US20060004185A1 - Peptide antibiotics and peptide intermediates for their prepartion - Google Patents
Peptide antibiotics and peptide intermediates for their prepartion Download PDFInfo
- Publication number
- US20060004185A1 US20060004185A1 US10/881,160 US88116004A US2006004185A1 US 20060004185 A1 US20060004185 A1 US 20060004185A1 US 88116004 A US88116004 A US 88116004A US 2006004185 A1 US2006004185 A1 US 2006004185A1
- Authority
- US
- United States
- Prior art keywords
- dab
- peptide
- fmoc
- thr
- protected
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 0 *NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=C(CC(=O)O)C=C2.*NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=C(S(=O)(=O)O)C=C2.*NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=CC=C2.*NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=CC=C2C(=O)O.CC Chemical compound *NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=C(CC(=O)O)C=C2.*NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=C(S(=O)(=O)O)C=C2.*NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=CC=C2.*NC(=O)OCC1C2=C(C=CC=C2)C2=C1C=CC=C2C(=O)O.CC 0.000 description 12
- STYAWVIANAKHSC-UHFFFAOYSA-N CC(=O)C(CCNP)NC(=O)C(N)C(C)O Chemical compound CC(=O)C(CCNP)NC(=O)C(N)C(C)O STYAWVIANAKHSC-UHFFFAOYSA-N 0.000 description 6
- NYUHDIDHOKTXKK-UHFFFAOYSA-N CC(=O)C(CCNP)NC(=O)C(NC(=O)C(N)CCNP)C(C)O Chemical compound CC(=O)C(CCNP)NC(=O)C(NC(=O)C(N)CCNP)C(C)O NYUHDIDHOKTXKK-UHFFFAOYSA-N 0.000 description 4
- HMUSXIXOEIDKGT-UHFFFAOYSA-N CC(=O)C(N)CCNP Chemical compound CC(=O)C(N)CCNP HMUSXIXOEIDKGT-UHFFFAOYSA-N 0.000 description 3
- CZOQGVXTIBASKJ-IEOVAKBOSA-N CC(=O)C(N)CCNP.[2HH] Chemical compound CC(=O)C(N)CCNP.[2HH] CZOQGVXTIBASKJ-IEOVAKBOSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0815—Tripeptides with the first amino acid being basic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06086—Dipeptides with the first amino acid being basic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
- C07K7/62—Polymyxins; Related peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- This invention relates to methods for preparing novel protected peptide intermediates from polymyxin B or from related lipopeptide antibiotics, which are used to readily prepare new families of antibiotics that have potent activity against gram-negative and gram-positive bacteria.
- Polymyxin B and the related colistin have been used in humans but their use has been previously restricted because of toxicity and the availability of the other less toxic and previously effective antibiotics cited above (Ref. 1). Polymyxin B and colistin are usually administered by intravenous or intramuscular dosing.
- Colistimethate sodium is a water soluble salt of colistin/formaldehyde/bisulfite and has been of particular therapeutic value in acute and chronic urinary tract infections caused by strains of Pseudomonas aeruginosa (PDR Generics).
- Polymyxin B sulfate is the drug of choice in the treatment of infections of the urinary tract, meninges, and bloodstream caused by susceptible strains of P. aeruginosa (PDR Generics). Aerosolized polymyxin B is an important component of therapeutic regimens used in the management of pseudomonal lung infections, characteristically found in cystic fibrosis (Ref. 2). Some renal toxicity was observed with recommended dosing of polymyxin B in patients.
- the total synthesis of polymyxin B and four analogs was accomplished by a combination of solid phase peptide syntheses to obtain linear structures followed by removal from the resin and condensation in solution at high dilution to obtain the cyclic peptides (Ref. 7).
- the derivatives were less active than polymyxin B and one compound contained an Fmoc group in the terminal acyl position.
- a more recent total synthesis of polymyxin B and a few closely related compounds was accomplished only by solid phase peptide synthesis (Ref. 15). Both of these solid phase total synthetic approaches can provide new derivatives of polymyxin but these methods appear limited compared to the methods of the present invention.
- the quantities of antibiotic produced are small and require large amounts of amino acid precursors.
- a primary object of the present invention is to provide potent and effective antibiotics for treating gram-negative bacteria, without limiting use because of toxicity.
- a further object of the present invention is to provide intermediates from polymyxin B or related lipopeptide antibiotics which can be used to prepare new families of antibiotics having potent activity against gram-negative and gram-positive bacteria.
- polymyxin B protected form of polymyxin B is polyanionic, water soluble as a salt, and readily reacts in aqueous solution with a particular deacylase enzyme produced by Actinoplanes utahensis. It has not been found to react with polymyxin deacylase or the other enzymes commonly used to produce the nonapeptides of polymyxin or colistin (Ref. 11).
- the deacylated protected polymyxin peptide designated the protected PBpeptide-3, has three amino acids in the side chain.
- the enzyme from Actinoplanes utahensis can be used as the whole broth from the fermentation, the washed cells, or a water-solubilized preparation.
- the water-solubilized enzyme preparation was obtained by a basic extraction of the washed cells and then the clear extract was adjusted to pH 7-8. This enzyme preparation is the easiest to use, can be freeze-dried to a powder form, and is the most efficient.
- This invention includes methods for preparing four novel protected peptide intermediates from polymyxin B or related lipopeptide antibiotics that are used to prepare new families of anti-biotics which have potent activity against gram-negative, and in some cases gram-positive bacteria.
- Polymyxin B can be isolated from the fermentation of Bacillus polymyxa according to procedures described in Ref. 14. New antibiotics can be prepared from not only polymyxin B but also from related antibiotics such as other polymyxins, colistin, circulin, and octapeptin (EM49, Ref. 13) by these procedures.
- the five basic groups of polymyxin B are protected with a sulfonic acid derivative of 9-fluorenylmethoxycarbonyl, as shown in the following structures of the protecting groups:
- the deacylated protected polymyxin peptide designated the protected PBpeptide-3, has three amino acids in the side chain.
- the enzyme preparation that deacylates the protected polymyxin B can be obtained as a water soluble freeze-dried powder which is relatively stable.
- the preparation of this enzyme is easily accomplished and requires a unique process involving fermentation of Actinoplanes utahensis, separating the cells from the fermentation, washing the cells with water, extracting the cells with a basic buffer at pH 10 for about 20 minutes, adjusting the extract to pH 7-8 and freeze-drying.
- the powdered form of the enzyme resulting from this process is relatively stable and can be readily re-dissolved in water for use. Further purification can be obtained by gel filtration chromatography.
- This enzyme readily deacylates the N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)] 5 polymyxin B to obtain the protected polymyxin B peptide.
- This soluble enzyme was also effective in current studies for deacylation of N-fluorenlymethoxycarbonyl) amphomycin, laspartomycin, and N-fluorenlymethoxycarbonyl)-A21978C which are not polymyxin.
- polymyxin-type antibiotics such as the polymyxins, octapeptins, colistin, or circulins
- the procedures described above can be used to prepare protected intermediates selected from a group consisting of the following or their corresponding salts:
- the present invention describes semisynthetic antimicrobial cyclopeptides and their methods of preparation from the PBpeptides or other related peptides derived from collistin, circulin or octapeptin.
- This invention describes four different PBpeptide intermediates which can be elaborated further to afford the new antimicrobial derivatives, illustrated in the following embodiment:
- Each of these novel PBpeptide intermediates can be used to make new series of potent antibacterial antibiotics by the general procedures described.
- the desired acids were converted to activated species and then coupled to the protected intermediate PBpeptides and deprotected to give the new antibiotics as represented by compounds 1, 2, 6, 7, 11 and 12 in Table 1.
- the protected intermediate PBpeptides could also be acylated directly with alkyl or aromatic isocyanates or isothiocyanates to give the corresponding ureas and thioureas and the products deprotected to give other new series of antibiotics as represented by compounds 3-5.
- the biological activities of some of the new antibiotics prepared from PBpeptides by these procedures are given in Table 1.
- the HSO 3 Fmoc or other acidic Fmoc protected peptides from colistin (Cpeptides), circulin A (CA peptides), octapeptin B (Obpeptin), octapeptin C (OCpeptides), polymyxin A (PApeptides), and polymyxin D (PDpeptides) can be derivatized to give new antibiotics and new antibiotic prodrugs by the described procedures.
- the new HSO 3 Fmoc derivatives of our new antibiotics are listed in Table 2, and would all be expected to be pro-drugs for their corresponding antibiotics.
- the pro-drug may act as a slow release mechanism for the pharmacokinetics of the antibiotic.
- Polymyxin B sulfate (1.0 g., 0.841 mmol.) was dissolved in a solution of 25 ml., saturated sodium bicarbonate, 25 ml. of water and 25 ml. of tetrahydrofuran.
- a solution of (2-sulfo)-9-fluorenylmethoxy-N-hydroxysuccinimide (2.0 g., 4.8 mmol.) in 25 ml. of tetrahydrofuran was added in several portions over 45 min.
- the reaction mixture was stirred at room temperature over night and diluted with 50 ml. of water, then acidified with 25 ml of 6N hydrochloric acid to give an oily precipitate.
- the deacylase is produced by culturing Actinoplanes utahensis NRRL 12052 under submerged aerobic fermentation conditions. Because single-colony isolates from a lyophile of the culture were heterogeneous for both morphology and enzyme production capability, selections were made to recover a stable, high-producing variant. Initially, multiple fermentations were carried out using inocula prepared from strain 12052. Vegetative growth from the flask yielding the best deacylating activity was plated on a differential agar (CM). Colonies were then selected for further evaluation. Generally, small colonies were better enzyme producers than the large colony types. Isolate No. 18 was selected as a small colony type and shown to be the best deacylase producer of all colonies selected.
- CM differential agar
- CM agar contained corn steep liquor 0.5%, Bacto peptone 0.5%, soluble starch 1.0%, NaCl 0.05%, CaCl 2 .2H 2 O 0.05% and Bacto agar 2.0%.
- the fermentation protocol employed is known (17).
- a high-producing, natural variant was used in this invention.
- the resulting mycelial suspension was transferred into 50 mL of PM3 medium in a 250-mL Erlenmeyer flask.
