Difco agar at 48 ℃ to give a 1X 10 cell suspension⁷Seeded cell density of cells/ml. 25ml of the cell suspension was poured into a sterile rectangular dish, and a special plastic cover fitted to the dish was fitted over the top of the dish while the medium was still molten. The cover has a standard 8 x 12 array of plastic spikes. The medium was gelled for 15 minutes at room temperature, the cap with the needle removed, and small concave blots were left on the gelled medium that had been in contact with the molten medium, these blots serving as loading areas for the sample.

The sample to be screened was dissolved in 100% Dimethylsulfoxide (DMSO) at a concentration of 300. mu.M. Samples were added to the sample-on-disk zone in 6. mu.l volumes. After the addition, the plate was incubated at 37 ℃ for 24 hours. Around the sample area, a clear annular zone is visible, which is outside the growth-inhibiting annular zone, bacteria grow irreducible and the agar medium becomes turbid due to bacterial growth. Under the same conditions DMSO alone did not produce any detectable growth inhibition. Chloramphenicol and DMSO were incubated in each dish as positive and negative controls.

The active substances obtained from the cultures produced by Nocardia sp (ATCC202099) were confirmed by Thin Layer Chromatography (TLC) bioimaging (biogram), the material of which included bioassay dishes (from Nunc-Nalge International) for agar diffusion analysis. The dish is 243X 18mm, and can provide 530cm²The microorganism culture section of (1). The TLC plate was placed on the bioassay dish and an agar cover was slowly poured over to cover the TLC plate and the bottom of the bioassay dish. The agar overlay comprised 200ml of Mueller-Hinton II agar base supplemented with 0.5% defibrinated sheep blood and a concentration of 1X 10⁶Cells/ml of drug-resistant enterococcus faecalis strain a 28152. After cooling, the TLC bioimages were incubated at room temperature for about 18 hours. After incubation, the blood agar becomes red-brown due to the activity of alpha-hemolytic cells in this enterococcus. If the compound inhibits this growth, it is seen that the dissolution is inhibitedThe very distinct red color zone produced by blood. The method takes into account the correlation between the desired biological activity and the appropriate chromatographic fractions, thus facilitating the confirmation of the activity of the active ingredient and its isolates.

This method for MREF detection involves the detection of two different contrast (contrast) agents. The first was 2, 3, 5-triphenyl-2H-tetrapyrrole chloride (0.003% final) supplemented to 200ml of Todd Hewitt agar base. The second method was to supplement 200ml of Mueller-Hinton agar with 0.5% sheep blood. Both methods showed clear areas of growth inhibition, however, the shorter the incubation time with blood agar, the better the resolution (resolution). The range of cell seeding concentrations was examined, but 1X 10⁶The results were optimal at cell/ml.

Separation and structural Properties

Purification of Nocardias p. fermentation produced nocardiasa antibiotic was determined by Multidrug Resistant Enterococcus Faecalis (MREF) agar diffusion analysis. Extracting with ethyl acetate, and performing solvent partition, Sephadex LH-20 and/or silica gel chromatography. These steps produce a mixture of nocathiacin antibiotics that show activity in the MREF agar diffusion assay. Finally, the purification of the single nocathiacin antibiotic is completed by normal phase or reverse phase high performance liquid preparative chromatography, and the spectral data show that the component belongs to the thiazole peptide antibiotic. The structures of nocathiacin I, II and III were arranged on the basis of 2D NMR cationic electrospray (electrospray) HRMS study and MS/MS data (see below).

Wherein W is

And R is OH (nocathiacin I) or R is H (nocathiacin II); and W is H and R is OH (nocathiacin III).