- This medium contained sucrose 2.0%, peanut meal 1.0%, K 2 HPO 4 0.12%, KH 2 PO 4 0.05% and MgSO 4 .7H 2 O 0.025% in tap water.
- the flask was incubated at a temperature of 30° C. for a period of 60 to 90 hrs.
- the harvest time was determined by an assay which involved HPLC analyses of the deacylation of (2-Sulfo-9-fluorenlymethoxycarbonyl)] 5 polymyxin B by the whole broth at different times during the fermentation.
- Precipitation Method Cells from 450 ml deacylase enzyme were washed 3 ⁇ with water, then brought back to original volume with 0.02 M ammonium phosphate buffer and adjusted to pH 8.0. N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)] 5 -polymyxin B, 897 mg, was added and the mixture was placed on a shaker at 174 rpm and maintained at 30° C. After five hours the mixture was separated by centrifuging. The clear decant was adjusted to pH 2.3 with 1 N HCl to induce precipitation and allowed to stand at room temperature.
- the precipitate was separated from the mixture, slurried in 80 ml water, adjusted to pH 6.5 to obtain a clear solution, and freeze-dried to obtain 400 mg of tan powder, the semi-purified salt of the protected peptide, N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl] 5 -polymyxin peptide [(NaSO 3 -Fmoc) 5 -PBP-3].
- the cells were extracted with methanol/water to obtain additional material.
- Resin Method Cells from 1 liter deacylase enzyme were washed 3 ⁇ with water, brought back to original volume with 0.02 M ammonium phosphate buffer, and combined with 2.0 g of N-(2-sulfo)-9-fluorenlymethoxycarbonyl)] 5 -polymyxin B. The mixture was placed on a shaker at 175 rpm and maintained at 30° C. for 17 hours. The mixture was then separated by centrifuge and the decant was combined with 20 ml of Amberchrom® CG-161m resin. The resin was washed with 150 ml water, 100 ml 10% CH 3 CN:H 2 O (3 ⁇ ), and 100 ml 20% CH 3 CN:H 2 O (2 ⁇ ).
- the peptide was then eluted 2 ⁇ with 30% CH 3 CN:H 2 O, evaporated to remove CH 3 CN, and freeze-dried to obtain 283 mg powder, the purified peptide.
- the remaining peptide was then eluted 3 ⁇ with 100 ml 50% CH 3 CN:H 2 O, which were combined evaporated and freeze-dried to obtain 460 mg powder, the purified peptide.
- the remaining peptide was extracted from the cells 6 ⁇ with MeOH: H 2 O, 100 ml each. The extracts were combined, brought to four liters with water, adjusted to pH 2.1 with sulfuric acid and combined with Amberchrom® CG-161m resin.
- the resin was rinsed with water and then the peptide was eluted with 100 ml 35% CH 3 CN:H 2 O, which was evaporated and freeze-dried to obtain 94 mg purified peptide. The remaining peptide was eluted with 50% CH 3 CN:H 2 O, evaporated to remove CH 3 CN, and freeze-dried to obtain 539 mg of the purified peptide.
- the [(2-Sulfo)-9-fluorenlymethoxycarbonyl)] 5— polymyxin B peptide[(HSO 3 -Fmoc) 5 -PBP-3)] was isolated from these procedures as tan powder, C 122 H 134 N 16 O 37 S 5 .
- N-phenylthiocarbamyl-(NaSO 3 -Fmoc) 5 -PBP-3 was dissolved in 0.20 mL dimethylformamide (DMF), 0.010 mL piperidine was added and stirred at RT for 60 min.
- the reaction mixture was diluted with 4 mL 0.10M ammonium acetate-0.050M acetic acid (pH 5.05) and 0.006 mL acetic acid and 4 mL MeOH.
- the clear solution was applied to a CM-Sepharose® column (10 ⁇ 20 mm, ca. 2 mL volume) which had been conditioned with 50% MeOH 0.05M in ammonium acetate buffer at pH 5.0.
- the sample-loaded column was rinsed with 4 mL 50% MeOH-0.05M ammonium acetate pH 5.0 then with 4 mL 0.05M ammonium acetate pH 5.0 buffer.
- Product was eluted with 8 mL of 0.27 M sodium sulfate at pH 2.3.
- the product was further purified by application onto a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) which was eluted with incrementally increasing concentrations of CH 3 CN 0.05M in pH 2.3 sodium sulfate; product was eluted with 20% CH 3 CN.
- Solvent was removed under vacuum from the product-containing fraction pool which was then desalted in similar fashion as in Example 5, pH adjusted to 6.3 then freeze dried. Yield: 2.7 mg of a white solid, PTC-PBP-3, C 54 H 87 N 17 O 12 S.
- N-phenylthiocarbamyl-(NaSO 3 -Fmoc) 5 -PBP-3 was dissolved in 0.30 mL of anhydrous trifluoroacetic acid (TFA) and heated in a 50° C. water bath for 15 min. TFA was evaporated with a stream of dry nitrogen and the residue was dissolved in 12 mL 0.20M ammonium phosphate at pH7.2 and 6 mL CH 3 CN containing 49 mg triglycine (as an acylating agent scavenger).
- TFA trifluoroacetic acid
- the product solution was first applied to a 0.5 g styrene-divinylbenzene resin cartridge (EnviChrom-P) which was eluted with 10 mL of 40% CH 3 CN 0.04M in ammonium phosphate at pH 7.2.
- Product-containing fractions were pooled ( ⁇ 18 mL), diluted with 12 mL distilled water, then applied to a fresh 0.5 g resin cartridge which was eluted with incrementally increasing concentrations of CH 3 CN about 0.05M in ammonium phosphate at pH 7.2.
- reaction mixture was diluted with 4 mL 0.25M ammonium sulfate at pH2.3 yielding a very milky mixture at pH 3.0 which was extracted with 4 mL ethylacetate; the product-containing aqueous phase was diluted with 4 mL distilled water.
- Product was initially isolated by size exclusion chromatography on a Sephadex® G-25 column (2.5 ⁇ 40 cm) eluted with 0.10M ammonium sulfate at pH 2.3.
- reaction mixture was diluted with 5 mL 0.20M ammonium sulfate at pH2.3 yielding a very milky mixture at pH 2.8 which was extracted with 5 mL ethylacetate.
- Product was initially isolated by size exclusion chromatography on a Sephadex® G-25 column (2.5 ⁇ 40 cm) eluted with 0.10M ammonium sulfate at pH 2.3.
- Product-containing fractions were pooled and further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) by elution with incrementally increasing concentrations of CH 3 CN about 0.10M in ammonium sulfate at pH 2.3; product was eluted with 30% CH 3 CN. Further purification was achieved using size exclusion chromatography on a Sephadex LH-20 column (2.5 ⁇ 40 cm) eluted with 0.026M ammonium acetate/0.053M acetic acid in MeOH. Product-containing fractions were evaporated under vacuum to near dryness, the residue dissolved in 10 mL distilled water, then desalted and freeze dried in similar fashion as in Example 5.
- EnviChrom-P 0.5 g styrene-divinylbenzene cartridge
- Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH 3 CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% CH 3 CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 4.7 mg of a white solid, Compound 7, C 59 H 90 N 16 O 14 .
- Product was isolated on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH 3 CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% and 30% CH 3 CN. Product-containing fractions were pooled, solvent removed under vacuum, 2 mL 1.0M ammonium acetate pH 5.0 buffer was added, pH adjusted to 5.0 by addition of ca. 0.6 mL 1.5M NH 4 OH, and then diluted with an equal volume of MeOH. Product was isolated via CM-Sepharose chromatography as in Example 7.
- Example 8 The procedure used in Example 8 to prepare the (NaSO 3 -Fmoc) 4 -PBP-2 from (NaSO 3 -Fmoc) 5 -PBP-3 was repeated starting with (NaSO 3 -Fmoc) 4 -PBP-2, 50 mg, to remove the threonine residue and yielding (NaSO 3 -Fmoc) 4 -PBP-1, 37 mg, as a white powder, with the expected HPLC relative retention time.
- (NaSO 3 -Fmoc) 4 -PBP-2 (5.0 mg) was dissolved in 1.4 mL of 0.02M sodium citrate buffer (pH6.4) and 0.006M in 2-mercaptoethylamine(MEA) hydrochloride.
- the enzyme solution was prepared by dissolving 10 units (0.9 mg) of cathepsin C (dipeptidyl aminopeptidase) (EC 3.4.14.1) in 11.0 mL of the citrate-MEA buffer. Enzyme solution (0.25 mL) was added to the peptide solution which was incubated at about 37° C. for about 24 hours. HPLC analysis indicated about 75% conversion to (NaSO 3 -Fmoc) 3 -PBP.
- the incubated solution was diluted with 11.0 mL 0.4M pH7.2 ammonium phosphate buffer, 3.0 mL distilled water and 0.5 mL CH 3 CN.
- the product was isolated on an ENVI-Chrom-P 0.5 g resin cartridge; product was eluted with 25% CH 3 CN, 0.05M in pH7.2 buffer.
- product-containing fractions (accounting for about 72% of the total product) were pooled, desalted and freeze dried as in Example 8. Yield: 1.0 mg of a pale yellow solid, 87% by HPLC (215 nm area %), with the expected HPLC relative retention time for the (NaSO 3 -Fmoc) 3 -PBP.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Novel protected cyclopeptide intermediates are prepared from polymyxin B are used to synthesize new peptide antibiotics. Intermediates are readily derivatized and deprotected to provide new families of antibiotics, which have potent anti-bacterial activity against gram-negative bacteria; but also are useful and potent against gram-positive bacteria.
Description
- This invention relates to methods for preparing novel protected peptide intermediates from polymyxin B or from related lipopeptide antibiotics, which are used to readily prepare new families of antibiotics that have potent activity against gram-negative and gram-positive bacteria.
- As and for the background for the present invention, the following references were used (throughout this description of the present invention, the numbers of the references will be referred to):
- 1. Evans, M. E., Feola, D. J., Rapp, R. P. 1999 Polymyxin B Sulfate and Colistin: Old Antibiotics for Emerging Multiresistant Gram-Negative Bacteria. The Annals of Pharmacotherapy. 33:960-967.
- 2. McCallister, S. M., Alpar H. O., Brown, M. R. W. 1999. Anti-microbial Properties of Liposomal Polymyxin B. Journal of Anti-microbial Chemotherapy 43:203-210.
- 3. Chihara, S., Ito, A., Yahata, M., Tobita, T., Koyama, Y. 1974. Chemical Synthesis and Characterization of n-Fattyacyl Mono-Aminoacyl Derivatives of Colistin Nonapeptide. Agr. Biol. Chem. 38:(10), 1767-1777.
- 4. Chihara S., Ito, A., Yahata M., Tobita, T., Koyama, Y. 1973. Chemical Synthesis and Characterization of α-N-Octanoyl and Other α-N-Acyl Nonapeptide Derivatives. Agr. Biol. Chem. 37: (12), 2709-2717.
- 5. Chihara, S., Ito, A., Yahata, M., Tobita, T., Koyama, Y. 1974. Chemical Synthesis, Isolation and Characterization of n-N-Fattyacyl Colistin Nonapeptide with Special Reference to the Correlation Between Antimicrobial Activity and Carbon Number of Fattyacyl Moiety. Agr. Biol. Chem. 38:(3), 521-529.
- 6. Weinstein, J., Afonso, A., Moss, E. J R., Miller, G. H. 1998. Selective Chemical Modifications of Polymyxin B. Bioorganic & Medicinal Chemistry Letters 8:3991-3996.
- 7. Tsubery, H., Ofek, I., Cohen, S., Fridkin, M. 2001. N-Terminal Modifications of Polymyxin B Nonapeptide and Their Effect on Antibacterial Activity. Peptides. 22: 1675-1681.
- 8. Mutter, M., and Bellof, D., A New Base-labile Anchoring Group for Polymer-supported peptide synthesis, Helv. Chim. Acta, 1984, 67, 2009.
- 9. Liu, Y.-Z., Ding, S.-H., Chu, J.-Y. and Felix, A. M., 1990, A Novel Fmoc-based Anchorage for the Synthesis of protected Peptide on Solid Phase, Int. J. Pept. Protein Res., 35, 95.
- 10. Markou, N., Apostolakos, Koumoudiou, C., Anthanasiou, M., Koutsoukou, A., Alamanos I., and Gregorakos, L., 2003, Intravenous colistin in the treatment of sepsis from multresistant Gran-negative bacilli in critically ill patients, Critical Care, 7, R78-R83.
- 11. Duwe, A. K., Rupar, C. A., Horsman, G. B., and Vas, S. I., 1986. In Vitro Cytotoxicity and Antibiotic Activity of Polymyxin B Nonapeptide, Antimicrobial Agents and Chemotherapy, 30:340-341.
- 12. Kurihara T., Takeda, H., and Ito, H. 1972, Compounds related to colistin. V. Synthesis and pharmacological activity of colistin analogs, Yakugaku Zasshi, 92:129-34.
- 13. Parker, W. L., Rathnum, M. L. 1975. EM49, A New Peptide Antibiotic IV. The Structure of EM49. The Journal of Antibiotics. 28:(5), 379-389.
- 14. Hausmann, W., et al., 1954, Polymyxin B1. Fractionation, molecular-weight determination, amino acid and fatty acid composition, J. Am. Chem. Soc. 76, 4892-4896.
- 15. DeVisser Kriek, N. M. A. J., van Hooft, P. A. V., Van Schepdael A., Fillipov, D. V., van der Marel, G. A., Overcleeft, H. S., van Boom, J. H., and Noort, D.m 2003, Synthesis of polymyxin B and analogues, J. Peptide Res. 61, 298-306.
- 16. Kimura, Y., Matsunaga, and Vaara, M., 1992, Polymyxin B Octapeptide and Polymyxin B Heptapeptide are Potent Outer Membrane Permeability-Increasing Agents. J. Antibiotics, 45m, 742-749.
- 17. Boeck L. D., Fukuda, D. S., Abbott, B. J., Debono, M. 1988. Deacylation of Actinoplanes utahensis. Journal of Antibiotics 41:(8), 1085-1092.
- 18. Kreuzman, A. J., Hodges, R. L., Swartling, J. R., Ghag, S. K., Baker, P. J., McGilvray, D., Yeh, W. K. 2000. Membrane-Associated Echinocandin B Deacylase of Actinoplanes utahensis: Purification, Characterization, Heterologous Cloning and Enzymatic Deacylation Reaction. Journal of Industrial Microbiology 24: 173-180.
- 19. Boeck, L. D., Fukuda, D. S., Abbott, B. J., Debono, M. 1989. Deacylation of Echinocandin B by Actinoplanes utahensis. Journal of Antibiotics 42:(3), 382-388.
- 20. Borders, D. B., Curran, W. V., Fantini, A. A., Francis, N. D., Jarolmen, H., and Leese, R. A., 2003 Derivatives of Laspartomycin and Preparation and Use Thereof, U.S. Pat. No. 6,511,962.
- Gram-negative bacteria that are resistant to the amino-glycosides, β-lactams, and fluoroquinolones are becoming more common. These bacteria are often susceptible to the polymyxins (Refs. 1, 10). Polymyxin B and the related colistin (polymyxin E) have been used in humans but their use has been previously restricted because of toxicity and the availability of the other less toxic and previously effective antibiotics cited above (Ref. 1). Polymyxin B and colistin are usually administered by intravenous or intramuscular dosing. Colistimethate sodium is a water soluble salt of colistin/formaldehyde/bisulfite and has been of particular therapeutic value in acute and chronic urinary tract infections caused by strains of Pseudomonas aeruginosa (PDR Generics). Polymyxin B sulfate is the drug of choice in the treatment of infections of the urinary tract, meninges, and bloodstream caused by susceptible strains of P. aeruginosa (PDR Generics). Aerosolized polymyxin B is an important component of therapeutic regimens used in the management of pseudomonal lung infections, characteristically found in cystic fibrosis (Ref. 2). Some renal toxicity was observed with recommended dosing of polymyxin B in patients. Neurotoxicity is seen most often in patients with compromised renal function, with an overall incidence of 7.3% reported in one large study with colistin (Ref. 1). When the acyl side chain along with the adjacent diaminobutric acid residue are removed from polymyxin B by an enzyme, the nonapeptide is obtained. The antibacterial activity of this compound is about 2-64 times less potent and the toxicity in cell culture is reduced by about 100 fold (Ref. 11). The in vivo toxicity of the nonapeptide of polymyxin B is significantly less than polymyxin B itself (16). A need for a better version of polymyxin is apparent since it is acceptable for limited use in humans at this time and it is not cross resistant with the aminoglycosides, β-lactams, and fluoroquinolones.
- There have been many studies to chemically modify polymyxin and colistin to obtain antibiotics with improved biological properties. Most of these studies were done before good characterization techniques such as HPLC, FABMS and ESIMS were available. In these studies, the nonapeptide has been derivatized by procedures with only some selectivity (Refs. 3, 4, 5, 10, 11, 12). Chromatographic purification methods were limited and many of the products seemed to be relatively impure. More recently, selective chemical modifications of polymyxin B were reported; however, no attempt was made to modify the acyl side chain and amino acids of the side chain (Ref. 6). Chemical selectivity among the basic amino groups of polymyxin B was obtained by pH control. The total synthesis of polymyxin B and four analogs was accomplished by a combination of solid phase peptide syntheses to obtain linear structures followed by removal from the resin and condensation in solution at high dilution to obtain the cyclic peptides (Ref. 7). The derivatives were less active than polymyxin B and one compound contained an Fmoc group in the terminal acyl position. A more recent total synthesis of polymyxin B and a few closely related compounds was accomplished only by solid phase peptide synthesis (Ref. 15). Both of these solid phase total synthetic approaches can provide new derivatives of polymyxin but these methods appear limited compared to the methods of the present invention. The quantities of antibiotic produced are small and require large amounts of amino acid precursors. Any scale up of these methods for clinical studies would be very difficult. The methods described below provide a much better, faster, and efficient synthesis. In addition, the new compounds obtained in our studies have a greater structure diversity with a variety of linkers between the side chain and the cyclic peptide.
- Accordingly, a primary object of the present invention is to provide potent and effective antibiotics for treating gram-negative bacteria, without limiting use because of toxicity.
- A further object of the present invention is to provide intermediates from polymyxin B or related lipopeptide antibiotics which can be used to prepare new families of antibiotics having potent activity against gram-negative and gram-positive bacteria.
- These and other objects of the present invention are provided by protecting the five basic groups of polymyxin B with a sulfonic acid derivative of 9-fluorenylmethoxycarbonyl. This protected form of polymyxin B is polyanionic, water soluble as a salt, and readily reacts in aqueous solution with a particular deacylase enzyme produced by Actinoplanes utahensis. It has not been found to react with polymyxin deacylase or the other enzymes commonly used to produce the nonapeptides of polymyxin or colistin (Ref. 11). The deacylated protected polymyxin peptide, designated the protected PBpeptide-3, has three amino acids in the side chain. By a sequence of modified Edman degradations or enzymatic reactions, it is possible to obtain the protected forms of other new intermediates designated either the protected PBpeptide, PBpeptide-1 or PBpeptide-2 containing zero, one or two amino acids, respectively, in the side chain.
- Other acidic protecting groups in other salt forms, which would provide water solubility for the deacylase reaction are possible. Examples of these protecting groups are the carboxylic acid derivatives (Refs. 12, 13) of FMOC. If standard protecting groups such as the FMOC are used, the products have no significant water solubility and an enzyme system in an organic solvent or mixed organic solvent would be required.
- The enzyme from Actinoplanes utahensis can be used as the whole broth from the fermentation, the washed cells, or a water-solubilized preparation. The water-solubilized enzyme preparation was obtained by a basic extraction of the washed cells and then the clear extract was adjusted to pH 7-8. This enzyme preparation is the easiest to use, can be freeze-dried to a powder form, and is the most efficient.