General procedure

Materials:

hexane, chloroform (ACS grade), and methanol, acetonitrile (chromatographically grade) were purchased from fisher scientific, inc. These solvents were not repurified and redistilled. The water used for the chromatographic experiments is deionized water passed through a microwell 4 cartridge reagent grade water system (10 megaohm Milli-Q water) in the chamber (in-house). Sephadex LH-20g was purchased from Pharmacia LKB. Uppsala, Sweden. Dicalite (diatomaceous earth) was produced by Grefco minerals, Torrance, Calif. LiChroprep silicon 60, 25-40 μm was purchased from EMSeparatohs, NJ, E.Merek, USA partner, Germany.

Thin Layer Chromatography (TLC)

Silica gel GHLF plates (model 10X 20cm, 250 μm) coated with thin layer chromatography were purchased from Analtech, Inc, Newark, DE. Components were spotted using a 2. mu.l microtube (with disposable pipette) and plates were developed in a well equilibrated with chloroform-methanol (9: 1V/V). The chromatographed fractions are visualized under UV long-wave light and/or after heating by spraying with concentrated sulfuric acid.

HPLC analysis

Purification of nocathiacin antibiotic was detected by HPLC analysis. APEX 5. mu. ODS column, diameter 4.6 mm. times.15 cm in length. (produced by Jones Chromatography, Lakewood). Hewlett Packard model 1100 liquid chromatography analysis, 254nm UV detection. A gradient system of acetonitrile and 0.01M potassium phosphate buffer pH3.5 was used, according to the method of D.J. hook et al (I.Chromatogr.385, 99(1987)), at a pump flow rate of 1.2 ml/min for the eluent.

HPLC preparation

The following components were used to set up the HPLC preparation system: beckman instruments Inc. (somersentnj), Beckman "gold system" 126 programmed solvent system apparatus; beckman 166 programmed detection device; beckman's gold System' translates 711U software; an IBMPS/255SX system controller; YMC (Wilmingtan, NC) HPLC preparative column (normal phase): PYA-silica gel 5 μm in diameter, aperture 120 °, diameter 20mm × length 150mm, with Diol 25 μ in diameter, aperture 120 °, diameter 10mm × length 10mm drop-in protection column); a mobile phase chloroform-methanol gradient with a flow rate of 10 ml/min was run at 290nm uv. Another column (reversed phase): YMC, ODS-AQ5 mu particle size, aperture 120 °, diameter 20mm x length 150mm, and ODS-A25 mu particle size, aperture 120 °, diameter 20mm x length 10mm drop protection column); mobile phase 0.1M acetic acid-acetone gradient with flow rate of 10 ml/min, 360nm uv detection.

Analytical device

Low resolution mass spectrometry was performed using a cationic electrospray Finnigan SSQ 7000 single quadrupole mass spectrometer. MS/MS measurement was carried out by using a cationic electrospray Finnigan SSQ 7000 tandem quadrupole mass spectrometer, and an argon collision gas or Finnigan LCQ ion capture mass spectrometer. High resolution MS data were determined using a Finnigan MAT 900 magnetic sector mass spectrometer, cationic electrospray type, ppg reference. Hewlett-Packard 8452A diode matrix spectrophotometer was used for UV spectroscopy and Perkin Elmer 2000 Fourier transform spectrometer was used for infrared measurements.¹H-NMR and¹³C-NMR data were obtained using Bruker DRX-500 with 500.13 and 125.76MHz respectively, using Nalorac microprobes. Chemical shifts are data relative to solvent (DMSO-d)₆，δ_H 2.49；δ_C39.6). Circular dichroism data were obtained using a JascoJ-720 spectropolarimeter.

Examples

The following examples illustrate the preparation of Nocathiacin antibiotics and their biological properties. Those reasonable variations which can be envisioned by those skilled in the art do not depart from the scope of the invention.

Fermentation and purification of Nocathiacin I, II

Preparation of Nocathiac by fermentationin I and II

4ml of the frozen Nocardia sp.ATCCC 202099 nutrient medium was removed and inoculated into 100ml of 500ml flasks of the following seed culture: deionized water containing 20g starch, 5g glucose, 3g N-C case, 2g yeast extract, 5g fish extract and 3g calcium carbonate per liter. The cultures were incubated for 3 days at 32 ℃ in a rotary shaker at 250 rpm. 4ml of the resulting culture was inoculated into 200 cells each containing 100ml of the followingProduction of500ml flask of culture medium: each L of deionized water contained 20g of glucose, 5g of peptone, 10g of starfish starch and 5g of Allophosite.Production ofThe cultures were incubated at 32 ℃ for 5 days in a rotary shaker at 250rpm and then processed to recover Nocathiacin I and II.