- This invention includes methods for preparing four novel protected peptide intermediates from polymyxin B or related lipopeptide antibiotics that are used to prepare new families of anti-biotics which have potent activity against gram-negative, and in some cases gram-positive bacteria. Polymyxin B can be isolated from the fermentation of Bacillus polymyxa according to procedures described in Ref. 14. New antibiotics can be prepared from not only polymyxin B but also from related antibiotics such as other polymyxins, colistin, circulin, and octapeptin (EM49, Ref. 13) by these procedures. The five basic groups of polymyxin B are protected with a sulfonic acid derivative of 9-fluorenylmethoxycarbonyl, as shown in the following structures of the protecting groups:
-
- No reaction has been found with polymyxin deacylase or the other enzymes commonly used to produce the nonapeptide of polymyxin or colistin (Refs. 10, 11). The deacylated protected polymyxin peptide, designated the protected PBpeptide-3, has three amino acids in the side chain. By a sequence of modified Edman degradations or enzymatic reactions, it is possible to obtain the protected forms of other new intermediates designated either the protected PBpeptide, PBpeptide-1 or PBpeptide-2 containing zero, one or two amino acids, respectively, in the side chain, shown above.
- Other acidic protecting groups in their salt forms which would provide water solubility for the deacylase reaction are possible. Examples of these protective groups are the carboxylic acid derivatives (Refs. 8, 9) of FMOC as shown above. If standard protective groups such as the FMOC are used, the products have no significant water solubility and an enzyme system in an organic solvent or mixed organic solvent would be required. The enzyme from the Actinoplanes utahensis can be used as the whole broth from the fermentation, the washed cells, or a water-solubilized preparation. The water-solubilized enzyme preparation is obtained by a basic extraction of the washed cells and then returning the clear extract to pH 7-8. This enzyme preparation is the easiest to use, can be freeze-dried to a powder form, and is the most efficient.
- The enzyme preparation that deacylates the protected polymyxin B can be obtained as a water soluble freeze-dried powder which is relatively stable. The preparation of this enzyme is easily accomplished and requires a unique process involving fermentation of Actinoplanes utahensis, separating the cells from the fermentation, washing the cells with water, extracting the cells with a basic buffer at pH 10 for about 20 minutes, adjusting the extract to pH 7-8 and freeze-drying. The powdered form of the enzyme resulting from this process is relatively stable and can be readily re-dissolved in water for use. Further purification can be obtained by gel filtration chromatography. This enzyme readily deacylates the N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)]5polymyxin B to obtain the protected polymyxin B peptide. This soluble enzyme was also effective in current studies for deacylation of N-fluorenlymethoxycarbonyl) amphomycin, laspartomycin, and N-fluorenlymethoxycarbonyl)-A21978C which are not polymyxin. The enzyme from Actinoplanes utahensis for deacylation of echinocandin, an antifungal antibiotic, was obtained in a water soluble form by subjecting the cells of A. utahensis to a high concentration of potassium chloride (Refs. 18, 19). This same soluble enzyme preparation was used to deacylate daptomycin which was used as a control (Ref. 18). The (tert-BOC)-A21978C was deacylated with whole broth of Actinoplanes utahensis (Ref. 17). Laspartomycin was also deacylated with whole broth of Actinoplanes utanhesis (Ref. 20).
- For the polymyxin-type antibiotics such as the polymyxins, octapeptins, colistin, or circulins the procedures described above can be used to prepare protected intermediates selected from a group consisting of the following or their corresponding salts:
-
- a) H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]n
- b) H-(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]n
- c) H-(X3)-peptide-[(2-sulfo)-9-Fmoc]n
- d) H-peptide-[(2-sulfo)-9-Fmoc]3
- wherein for Case 1) H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]n
- H is hydrogen, X1 is L-Dab or another amino acid, X2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid, and n=3-6;
- for Case 2) H-(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]n
- H is hydrogen, X2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid, and n=3-5;
- for Case 3) H-(X3)-peptide-[(2-sulfo)-9-Fmoc]n
- H is hydrogen X3 is L-Dab or D-Dab or another amino acid and n=3-4; and
- for Case 4) H-peptide-[(2-sulfo)-9-Fmoc]3
- H is hydrogen.
- The present invention describes semisynthetic antimicrobial cyclopeptides and their methods of preparation from the PBpeptides or other related peptides derived from collistin, circulin or octapeptin. This invention describes four different PBpeptide intermediates which can be elaborated further to afford the new antimicrobial derivatives, illustrated in the following embodiment:
-
- 1) A-(X1)(X2)(X3)-PBpeptide
- 2) A-(X2)(X3)-PBpeptide
- 3) A-(X3)-PBpeptide
- 4) A-PBpeptide
- Wherein for Case 1) A-(X1)(X2(X3)-PBpeptide
- A=R′—(C═O)—, R′—SO1—, R′—(C═NH)—, R′—NH—(C═S)—, R′—NH—(C═O)—, R′—CH—, R′—O—(C═O)—, where R′ is alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, or heterocyclic and X1 is L-Dab or another amino acid, X2 is L-Thr or another amino acid, and X3 is L-Dab or another amino acid.
- For Case 2) A-(X2)(X3)-PBpeptide
- “A” is the same as described for Case 1, X2 is L-Thr or another amino acid and X3 is L-Dab or another amino acid.
- For Case 3) A-(X3)-BPpeptide
-
- “A” is the same as described in Case 1 and X3 is L-Dab or another amino acid.
- For Case 4) A-PBpeptide, “A” is same as described for Case 1.
- Each of these novel PBpeptide intermediates can be used to make new series of potent antibacterial antibiotics by the general procedures described. The desired acids were converted to activated species and then coupled to the protected intermediate PBpeptides and deprotected to give the new antibiotics as represented by compounds 1, 2, 6, 7, 11 and 12 in Table 1. The protected intermediate PBpeptides could also be acylated directly with alkyl or aromatic isocyanates or isothiocyanates to give the corresponding ureas and thioureas and the products deprotected to give other new series of antibiotics as represented by compounds 3-5. The biological activities of some of the new antibiotics prepared from PBpeptides by these procedures are given in Table 1.
- Similar to the derivatives of the polymyxin B peptides (PBpeptides), the HSO3 Fmoc or other acidic Fmoc protected peptides from colistin (Cpeptides), circulin A (CA peptides), octapeptin B (Obpeptin), octapeptin C (OCpeptides), polymyxin A (PApeptides), and polymyxin D (PDpeptides) can be derivatized to give new antibiotics and new antibiotic prodrugs by the described procedures. The structure for these peptides differ from the PBpeptide by the following:
Side chain cyclic peptide PBpeptide Dab-Thr-Dab- [Dab-Dab-D-Phe-Leu-Dab-Dab-Thr-] Cpeptide Dab-Thr-Dab- [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr-] CApeptide Dab-Thr- Dab - [Dab-Dab-D-Phe-Ileu-Dab-Dab-Thr-] PApeptide Dab-Thr-D-Dab- [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr-] PDpeptide Dab-Thr-D-Ser- [Dab-Dab-D-Leu-Thr-Dab-Dab-Thr-] OBpeptide D-Dab- [ Dab-Dab-D-Leu-Leu-Dab-Dab-Leu-] OCpeptide D-Dab- [ Dab-Dab-D-Leu-Phe-Dab-Dab-Leu-] - The various new antibiotics described in Table 1 all had good activity against Escherichia coli which is a gram-negative bacterium. The most potent were compounds 1, 4 and 5 which had minimum inhibitory concentrations of the antibiotics at 0.6 micrograms per milliliter. Compounds 2 and 10 were somewhat less potent against Escherichia coli but also had significant activity against Staphylococcus aureus which is a gram-positive bacterium. Therefore some of the new antibiotics can have activity against both gram- and negative and gram-positive bacteria.
TABLE 1 Minimum Inhibitory Concentrations (MIC) for New Peptide Antibiotics Com- E. coli Staph pound R X1 X2 X3 MIC* MIC* Poly- C8H17CO— Dab Thr Dab 0.6 >10 myxin B 1 n-C9H19CO— Dab Thr Dab 0.6 2 n-C10H21CO-PAPA-** Dab Thr Dab 1.25 2.5 3 n-C8H17NHCO— Dab Thr Dab 1.25 10 4 phenyl-NHCS— Dab Thr Dab 0.6 >10 5 phenyl-NHCO— Dab Thr Dab 0.6 >10 6 phenyl-CO— Dab Thr Dab 1.25 >10 7 2-naphthyl-OCH2—CO— Dab Thr Dab 1.25 >10 8 4-CH3—C6H4—SO2— Dab Thr Dab 9 n-C8H17NHCO— — Thr Dab 2.5 >10 10 n-C10H21SO2— Gly Thr Dab 2.5 5 11 n-C9H19CO— Lys Thr Dab 2.5 10 12 n-C9H19CO— Phe Thr Dab
*MIC values were determined by serial twofold broth dilution method using Escherichia coli, ATCC #26, and Staphylococcus aureus Smith as assay organisms which were grown in Mueller Hinton broth.
**p-aminophenylacetyl
- Another variation or series of new antibiotics is represented by the sulfonyl derivatives (Table 1, compounds 8 and 10). The side chains from these compounds are not attached to the PBpeptides by an acyl group but instead a sulfonyl group. Other linkers such as ureas or thioureas have been made and resulted in compounds with good antibacterial activity. Perhaps the most unexpected result was the potent activity of the aromatic acyl side chains and the aromatic groups linked through urea and thiourea linkages (compounds 4 and 5) as shown in Table 1.