Example 1: fermentation from Shake flasks

Preparation of crude extract

Nocardia sp.ATCC-202099(20L.) fermentation medium (whole medium including mycelia) was extracted with about 8L of ethyl acetate by vigorous shaking for 0.5 hour. The two-phase mixture was mixed with a 3L. (1Kg) precoat and then filtered under vacuum through a large Coors Buchner funnel (internal diameter 27cm, external diameter 28.5cm, depth 9 cm). The grayish yellow ethyl acetate extract was separated and evaporated to dryness under vacuum using a rotary evaporator to give about 7.2g of residue a.

Liquid-liquid distribution residue A

Residue A (7.2g) was dissolved in methanol containing 10% water. The solution was transferred to a separation funnel and extracted 4 times with the same volume of hexane. The hexane layer was removed. The methanol phase was diluted to 35% water in methanol by adding 38ml of water and extracted 3 times with the same volume of chloroform. Chloroform was saturated with 35% aqueous methanol. The hexane, chloroform and methanol extracts were evaporated to dryness in vacuo using a rotary evaporator. Nocathiacin antibiotic was detectable mainly in chloroform (1.3g residue B).

SeChromatographic separation of residue B from padex LH-20

Residue B (1.3g) was dissolved in 10ml 1: 1 chloroform-methanol and applied to a 3X 100cm Glenco column packed with 100g _ Sepadex LH-20 in 1: 1 chloroform-methanol suspension. The first 75ml was discarded and fractions were collected every 8-10ml at a flow rate of 2-3 ml/min. The fractions were solidified in silica gel TLC profiles (chloroform-methanol 9: 1, long-wave UV and/or ceric sulfate spray). In this way, a yellow fluorescent Nocathiacin antibiotic mixture was detected in fractions 10-15. The fractions were combined and evaporated to dryness (284mg residue C).

Silica gel liquid chromatography of residue C

The enriched fraction of the Nocathiacin antibiotic (residue C) was pre-adsorbed onto 2g of merck LiChroprep silica gel 60 and then fed to a 2.5X 115cm glazed filter funnel packed with this adsorbent. Elute under house vacuum first with 1: 1 hexane-chloroform (100ml), then with chloroform and gradually increasing chloroform ratios (e.g., 2.5, 5, 7.5, 10, 12.5, 15, and 25% methanol/chloroform solutions, 100ml each). The fractions were solidified in silica gel TLC profiles (chloroform-methanol 9: 1, long-wave UV and/or ceric sulfate spray). In this way, the Nocathiacin I antibiotic was detected in 5-12.5% methanol/chloroform fractions. Nocathiacin II antibiotics were detected in 15-15% methanol/chloroform fractions. The fractions were combined and evaporated to dryness (residue D, Nocathiacin I, 230mg), (residue E, Nocathiacin II, 49mg)

Isolation of Nocathiacin I and II

The residue D and E were further purified using a preparative HPLC system (YMC ODS-AQ C18 column) of Backman gold system. The typical sample size is 25-50 mg/50-100. mu.l of dimethyl sulfoxide solution. Elution with chloroform followed by a pre-programmed cone gradient to 8: 2 chloroform: methanol for 30 minutes. The flow rate was 10 ml/min. Ultraviolet detection at 290 nm. Nocathiacin obtained by this methodI(Peak with retention time of 17 minutes, Total yield 56mg) and NocathiacinII(peak retention time 19.5 min, total yield 6.7mg)。