TABLE 2 New Antibiotics and Intermediates Compound R X1* X2 X3* P (P)5Polymyxin B C8H17CO— Dab-P Thr Dab-P HSO3-Fmoc- 1 n-C9H19CO— Dab Thr Dab H 1P n-C9H19CO— Dab-P Thr Dab-P HSO3-Fmoc- 2 n-C10H21CO-PAPA-** Dab Thr Dab H 2P n-C10H21CO-PAPA-** Dab-P Thr Dab-P HSO3-Fmoc- 3 n-C8H17NHCO— Dab Thr Dab H 3P n-C8H17NHCO— Dab-P Thr Dab-P HSO3-Fmoc- 4 phenyl-NHCS— Dab Thr Dab H 4P phenyl-NHCS— Dab-P Thr Dab-P HSO3-Fmoc- 5 phenyl-NHCO— Dab Thr Dab H 5P phenyl-NHCO— Dab-P Thr Dab-P HSO3-Fmoc- 6 phenyl-CO— Dab Thr Dab H 6P phenyl-CO— Dab-P Thr Dab-P HSO3-Fmoc- 7 2-naphthyl-OCH2—CO— Dab Thr Dab H 7P 2-naphthyl-OCH2—CO— Dab-P Thr Dab-P HSO3-Fmoc- 8 4-CH3—C6H4—SO2— Dab Thr Dab H 8P 4-CH3—C6H4—SO2— Dab-P Thr Dab-P HSO3-Fmoc- 9 n-C8H17NHCO— — Thr Dab H 9P n-C8H17NHCO— — Thr Dab-P HSO3-Fmoc- 10 n-C10H21SO2— Gly Thr Dab H 10P n-C10H21SO2— Gly Thr Dab-P HSO3-Fmoc- 11 n-C9H19CO Lys Thr Dab H 11P n-C9H19CO Lys-P Thr Dab-P HSO3-Fmoc- 12 n-C9H19CO Phe Thr Dab H 12P n-C9H19CO Phe Thr Dab-P HSO3-Fmoc-
*Dab-P is 4-N—(HSO3-Fmoc)-diaminobutyryl, Lys-P is 6-N—(HSO3-Fmoc)-lysyl
**PAPA is p-aminophenylacetyl
All amino acids are the L-isomers unless indicated otherwise.
- The new antibiotics and the new protected antibiotics that were prepared are summarized in Table 2.
- Compounds designated 1P, 2P, 3P, 4P, 5P, 6P, 7P, 8P, 9P, 10P, 11P and 12P in Table 2 have the protecting group and would be expected to be pro-drugs. The protected antibiotics according to the present invention are most likely pro-drugs based on work with the corresponding derivatives of insulin and gentamicin. [See Gershonov, E., Goldwaser, I., Fridkin, M., Shechter, Y 2000. A Novel Approach for a Water-Soluble Long-Acting Insulin ProDrug; Design, Preparation, and Analysis of [(2-Sulfo)-9-Fluorenylmethoxycarbonyl]3-Insulin. Journal of Medicinal Chemistry 43:(13), 2530-2537; and Schechter, Y., Tsubery, H. Fridkin, M. 2002 N-[(2-Sulfo)-9-Fluorenylmethoxycarbonyl]3-Gentamicin, Is A Long-Acting Prodrug Derivative. Journal of Medicinal Chemistry 45: (19), 4264-4270]. The HSO3 Fmoc derivatives of insulin, a hormone, and gentamicin, an antibiotic active against gram-negative bacteria, undergo deprotection after they are injected into an animal such as a rat. This liberates the biologically active compound. The new HSO3Fmoc derivatives of our new antibiotics are listed in Table 2, and would all be expected to be pro-drugs for their corresponding antibiotics. The pro-drug may act as a slow release mechanism for the pharmacokinetics of the antibiotic.
- The following Examples are provided to enable a complete understanding of the method of preparation, the intermediates for the preparation of antibiotics and the products obtained from the method, according to the present invention:
- Polymyxin B sulfate (1.0 g., 0.841 mmol.) was dissolved in a solution of 25 ml., saturated sodium bicarbonate, 25 ml. of water and 25 ml. of tetrahydrofuran. A solution of (2-sulfo)-9-fluorenylmethoxy-N-hydroxysuccinimide (2.0 g., 4.8 mmol.) in 25 ml. of tetrahydrofuran was added in several portions over 45 min. The reaction mixture was stirred at room temperature over night and diluted with 50 ml. of water, then acidified with 25 ml of 6N hydrochloric acid to give an oily precipitate. The mixture was chilled and the aqueous layer was decanted and the oily residue was dissolved in 100 ml. of ethanol. The ethanol was evaporated under vacuum (35° C.) and the resulting solid was triturated with ethyl acetate, filtered and dried to afford 1.74 g. of product. HPLC with a gradient on a reverse phase column showed a single peak, N-[2-(sulfo)-9-fluorenlymethoxycarbonyl)]5-polymyxin B, C131H150N16O38S5, when the column eluent was monitered at 215 nm. ESIMS: calc. m/z for C131H152N16O38S5, (M+2H)+2=1358.4. Found 1358.5
- The above procedure can be used with polymyxin free base and 2(sulfo)-9-fluorenylmethoxycarbonyl chloride with similar results.
- The deacylase is produced by culturing Actinoplanes utahensis NRRL 12052 under submerged aerobic fermentation conditions. Because single-colony isolates from a lyophile of the culture were heterogeneous for both morphology and enzyme production capability, selections were made to recover a stable, high-producing variant. Initially, multiple fermentations were carried out using inocula prepared from strain 12052. Vegetative growth from the flask yielding the best deacylating activity was plated on a differential agar (CM). Colonies were then selected for further evaluation. Generally, small colonies were better enzyme producers than the large colony types. Isolate No. 18 was selected as a small colony type and shown to be the best deacylase producer of all colonies selected. This isolate was routinely used for the production of the deacylase enzyme. CM agar contained corn steep liquor 0.5%, Bacto peptone 0.5%, soluble starch 1.0%, NaCl 0.05%, CaCl2.2H2O 0.05% and Bacto agar 2.0%.
- The fermentation protocol employed is known (17). A high-producing, natural variant was used in this invention. A stock culture of the NRRL 12052 variant, preserved in 20% glycerol at −70° C., was introduced into a 25×150 mm test tube with a glass rod and Morton closure containing 10 mL of a medium composed of sucrose 2.0%, pre-cooked oatmeal 2.0%, distiller's grains and solubles 0.5%, yeast extract 0.25%, K2HPO4 0.1%, KCl 0.05%, MgSO4.7H2O 0.05% and FeSO4.7H2O 0.0002% in deionized water. After incubation at 30° C. for 72 hrs on a rotary shaker orbiting at 250 rpm the resulting mycelial suspension was transferred into 50 mL of PM3 medium in a 250-mL Erlenmeyer flask. This medium contained sucrose 2.0%, peanut meal 1.0%, K2HPO4 0.12%, KH2PO4 0.05% and MgSO4.7H2O 0.025% in tap water. The flask was incubated at a temperature of 30° C. for a period of 60 to 90 hrs. The harvest time was determined by an assay which involved HPLC analyses of the deacylation of (2-Sulfo-9-fluorenlymethoxycarbonyl)]5 polymyxin B by the whole broth at different times during the fermentation.
- Precipitation Method: Cells from 450 ml deacylase enzyme were washed 3× with water, then brought back to original volume with 0.02 M ammonium phosphate buffer and adjusted to pH 8.0. N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)]5-polymyxin B, 897 mg, was added and the mixture was placed on a shaker at 174 rpm and maintained at 30° C. After five hours the mixture was separated by centrifuging. The clear decant was adjusted to pH 2.3 with 1 N HCl to induce precipitation and allowed to stand at room temperature. The precipitate was separated from the mixture, slurried in 80 ml water, adjusted to pH 6.5 to obtain a clear solution, and freeze-dried to obtain 400 mg of tan powder, the semi-purified salt of the protected peptide, N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl]5-polymyxin peptide [(NaSO3-Fmoc)5-PBP-3]. The cells were extracted with methanol/water to obtain additional material.
- Resin Method: Cells from 1 liter deacylase enzyme were washed 3× with water, brought back to original volume with 0.02 M ammonium phosphate buffer, and combined with 2.0 g of N-(2-sulfo)-9-fluorenlymethoxycarbonyl)]5-polymyxin B. The mixture was placed on a shaker at 175 rpm and maintained at 30° C. for 17 hours. The mixture was then separated by centrifuge and the decant was combined with 20 ml of Amberchrom® CG-161m resin. The resin was washed with 150 ml water, 100 ml 10% CH3CN:H2O (3×), and 100 ml 20% CH3CN:H2O (2×). The peptide was then eluted 2× with 30% CH3CN:H2O, evaporated to remove CH3CN, and freeze-dried to obtain 283 mg powder, the purified peptide. The remaining peptide was then eluted 3× with 100 ml 50% CH3CN:H2O, which were combined evaporated and freeze-dried to obtain 460 mg powder, the purified peptide. The remaining peptide was extracted from the cells 6× with MeOH: H2O, 100 ml each. The extracts were combined, brought to four liters with water, adjusted to pH 2.1 with sulfuric acid and combined with Amberchrom® CG-161m resin. The resin was rinsed with water and then the peptide was eluted with 100 ml 35% CH3CN:H2O, which was evaporated and freeze-dried to obtain 94 mg purified peptide. The remaining peptide was eluted with 50% CH3CN:H2O, evaporated to remove CH3CN, and freeze-dried to obtain 539 mg of the purified peptide. The [(2-Sulfo)-9-fluorenlymethoxycarbonyl)]5—polymyxin B peptide[(HSO3-Fmoc)5-PBP-3)] was isolated from these procedures as tan powder, C122H134N16O37S5. These materials appeared to be about 75% pure when analyzed by HPLC with the column eluent monitored at 215 nm. ESIMS: calc. m/z for C122H136N16O37S5: (M+2H)+2=1288.4; Found: 1288.
- Water washed cells from 250 ml of Actinoplanes utahensis fermentation were combined with 125 ml 0.02 M ammonium phosphate buffer, adjusted to pH 10.1 and stirred thirty minutes. The solution of the enzyme was separated by centifuge and adjusted to pH 8.0. This solution could be used directly for deacylations or freeze-dried to obtain a powder form for storage. The decant containing the solubilized enzyme at pH 8.0, was combined with 100 mg (HSO3-Fmoc)5-polymyxin B dissolved in 10 ml of CH3CN:H2O (1:1), and placed on a shaker at 175 rpm, 84° C. After two hours the completed reaction was removed from the shaker and adjusted to pH 2.0. The precipitate was mixed with 40 ml methanol and the soluble product was separated from a dark precipitate. The solution containing 80 mg of the (HSO3-Fmoc)5-PBP-3 was evaporated to 2 ml and added to 10 ml of EtOAc to precipitate the (HSO3-Fmoc)5-PBP-3 as a tan powder.
- About 143 mg of partially purified (NaSO3-Fmoc)5-PBP-3 was dissolved in 40 mL of 20% CH3CN 0.05M in sodium phosphate at pH 6.7. Insolubles were removed by centrifugation. The decant was applied to a styrene-divinylbenzene resin cartridge (Supelco EnviChrom-P®, 25×35 mm) which had been slurry packed and rinsed with 20 mL 20% CH3CN-0.05M pH 6.7 buffer. Flow rate was about 2 mL/min at RT. The cartridge was eluted with incrementally increasing concentrations of CH3CN about 0.05M in sodium phosphate at pH 6.7. Collected fractions were evaluated by analytical HPLC. The desired product was eluted with 33% and 40% CH3CN eluents. Product-containing fractions were pooled and CH3CN removed under vacuum. The product pool was desalted by adsorption onto a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) which was then rinsed with four 1.0 mL portions of distilled water. Product was stripped from the cartridge with 16 mL of 67% CH3CN, solvent evaporated under vacuum, pH adjusted to about 5.8 with dilute NaOH, then freeze dried. Yield: 72 mg of a light tan solid, (NaSO3-Fmoc)5-PBP-3, C122H134N16O37S5. ESIMS: calc. m/z for C122H136N16O37S5: (M+2H)+2=1288.4. Found: 1288.
- About 3.9 mg of purified (NaSO3-Fmoc)5-PBP-3 was dissolved in 0.20 mL of 75% MeOH 0.25M in pH 8.8 potassium borate, 0.003 mL phenylisothiocyanate was added and stirried at RT. After about 90 min, the reaction mixture was diluted with 4 mL of 0.4M ammonium phosphate at pH 7.2. The product solution was applied to a 0.5 g styrene-divinylbenzene resin cartridge (EnviChrom-P) which was rinsed with 6 mL of 20% CH3CN 0.10M in sodium phosphate at pH 6.7. Product was eluted using 6 mL of 40% CH3CN 0.05M in sodium phosphate at pH 6.7. Solvent was evaporated under vacuum and the product was desalted in similar fashion as in Example 5. Yield: 3.5 mg of a white solid, N-phenylthiocarbamyl-(NaSO3-Fmoc)5-PBP-3, C129H137N17O37S6. ESIMS: calc. m/z for C129H139N17O37S6: (M+2H)+2=1355.4. Found: 1355.
- About 8.4 mg of N-phenylthiocarbamyl-(NaSO3-Fmoc)5-PBP-3 was dissolved in 0.20 mL dimethylformamide (DMF), 0.010 mL piperidine was added and stirred at RT for 60 min. The reaction mixture was diluted with 4 mL 0.10M ammonium acetate-0.050M acetic acid (pH 5.05) and 0.006 mL acetic acid and 4 mL MeOH. The clear solution was applied to a CM-Sepharose® column (10×20 mm, ca. 2 mL volume) which had been conditioned with 50% MeOH 0.05M in ammonium acetate buffer at pH 5.0. The sample-loaded column was rinsed with 4 mL 50% MeOH-0.05M ammonium acetate pH 5.0 then with 4 mL 0.05M ammonium acetate pH 5.0 buffer. Product was eluted with 8 mL of 0.27 M sodium sulfate at pH 2.3. The product was further purified by application onto a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) which was eluted with incrementally increasing concentrations of CH3CN 0.05M in pH 2.3 sodium sulfate; product was eluted with 20% CH3CN. Solvent was removed under vacuum from the product-containing fraction pool which was then desalted in similar fashion as in Example 5, pH adjusted to 6.3 then freeze dried. Yield: 2.7 mg of a white solid, PTC-PBP-3, C54H87N17O12S.
- FABMS: calc. for C54H88N17O12S:(M+H)+=1198.7. Found: 1198.5(M+H)+, 1220.4 (M+Na)+.
- About 2.9 mg N-phenylthiocarbamyl-(NaSO3-Fmoc)5-PBP-3 (from Example 6) was dissolved in 0.30 mL of anhydrous trifluoroacetic acid (TFA) and heated in a 50° C. water bath for 15 min. TFA was evaporated with a stream of dry nitrogen and the residue was dissolved in 12 mL 0.20M ammonium phosphate at pH7.2 and 6 mL CH3CN containing 49 mg triglycine (as an acylating agent scavenger). To remove potentially reactive intermediates, the product solution was first applied to a 0.5 g styrene-divinylbenzene resin cartridge (EnviChrom-P) which was eluted with 10 mL of 40% CH3CN 0.04M in ammonium phosphate at pH 7.2. Product-containing fractions were pooled (˜18 mL), diluted with 12 mL distilled water, then applied to a fresh 0.5 g resin cartridge which was eluted with incrementally increasing concentrations of CH3CN about 0.05M in ammonium phosphate at pH 7.2. Product-containing fractions were pooled (˜12 mL), 8 mL of 0.54M sodium sulfate pH 2.3 buffer added, and the product was desalted in similar fashion as in Example 5. The solution pH was adjusted to 5.9 and the sample freeze dried. Yield: 1.8 mg of a white solid, (NaSO3-Fmoc)4-PBP-2, C103H116N14O31S4 ESIMS: calc. m/z for C103H118N14O31S4 (M+2H)+2=1087.3. Found: 1087.
- About 21.8 mg of partially purified (NaSO3-FMoc)5-PBP-3 was dissolved in 0.20 mL DMF, 0.020 mL distilled water, and 0.030 mL saturated NaHCO3 (pH-8.9); 4.5 mg n-decanoyl-N-hydroxysuccinimide (C10—OSu) was added and stirred at RT for 55 min (about 82% conversion by HPLC). An additional 1.8 mg of C10—OSu was added; after 20 min at RT conversion to Compound 1P was at least 95%. To the reaction mix was added 0.010 mL piperidine. After 35 min at RT the reaction mixture was diluted with 4 mL 0.25M ammonium sulfate at pH2.3 yielding a very milky mixture at pH 3.0 which was extracted with 4 mL ethylacetate; the product-containing aqueous phase was diluted with 4 mL distilled water. Product was initially isolated by size exclusion chromatography on a Sephadex® G-25 column (2.5×40 cm) eluted with 0.10M ammonium sulfate at pH 2.3. Product-containing fractions were pooled and further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) by elution with incrementally increasing concentrations of CH3CN about 0.05M in sodium phosphate at pH 6.7; product was eluted with 25% CH3CN. Product was desalted and freeze dried in similar fashion as in Example 5. Yield: 2.5 mg of a white solid, Compound 1, C57H100N16O13. FABMS: calc. for C57H101N16O13, (M+H)+=1217.8. Found: 1217(M+H)+, 1239(M+Na)+.
- About 18.5 mg of purified (NaSO3-Fmoc)5-PBP-3 was dissolved in 0.20 mL DMF and 0.030 mL saturated NaHCO3 (pH˜8.9); 5.8 mg N-(n-decanoyl)-p-aminophenylacetyl-N-hydroxysuccinimide (C10-PAPA-OSu) was added and stirred at RT for 50 min (about 69% conversion by HPLC). An additional 2.5 mg of C10-PAPA-OSu was added; after 30 min at RT conversion to Compound 202P was about 87%. To the reaction mix was added 0.010 mL piperidine. After 25 min at RT the reaction mixture was diluted with 5 mL 0.20M ammonium sulfate at pH2.3 yielding a very milky mixture at pH 2.8 which was extracted with 5 mL ethylacetate. Product was initially isolated by size exclusion chromatography on a Sephadex® G-25 column (2.5×40 cm) eluted with 0.10M ammonium sulfate at pH 2.3. Product-containing fractions were pooled and further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) by elution with incrementally increasing concentrations of CH3CN about 0.10M in ammonium sulfate at pH 2.3; product was eluted with 30% CH3CN. Further purification was achieved using size exclusion chromatography on a Sephadex LH-20 column (2.5×40 cm) eluted with 0.026M ammonium acetate/0.053M acetic acid in MeOH. Product-containing fractions were evaporated under vacuum to near dryness, the residue dissolved in 10 mL distilled water, then desalted and freeze dried in similar fashion as in Example 5. Yield: 2.5 mg of a white solid, Compound 2, C65H107N17O14. FAMS: calc. for C65H108N17O14, (M+H)+=1350.8. Found: 135I(M+H)+, 1373(M+Na)+.
- About 10.2 mg of purified (NaSO3-Fmoc)5-PBP-3 was dissolved in 0.20 mL DMF and 0.020 mL distilled water; 0.003 mL octylisocyanate was added and stirred at RT for 60 min (about 31% conversion by HPLC). To the reaction mixture was added 0.020 mL saturated NaHCO3 pH 8.9; after 20 min at RT conversion to Compound 3P was about 91%. After another 35 min at RT 0.010 mL piperidine was added. After 30 min at RT the reaction mixture was diluted with 4 mL 0.026M ammonium acetate/0.053M acetic acid in MeOH and the product was isolated by size exclusion chromatography on a Sephadex LH-20 column as in Example 10. Product-containing fractions were evaporated under vacuum to near dryness, the residue dissolved in 8 mL distilled water, then desalted and freeze dried in similar fashion as in Example 5. Yield: 2.3 mg of a white solid, Compound 3, C56H99N17O13. FABMS: calc. for C56H100N17O13, (M+H)+=1218.8. Found: 1219(M+H)+, 1241(M+Na)+.
- About 19.8 mg of purified (NaSO3-Fmoc)5-PBP-3 (ca. 85%) was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO3 pH 8.7; 0.005 mL phenylisocyanate was added and stirred at RT to obtain Compound 5P. After 45 min 0.020 mL of piperidine was added. After 45 min at RT the reaction mixture was diluted with 4 mL 0.10M ammonium acetate-0.05M acetic acid and 0.012 mL acetic acid and 4 mL MeOH yielding a clear solution at apparent pH 6.2. The product was isolated on a CM-Sepharose column as in Example 7. The product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH3CN 0.05M in sodium sulfate at pH 2.3; product was eluted with 20% CH3CN. Product-containing fractions were desalted and freeze dried in similar fashion as in Example 5. Yield: 4.4 mg of a white solid, Compound 5, C54H87N17O13. FABMS: calc. for C54H88N17O13, (M+H)+=1182.7. Found: 1183(M+H)+, 1205(M+Na)+, 1221(M+K)+.