Purification scheme

The following table describes the purification scheme of Nocathiacin antibiotics:

purification of Nocathiacins I and II

Example 1 fermentation from Shake flasks

Physico-chemical Properties II III of Nocathiacin I

The form is as follows: grayish yellow amorphous solid

The molecular formula is as follows: c₆₁H₆₀N₁₄O₁₈S₅

Molecular weight: 1436

Mass spectrum: HR-ESIMS [ M + H ]]⁺m/z 1437.285

ESI-MS/MS fragmentation ions：m/z1266，1248，

1221，1204，1186，1154，788

Infrared spectrum: dominant wavelength (cm)^-1)3392，3108，2932，1740，

1721，1694，1670，1640，1533，1478，1420，1384，1320，

1250，1207，1128，1091，1037，1014，751.

Ultraviolet spectrum: lambda [ alpha ]_max(MeOH)222，290，364nm(logε4.89，4.52，

4.17)

Circular dichroism spectrum: CD λ nm (. DELTA.. di-elect cons) (MeOH)212(+34.1), 239(-50.5),

267(+20.8)，307(-8.7)，364(+5.5)

HPLC retention time 25.6 min, (C18; 0.01M potassium phosphate buffer pH3.5

(Rt) -acetonitrile gradient elution (J chromatogr.385, 99, (1987))

¹H-NMR chemical shifts (relative to the solvent DMSO-d)₆Signal δ 2.49):

δ10.05(1H，s)，9.07(1H，s)，8.65(1H，s)，8.57(1H，

d，J＝8.0Hz)，8.51(1H，s)，8.50(1H，s)，8.24(1H，s)，

8.07(1H，s br)，7.89(1H，d，J＝11.2Hz)，7.86(2H，s)，

7.70(1H，d，J＝8.4Hz)，7.61(1H，s br)，7.34(2H，m)，

7.16(1H，d，J＝6.9Hz)，6.40(1H，s)，5.98(1H，d，

J＝12.0Hz)，5.77(1H，s)，5.73(1H，dd，J＝10.9，4.4

Hz)，5.70(1H，d，J＝8.7Hz)，5.22(1H，m)，5.02(1H，

d，J＝11.4Hz)，4.94(1H，d，J＝3.7Hz)，4.75(1H，d，

J＝10.2Hz)，4.51(1H，d，J＝11.0Hz)，4.29(1H，d，

J＝9.6Hz)，4.19(1H，m)，4.13(1H，d，J＝10.1Hz)，

4.00(1H，d，J＝95Hz)，3.88(3H，s)，3.78(1H，d br，

J＝5.9Hz)，2.52(6H，s)，2.49(1H，m)，2.11(1H，s br)，

1.99(3H，s)，1.95(1H，m)，1.81(1H，d，J＝13.9Hz)，

1.43(3H，s)，1.21(1H，m)，1.16(3H，s，br)，0.59(3H，

d，J＝6.3Hz).

¹³C-NMR chemical shifts (relative to the solvent DMSO-d)₆Signal δ 39.6):

δ171.8，168.1，167.7，167.4，167.0，165.2，164.0，

163.1，161.5，161.1，160.5，160.3，159.1，158.6，154.6，

151.9，149.7，149.6，148.8，145.8，142.5，135.3，135.0，

134.5，129.9，128.1，127.0，126.3，125.5，124.0，123.1，

119.8，119.4，112.7，111.2，109.7，103.7，95.1，79.3，

70.6，68.4，67.5，66.1，65.1，64.5，63.3，56.1，55.5，50.2，

49.8，44.4，40.0，30.5，18.0，13.1.

physico-chemical Properties of Nocathiacin II

The form is as follows: grayish yellow amorphous solid

The molecular formula is as follows: c₆₁H₆₀N₁₄O₁₇S₅

Molecular weight: 1420

Mass spectrum: HR-ESIMS [ M + H ]]⁺m/z 1421.297

ESI-MS/MS fragmentation ions：m/z 1250，1206，

1188，1170，1156，1138，788

Infrared spectrum: dominant wavelength (cm)^-1)3387，1725，1656，1534，

1478，1423，1318，1249，1192，1087，1014，886，793，

751.