- About 20.4 mg of purified (NaSO3-Fmoc)5-PBP-3 (ca. 85%) was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO3 pH 8.0; 8.4 mg of benzoyl-N-hydroxysuccinimide was added and stirred at 29° C. to obtain Compound 6P. After 60 min 0.020 mL of piperidine was added. After 20 min at RT the reaction mixture was diluted with 4 mL 0.10M ammonium acetate-0.05M acetic acid and 0.013 mL acetic acid and 4 mL MeOH yielding a clear solution. The product was isolated on a CM-Sepharose column as in Example 7. The product was further purified by preparative HPLC using a Delta-Pak® C18 column (25×210 mm, Waters Corp.) eluted with an isopropanol gradient (18%-23% over 100 min, linear, at 5 mL/min) buffered with 0.05M sodium sulfate at pH2.5. Product-containing fractions were pooled, solvent removed under vacuum, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 1.6 mg of a white solid, Compound 6, C54H86N16O13. FABMS: calc. for C54H87N16O13, (M+H)+=1166.7. Found: 1167(M+H)+, 1189(M+Na)+.
- About 19.0 mg of purified (NaSO3-Fmoc)5-PBP-3 was dissolved in 0.40 mL of 75% MeOH 0.25M in pH 8.8 potassium borate; 6.3 mg of 2-naphthoxyacetyl-N-hydroxysuccinimide was added and stirred at RT to produce Compound 7P. After 35 min 0.020 mL piperidine was added. After 30 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH3CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% CH3CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 4.7 mg of a white solid, Compound 7, C59H90N16O14.
- FABMS: calc. for C59H91N16O4, (M+H)+=1247.7. Found: 1247(M+H)+, 1269(M+Na)+.
- About 21.7 mg of purified (NaSO3-Fmoc)5-PBP-3 was dissolved in 0.50 mL of 75% MeOH 0.25M in pH 8.8 potassium borate; 5.1 mg of 4-methylphenylsulfonyl chloride (tosyl chloride) was added and stirred at RT to produce Compound 8P. After 60 min 0.020 mL piperidine was added. After 30 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH3CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 20% CH3CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 4.9 mg of a white solid, Compound 8, C54H88N16O14S. FABMS: calc. for C54H89N16O14S (M+H)+=1217.6, Found: 1217(M+H)+, 1239(M+Na)+.
- About 10.1 mg of purified (NaSO3-Fmoc)4-PBP-2 was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO3 pH 8.7; 0.003 mL octylisocyanate was added and stirred at RT to produce Compound 9P. After 45 min at RT 0.020 mL piperidine was added. After 40 min at RT the reaction mixture was diluted with 10 mL 20% CH3CN containing 0.014 mL H2SO4. Product was isolated on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH3CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% and 30% CH3CN. Product-containing fractions were pooled, solvent removed under vacuum, 2 mL 1.0M ammonium acetate pH 5.0 buffer was added, pH adjusted to 5.0 by addition of ca. 0.6 mL 1.5M NH4OH, and then diluted with an equal volume of MeOH. Product was isolated via CM-Sepharose chromatography as in Example 7. The product was further purified on a 0.5 g styrene-divinylbenzene cartridge as above but using eluents at pH 9.9 (0.10M NH4OH-0.01M (NH4)2SO4); product eluted with 30% CH3CN. Product-containing fractions were pooled, solvent removed under vacuum, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 2.7 mg of a white solid, C52H92N16O11. FABMS: calc. for C52H93N16O11, (M+H)+=1118.7, Found: 1119(M+H)+, 1141(M+Na)+, 1157(M+K)+.
- About 9.7 mg of purified (NaSO3-Fmoc)4-PBP-2 was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO3 pH 8.7; 5.2 mg of n-decylsulfonamidoglycyl-N-hydroxy succinimide was added and stirred at RT to produce Compound 10P. After 45 min 0.020 mL piperidine was added. After another 40 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH3CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 33% CH3CN. Product-containing fractions were pooled, solvent removed under vacuum, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 1.5 mg of a white solid, Compound 10, C55H97N15O14S. FABMS: calc. for C55H98N15O14S, (M+H)+=1224.7. Found: 1224.5(M+H)+, 1246.5(M+Na)+.
- About 10.3 mg of purified (NaSO3-Fmoc)4-PBP-2 was dissolved in 0.20 mL of DMF and 0.020 mL of saturated NaHCO3 pH8.3; 8.7 mg of 6-N-Fmoc-2-N-(n-decanoyl)-lysyl-N-hydroxysuccinimide was added and stirred at RT to produce Compound 11P. After 50 min 0.020 mL piperidine was added. After 45 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH3CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% CH3CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 2.0 mg of a white solid, Compound 11, C59H104N16O13. FABMS: calc. for C59H105N16O13, (M+H)+=1245.8, Found: 1245(M+H)+, 1267(M+Na)+.
- About 10 mg of purified (NaSO3-Fmoc)4-PBP-2 was dissolved in 0.40 mL of 75% MeOH 0.25M in pH 8.8 potassium borate; 6 mg of N-(n-decanoyl)-phenylalanyl-N-hydroxysuccinimide was added and stirred at RT to produce Compound 12P. After 50 min 0.020 mL piperidine was added. After 45 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH3CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 33% CH3CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 3.0 mg of a white solid, Compound 12, C62H101N15O13. FABMS: calc. for C62H102N15O13 (M+H)+=1264.8. Found: 1265(M+H)+, 1287(M+Na)+.
- The procedure used in Example 8 to prepare the (NaSO3-Fmoc)4-PBP-2 from (NaSO3-Fmoc)5-PBP-3 was repeated starting with (NaSO3-Fmoc)4-PBP-2, 50 mg, to remove the threonine residue and yielding (NaSO3-Fmoc)4-PBP-1, 37 mg, as a white powder, with the expected HPLC relative retention time.
- (NaSO3-Fmoc)4-PBP-2 (5.0 mg) was dissolved in 1.4 mL of 0.02M sodium citrate buffer (pH6.4) and 0.006M in 2-mercaptoethylamine(MEA) hydrochloride. The enzyme solution was prepared by dissolving 10 units (0.9 mg) of cathepsin C (dipeptidyl aminopeptidase) (EC 3.4.14.1) in 11.0 mL of the citrate-MEA buffer. Enzyme solution (0.25 mL) was added to the peptide solution which was incubated at about 37° C. for about 24 hours. HPLC analysis indicated about 75% conversion to (NaSO3-Fmoc)3-PBP. The incubated solution was diluted with 11.0 mL 0.4M pH7.2 ammonium phosphate buffer, 3.0 mL distilled water and 0.5 mL CH3CN. The product was isolated on an ENVI-Chrom-P 0.5 g resin cartridge; product was eluted with 25% CH3CN, 0.05M in pH7.2 buffer. After HPLC evaluation, product-containing fractions (accounting for about 72% of the total product) were pooled, desalted and freeze dried as in Example 8. Yield: 1.0 mg of a pale yellow solid, 87% by HPLC (215 nm area %), with the expected HPLC relative retention time for the (NaSO3-Fmoc)3-PBP.
- While the foregoing description of the preferred and and alternative embodiments of the present invention is considered sufficient, the only limitations thereof are in the following claims:
Claims (11)
1. A method for preparing an intermediate for use in the synthesis of a new peptide antibiotic, including the steps of:
(a) Protecting the amino groups of the polymyxins or other related antibiotics chosen from the group consisting of colistin, a circulin, and an octapeptin, with (2-sulfo)-9-fluorenylmethoxycarbonyl or another acidic derivative of 9-fluorenylmethoxycarbonyl;
(b) Treating the product from the reaction of step (a) with a deacylase to provide a protected peptide intermediate; and
(c) Using a modified Edman degradation method or peptidase enzymatic reaction to obtain another protected intermediate peptide by reducing in size by one to three amino acids in the exocyclic peptide side chain of the protected peptide.
2. A method for producing an antibiotic active against gram-negative and gram-positive bacteria, including strains resistant to clinically used antibiotics, comprising the steps of:
(a) Protecting the amino groups of the polymyxins or other related antibiotics chosen from the group consisting of colistin, a circulin, and an octapeptin, with (2-sulfo)-9-fluorenylmethoxycarbonyl or another acidic derivative of 9-fluorenylmethoxycarbonyl;
(b) Treating the product from the reaction of step (a) with a deacylase to provide a protected peptide intermediate;
(c) Using a modified Edman degradation method or peptidase enzymatic reaction to obtain another protected intermediate peptide by reducing in size by one to three amino acids in the exocyclic peptide side chain of the protected peptide.
(d) Chemically modifying the intermediate to produce a protected antibacterial derivative; and
(e) Removing the acidic protecting groups to produce the antibiotic.
3. An intermediate, which is a chemically protected form of a peptide derived from the polymyxins, octapeptins, colistin, or circulins, and selected from a group consisting of the following, or their corresponding salts:
Case 1) H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]n
Case 2) H-(X2) (X3)-peptide-[(2-sulfo)-9-Fmoc]n
Case 3) H-(X2)-peptide-[(2-sulfo)-9-Fmoc]n
Case 4) H-peptide-[(2-sulfo)-9-Fmoc]3
wherein for Case 1) H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]n
H is hydrogen, X1 is L-Dab or another amino acid, 2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid, and n=3-6;
for Case 2) H-(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]n
H is hydrogen, X2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid, and n=3-5;
for Case 3) H-(X3)-peptide-[(2-sulfo)-9-Fmoc]n
H is hydrogen X3 is L-Dab or D-Dab or another amino acid and n=3-4; and
for Case 4) H-peptide-[(2-sulfo)-9-Fmoc]3
H is hydrogen.
4. Acidic protected peptide intermediates, derived from the corresponding protected polymyxin B, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably HSO3-Fmoc and the protected peptide intermediates have the structure:
5. Acidic protected peptide intermediates, derived from the corresponding protected colistin, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably. HSO3-Fmoc- and the protected peptide intermediates have the structure:
6. Acidic protected peptide intermediates, derived from the corresponding protected circulin A, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably HSO3-Fmoc- and the protected intermediates have the structure:
7. Acidic protected peptide intermediates, derived from the corresponding protected octapeptin, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably HSO3-Fmoc- and the protected peptide intermediates have the structure:
8. The protected peptide intermediate of claim 7 , wherein another component of the octapeptin antibiotic includes L-phenylalanine instead of L-leucine at the 5-position and wherein the component forms a similar, but alternative, protected peptide intermediate.