Ultraviolet spectrum: lambda [ alpha ]_max(MeOH)220，295，364nm(logε5.01，4.67，

4.36)

Circular dichroism spectrum: CD λ nm (. DELTA.. di-elect cons) (MeOH)211(+41.5), 235(-56.1),

258(+21.0)，277(+13.4)，307(-10.0)，364(+5.6)

HPLC retention time 21.1 min, (C18; 0.01M potassium phosphate buffer pH 3.5-

(Rt) gradient elution with acetonitrile (J chromatogr.385, 99, (1987))

¹H-NMR chemical shifts (relative to the solvent DMSO-d)₆Signal δ 2.49);

δ9.10(1H，s)，8.59(1H，d，J＝8.5Hz)，8.51(1H，s)，

8.45(1H，s)，8.22(1H，s)，8.15(1H，s)，8.05(1H，s

br)，7.69(1H，sbr)，7.66(1H，s)，7.51(1H，d，J＝8.3

Hz)，7.47(1H，s)，7.30(1H，s)，7.25(1H，dd，J＝8.0，

7.2Hz)，7.12(1H，d，J＝7.1Hz)，7.02(1H，d，J＝8.4

Hz)，6.49(1H，s)，6.03(1H，d，J＝12.1Hz)，5.78(1H，

m)，5.70(1H，d，J＝8.6Hz)，5.66(1H，s)，5.30(1H，d，

J＝7.7Hz)，5.00(1H，d，J＝13.0Hz)，4.98(1H，d，

J＝10.5Hz)，4.94(1H，d，J＝4.8Hz)，4.83(1H，d，

J＝11.2Hz)，4.72(1H，m)，4.37(1H，d，J＝9.6Hz)，

4.23(1H，m)，4.11(1H，d，J＝9.9Hz)，4.03(1H，d，

J＝7.7Hz)，3.89(3H，s)，3.75(1H，m)，2.49(6H，s)，

2.14(1H，m)，2.01(3H，s)，1.94(1H，m)，1.79(1H，

d，J＝14.2Hz)，1.40(3H，s)，1.23(1H，m)，1.04(3H，

s，br)，0.54(3H，d，J＝6.5Hz)。

¹³C-NMR chemical shifts (relative to the solvent DMSO-d)₆Signal δ 39.6):

δ172.0，168.2，167.1，167.0，165.5，165.3，163.2，

162.9，161.6，161.2，160.6，160.1，158.7，156.3，149.9，

149.0，148.2，145.9，138.8，136.9，134.3，128.8，127.9，

127.2，126.6，125.6，125.2，124.7，124.1，123.9，122.8，

117.9，116.3，115.6，109.9，101.9，95.1.，79.0，70.7，

68.4，67.6，66.3，66.0，64.0，62.5，56.1，55.1，51.4，50.6，

44.4，40.5，30.6，18.1，18.0，13.0.

biological evaluation of Nocathiacin antibiotics

Example 2: antibacterial Activity of Nocathiacin I and II

To demonstrate their antibacterial activity, the Minimum Inhibitory Concentrations (MIC) of the Nocathiacin antibiotics of the present invention against various bacteria were determined by the conventional medium dilution method (serial dilution with nutrient medium (Difcos)). The results are shown in table 1, which indicates that Nocathiacin antibiotics can be used to treat bacterial infections.

TABLE 1

Bacterial name Strain number MIC (ug/ml)

Nocathiacin I Nocathiacin II

Streptococcus pneumoniae A95850.0010.015

Streptococcus pneumoniae/penicillin A278810.0010.015

intermediate

Streptococcus pneumoniae/penicillin resistant A282720.0010.015

Streptococcus pyogenes A96040.0010.125

Enterococcus faecalis A206880.030.5

Enterococcus faecalis A275190.030.5

Enterococcus faecalis + 50% calf serum A206880.250.5

Enterococcus faecalis A248850.0150.5

Enterococcus faecalis/S.thiotolerans SC158290.0250.5

Peptide (10ug/ml)

Enterococcus avium A274560.0150.5

Staphylococcus aureus A95370.0010.06

Staphylococcus aureus/beta-Penicillium A150900.0070.5

Enzyme positive

Staphylococcus aureus + 50% Calf A150900.0150.5

Serum

Staphylococcus aureus A244070.0070.5

/QC/ATCC#29213

Staphylococcus aureus/tolteromethylene A272180.0030.5

Oxacillin

Staphylococcus aureus + 50% Calf A272180.0070.5

Serum

Staphylococcus aureus/tolteromyces A272230.0030.125

Oxacillin

Staphylococcus aureus + 50% Calf A272230.0010.125

Serum

Micrococcus luteus A98520.0030.125

Bacillus subtilis A9506A 0.0010.125

Staphylococcus epidermidis A245480.0030.125

Lysostaphin A272980.030.25

Escherichia coli A15119 > 128

Escherichia coli A22292 > 128

Escherichia coli/AcrA: Kan A28901 > 128

Salmonella enteritidis A9531 & gt 128

Moraxella catarrhalis/beta-penicillium A223440.060.125

Enzyme positive

Moraxella catarrhalis/beta-penicillium A254090.060.125

Enzyme positive

Haemophilus influenzae/beta-penicillin A20191 > 128

Enzyme negative

Haemophilus influenzae/beta-penicillin a20183 & gt 128

Enzyme negative

Haemophilus influenzae/beta-penicillin A21515 > 128

Enzyme positive

Salmonella/WT A27207 & gt, 128

Salmonella/RE A27208 > 128

Example 3: in vivo antibacterial Activity of Nocathiacin I in a model of systemic Staphylococcus aureus infection

The in vivo antibacterial activity of Nocathiacin I was evaluated using a female ICR mouse systemic infection model. 6.5X 10 for animals⁶Staphylococcus aureus infection of CFU, which was previously cultured overnight suspended in 7% mucin. Nocathiacin I was dissolved in the test formulation consisting of 10% dimethyl sulfoxide, 5% Tween 80 and 85% water. The solution was administered subcutaneously at a total dose of 100mg/kg (2X 50mg/kg at 1 and 4 hours post infection). All 9 mice survived during the experiment and were free of toxic symptoms. Nocathiacin I was found to have a PD50 of less than 6.25 mg/kg.

Preparation of Nocathiacin by fermentationIII

Production of Nocathiacin III may be carried out at any temperature conducive to the growth of the culture, such as 16 ℃ and 40 ℃, but is preferably carried out at 22-26 ℃. The liquid medium is necessarily incubated for a period of time necessary to ensure that production of Nocathiacin III is sufficiently performed, typically monitored by HPLC for 1-5 days, with a rotational shaker speed during production of 50-300rpm, preferably 150-200 rpm.

The product Nocathiacin III can be recovered from the culture medium in the same manner as other known biologically active substances. Nocathiacin III was obtained by extraction of the culture medium with a conventional solvent such as ethyl acetate, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin) and a conventional adsorbent (e.g., activated carbon, silica gel, cellulose and alumina), crystallization, recrystallization, and/or reverse phase preparative HPLC purification.

The following example illustrates the fermentative preparation of Nocathiacin III. Those reasonable variations which can be envisioned by those skilled in the art do not depart from the scope of the invention.

Fermentation of

Example 4:

4ml of the frozen Nocardia sp.ATCCC 202099 nutrient medium was taken and inoculated into 100ml 500ml flasks of the following seed medium: each ml of deionized water contained 20g of Japanese soluble starch, 5g of glucose, 3g N-C case, 2g of yeast extract, 5g of fish meat extract and 3g of calcium carbonate. The cultures were incubated for 3 days at 28 ℃ in a rotary shaker at 250 rpm. 4ml of the resulting culture were inoculated into 10 500ml flasks containing 100ml of the following production medium: each ml of deionized water contained 10g HY yeast 412, 20g glucose and 10g Nutrisoy. The production cultures were incubated for 2 days at 28 ℃ in a rotary shaker at 250rpm, and Nocathiacin III was recovered.

Example 5:

4ml of the frozen Nocardia sp.ATCC202099 nutrient medium was removed and inoculated into 100ml of a 500ml flask of the following seed broth: each ml of deionized water contained 20g of Japanese soluble starch, 5g of glucose, 3g N-C case, 2g of yeast extract, 5g of fish meat extract and 3g of calcium carbonate. The cultures were incubated for 3 days at 28 ℃ in a rotary shaker at 250 rpm. 4ml of the resulting culture were taken and inoculated into 5 500ml flasks containing 100ml of fresh seed medium, respectively, and the culture was incubated for 3 days at 28 ℃ in a rotary shaker at 250 rpm. The media in the 5 flasks were mixed and then 4ml of the combined culture was inoculated into 100 500ml flasks containing 100ml of the following production medium: each ml of deionized water contained 10g HY yeast 412, 20g glucose and 10g Nutrisoy. The production cultures were incubated at 24 ℃ for 4 days in a rotary shaker at 180rpm, and Nocathiacin III was recovered.

Example 6: isolation of Nocathiacin III

Preparation of crude extract

Nocardia sp. atcc202099 fermentation medium (including mycelium) was extracted with about 1L of ethyl acetate with vigorous shaking. The biphasic mixture was centrifuged. The aqueous phase was extracted with additional ethyl acetate (0.5L). The gray yellow ethyl acetate extracts were combined and evaporated to dryness in vacuo using a rotary evaporator to give approximately 198mg of residue a.

Liquid-liquid distribution residue A

Residue A (198mg) was dissolved in 10ml of 10% water/methanol. The solution was transferred to a separation funnel and extracted 3 times with the same volume of hexane. The hexane layer was removed. The methanol phase was diluted to 35% water in methanol by adding 3.8ml of water and extracted 3 times with the same volume of chloroform. Chloroform was saturated with 35% aqueous methanol. The hexane, chloroform and methanol extracts were evaporated to dryness in vacuo using a rotary evaporator. Nocathiacin antibiotic was detectable mainly in chloroform, (106mg residue B).

Isolation of Nocathiacin III

Residue B was further purified using a preparative HPLC system (YMC ODS-AQ C18 column) of Backman gold System. A typical sample size is 50 mg/250. mu.l of dimethyl sulfoxide solution. Elution was carried out with a linear gradient of 0.1M ammonium acetate-acetonitrile (55: 45 v/v) to acetonitrile. The flow rate was 10 ml/min. And detecting by ultraviolet 360 hm. Nocathiacin III (peak with retention time 9 min, total yield 12mg) was obtained in this way.

Physico-chemical characteristics of Nocathiacin III

The form is as follows: pale yellow amorphous solid

The molecular formula is as follows: c₅₂H₄₃N₁₃O₁₆S₅

Molecular weight: 1265

Mass spectrum: HR-ESIMS [ M + H ]]⁺m/z 1266.162

ESI-MS/MS fragmentation ions；m/z 1248，1222，

1204，1186，788

Infrared spectrum: dominant wavelength (cm)^-1)3382，1720，1667，1643sh，

1534，1510，1477，1420，1250，1207，1126，1015，750.

Ultraviolet spectrum: lambda [ alpha ]_max(MeOH)nm 224，290，364(logε4.85，4.52，

4.11).

Circular dichroism spectrum: CD λ nm (. DELTA.. di-elect cons) (MeOH)212(+38.7), 238(-47.6),

266(+21.2)，305(-11.4)，362(+5.1).

HPLC retention time 19.3 min, (C18; 0.01M potassium phosphate buffer pH 3.5-

(Rt) gradient elution with acetonitrile (J chromatogr.385, 99, (1987))

¹H-NMR chemical shifts (relative to the solvent DMSO-d)₆Signal δ 2.49):

δ10.05(1H，s)，8.93(1H，s)，8.59(1H，s)，8.51(1H，

s)，8.46(1H，s)，8.37(1H，d，J＝8.9Hz)，8.20(1H，s)，

8.05(1H，s)，7.91(1H，d，J＝10.9 Hz)，7.77(1H，s br)，

7.74(1H，s br)，7.69(1H，d，J＝8.4Hz)，7.60(1H，s)，

7.38(1H，d，J＝7.5Hz)，7.34(1H，dd，J＝8.2，7.2Hz)，

7.18(1H，d，J＝7.0Hz)，6.37(1H，s)，6.07(1H，d，

J＝7.1Hz)，5.92(1H，d，J＝12.2Hz)，5.89(1H，d，

J＝10.3Hz)，5.74(1H，dd，)＝10.8，4.8H2)，5.72(1H，

s)，5.24(1H，m)，5.02(1H，d，J＝12.5Hz)，4.71(1H，

d，J＝10.4Hz)，4.54(1H， d，J＝11.2Hz)，4.50(1H，m)，

4.20(1H，m)，4.13(1H，d，J＝10.4Hz)，4.02(1H，dd，

J＝9.4，7.2Hz)，3.87(3H，s)，3.73(1H，d，J＝9.6Hz)，

1.97(3H，s)，1.15(3H，s br).

¹³C-NMR chemical shifts (relative to the solvent DMSO-d)₆Signal δ 39.6):

δ174.5，172.2，168.1，167.8，166.6，165.3，164.4，

163.0，161.2，160.4，159.3，158.8，154.7，152.9，149.6，

148.8，146.0，141.6，135.7，135.0，134.5，12.9.9，128.6，

127.1，126.4，126.3，125.6，125.3，124.2，123.1，119.7，

119.5，112.5，111.3，109.8，103.7，81.4，67.9，67.3，

65.3，64.5，63.5，56.2，55.7，49.7，49.5，18.0，13.2.

example 7: antibacterial Activity of Nocathiacin

To demonstrate its antibacterial activity, the Minimum Inhibitory Concentration (MIC) of Nocathiacin III antibiotics against various bacteria was determined by the conventional medium dilution method (serial dilution with nutrient medium (Difcos)). The results are shown in Table 2, which indicates that the Nocathiacin III antibiotic is useful for the treatment of bacterial infections.

TABLE 2

Bacterial name Strain number MIC (ug/ml)

NocathiacinIII

Streptococcus pneumoniae A9585 is less than or equal to 0.002

Streptococcus pneumoniae/penicillin intercediate A27881 is less than or equal to 0.002

Streptococcus pneumoniae/penicillin-resistant A28272 less than or equal to 0.002

Enterococcus faecalis A206880.03

Enterococcus faecalis A275190.03

Enterococcus faecalis + 50% calf serum A206880.25

Enterococcus faecalis A248850.03

Enterococcus avium A274560.03

Staphylococcus aureus/beta-penicillinase yang A150900.007

Property of (2)

Staphylococcus aureus + 50% calf serum A150900.03

Staphylococcus aureus/QC/ATCC # 29213A 244070.007

Staphylococcus aureus/Penicillium allomethidahliae A272230.007

Vegetable extract

Staphylococcus aureus + 50% calf serum A272230.007

Staphylococcus epidermidis A245480.007

Staphylococcus haemolyticus A272980.007

Moraxella catarrhalis/beta-penicillinase A223440.06

Property of (2)

Moraxella catarrhalis/beta-penicillinase A254090.06

Property of (2)

Haemophilus influenzae/beta-penicillinase negative A20191 > 64

Haemophilus influenzae/penicillinase negative A20183 > 64

Haemophilus influenzae/beta-penicillinase positive A21515 > 64

Claims (1)

1. A biologically pure culture of the Nocardia species of the microorganism having accession number ATCC-202099.