9. An antibacterial compound or protected compound prepared from a chemically protected form of PBpeptide having the following structure where p equals the protective group HSO3-Fmoc- or hydrogen:
*Dab-P is 4-N—(HSO3-Fmoc)-diaminobutyryl, Lys-P is 6-N—(HSO3-Fmoc)-lysyl
**PAPA is p-aminophenylacetyl
All amino acids are the L-isomers unless indicated otherwise.
10. Peptide antibiotics having the following structure and concentration for use against gram-positive and gram-negative bacteria:
*MIC values were determined by serial twofold broth dilution method using Escherichia coli, ATCC #26, and Staphylococcus aureus Smith as assay organisms which were grown in Mueller Hinton broth.
**p-aminophenylacetyl
11. An antibiotic prepared from an intermediate, which is a chemically protected form of a peptide derived from the polymyxins, octapeptins, colistin, or circulins, said antibiotic selected from a group consisting of the following, or their corresponding salts:
Case 1) A-(X1)(X2)(X3)-peptide
Case 2) A-(X2)(X3)-peptide
Case 3) A-(X3)-peptide
Case 4) A-peptide
wherein for Case 1) A-(X1)(X2)(X3)-peptide
A=R′—(C═O)—, R′—SO2—, R′—(C═NH)—, R′—NH—(C═S)—, R′—NH—(C═O)—, R′—O—(C═O)—, R′CH2—
where R′ is alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, or heterocyclic and X1 is L-Dab or another amino acid, X2 is L-Thr or another amino acid, and X3 is L-Dab, D-Dab or another amino acid, excluding colistin peptides where X1 is an aliphatic amino acid and where R′(C—O)— is alkyl,
for Case 2) A-(X2)(X3)-peptide
“A” is the same as described for Case 1, X2 is L-Thr or another amino acid and X3 is L-Dab, D-Dab or another amino acid, excluding N-acyl colistin nonapeptide derivatives where R′ is alkyl, aryl, or cycloalkyl,
for Case 3) A-(X3)-peptide
“A” is the same as described in Case 1, and X3 is L-Dab, D-Dab or another amino acid, excluding R′—(CO)— where R′ is alkyl for octapeptin peptides,
for Case 4) A-peptide
“A” is the same as described for Case 1.
Priority Applications (20)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/881,160 US20060004185A1 (en) | 2004-07-01 | 2004-07-01 | Peptide antibiotics and peptide intermediates for their prepartion |
PCT/US2005/023343 WO2006083317A2 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
CA002571944A CA2571944A1 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
AU2005326770A AU2005326770B2 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
MXPA06015239A MXPA06015239A (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same. |
EP05856867A EP1761554A2 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
US11/630,847 US8889826B2 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
RU2007103811/04A RU2428429C2 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and production methods thereof |
KR1020077002649A KR20070047770A (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
NZ579261A NZ579261A (en) | 2004-07-01 | 2005-07-01 | Peptide Antibodies And Methods For Making Same |
JP2007519447A JP2008505858A (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and method for producing the same |
CNA2005800294199A CN101010336A (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and their preparation methods |
EP10184953A EP2332965A1 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
NZ552730A NZ552730A (en) | 2004-07-01 | 2005-07-01 | Peptide antibodies and methods for making same |
BRPI0512941-9A BRPI0512941A (en) | 2004-07-01 | 2005-07-01 | peptide antibiotics and methods to do the same |
ZA200610818A ZA200610818B (en) | 2004-07-01 | 2006-12-21 | Peptide antibiotics and methods for making same |
IL180458A IL180458A0 (en) | 2004-07-01 | 2006-12-31 | Peptide antibiotics and methods for making same |
NO20070563A NO20070563L (en) | 2004-07-01 | 2007-01-30 | Peptide antibiotics and methods for their preparation. |
RU2011115077/04A RU2011115077A (en) | 2004-07-01 | 2011-04-15 | PEPTIDE ANTIBIOTICS AND METHODS FOR PRODUCING THEM |
JP2011168634A JP2011256189A (en) | 2004-07-01 | 2011-08-01 | Peptide antibiotic and method for making the same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/881,160 US20060004185A1 (en) | 2004-07-01 | 2004-07-01 | Peptide antibiotics and peptide intermediates for their prepartion |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060004185A1 true US20060004185A1 (en) | 2006-01-05 |
Family
ID=35514884
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/881,160 Pending US20060004185A1 (en) | 2004-07-01 | 2004-07-01 | Peptide antibiotics and peptide intermediates for their prepartion |
US11/630,847 Expired - Fee Related US8889826B2 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/630,847 Expired - Fee Related US8889826B2 (en) | 2004-07-01 | 2005-07-01 | Peptide antibiotics and methods for making same |
Country Status (15)
Country | Link |
---|---|
US (2) | US20060004185A1 (en) |
EP (2) | EP1761554A2 (en) |
JP (2) | JP2008505858A (en) |
KR (1) | KR20070047770A (en) |
CN (1) | CN101010336A (en) |
AU (1) | AU2005326770B2 (en) |
BR (1) | BRPI0512941A (en) |
CA (1) | CA2571944A1 (en) |
IL (1) | IL180458A0 (en) |
MX (1) | MXPA06015239A (en) |
NO (1) | NO20070563L (en) |
NZ (2) | NZ552730A (en) |
RU (2) | RU2428429C2 (en) |
WO (1) | WO2006083317A2 (en) |
ZA (1) | ZA200610818B (en) |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007142611A1 (en) * | 2006-06-02 | 2007-12-13 | Biotika A.S. | Method of polymyxin b recovery from fermentation broth |
WO2008017734A1 (en) | 2006-08-11 | 2008-02-14 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US20080287345A1 (en) * | 2006-08-11 | 2008-11-20 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
WO2009098357A1 (en) * | 2008-02-08 | 2009-08-13 | Northern Antibiotics Oy | Short fatty acid tail polymyxin derivatives and uses thereof |
US20090215677A1 (en) * | 2008-02-08 | 2009-08-27 | Martti Sakari Vaara | Polymyxin derivatives and uses thereof |
US20090239792A1 (en) * | 2008-02-08 | 2009-09-24 | Martti Sakari Vaara | Short fatty acid tail polymyxin derivatives and uses thereof |
US20130053305A1 (en) * | 2010-03-10 | 2013-02-28 | Universidad De Barcelona | Peptide compounds that can be used as antibacterial agents |
US9096649B2 (en) | 2008-02-08 | 2015-08-04 | Northern Antibiotics Ltd. | Polymyxin derivatives and uses thereof |
WO2016100578A2 (en) | 2014-12-16 | 2016-06-23 | Micurx Pharmaceuticals, Inc. | Antimicrobial polymyxins for treatment of bacterial infections |
EP3045469A1 (en) | 2015-01-15 | 2016-07-20 | Northern Antibiotics Oy | Polymyxin derivative and uses thereof |
US9763996B2 (en) | 2015-01-16 | 2017-09-19 | Northern Antibiotics, Ltd. | Polymyxin derivative and uses thereof |
EP3126376A4 (en) * | 2014-04-01 | 2017-10-25 | Monash University | Polymyxin derivatives as antimicrobial compounds |
US10234460B2 (en) | 2013-08-26 | 2019-03-19 | The University Of Tokyo | Macrocyclic peptide, method for producing same, and screening method using macrocyclic peptide library |
EP3556769A4 (en) * | 2016-12-16 | 2021-03-03 | Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences | Polymyxin derivative, preparation method and application thereof |
CN113174398A (en) * | 2021-04-22 | 2021-07-27 | 浙江工业大学 | Expression cassette for recombinant expression of echinocandin B deacylase and application |
WO2021150792A1 (en) | 2020-01-21 | 2021-07-29 | Micurx Pharmaceuticals, Inc. | Novel compounds and composition for targeted therapy of kidney-associated cancers |
US11225505B2 (en) | 2015-09-29 | 2022-01-18 | Monash University | Antimicrobial polymyxin derivative compounds |
US11819532B2 (en) | 2018-04-23 | 2023-11-21 | Hoffmann-La Roche Inc. | Peptide macrocycles against Acinetobacter baumannii |
US12012466B2 (en) | 2015-10-27 | 2024-06-18 | Hoffmann-La Roche Inc. | Peptide macrocycles against Acinetobacter baumannii |
Families Citing this family (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060004185A1 (en) | 2004-07-01 | 2006-01-05 | Leese Richard A | Peptide antibiotics and peptide intermediates for their prepartion |
EP2261238B1 (en) * | 2008-03-14 | 2014-05-07 | Astellas Pharma Inc. | Cyclic compound and salt thereof |
WO2010075416A1 (en) * | 2008-12-23 | 2010-07-01 | Biosource Pharm, Inc. | Antibiotic compositions for the treatment of gram negative infections |
WO2010130007A1 (en) * | 2009-05-14 | 2010-11-18 | Monash University | Antimicrobial compounds |
US8415307B1 (en) | 2010-06-23 | 2013-04-09 | Biosource Pharm, Inc. | Antibiotic compositions for the treatment of gram negative infections |
WO2012051663A1 (en) * | 2010-10-21 | 2012-04-26 | Monash University | Antimicrobial compounds |
WO2012168820A1 (en) * | 2011-06-08 | 2012-12-13 | Pfizer Inc. | Polymyxin derivatives useful as antibacterial agents |
WO2013072695A1 (en) | 2011-11-18 | 2013-05-23 | Novacta Biosystems Limited | Polymyxin derivatives |
EP2679236A1 (en) | 2012-06-29 | 2014-01-01 | Zentrum für biomedizinische Technologie der Donau- Universität Krems | Metering guide for lipopeptides that bind endotoxin |
KR102354902B1 (en) | 2013-05-22 | 2022-01-24 | 에버레스트 메디신즈 (싱가포르) 피티이. 리미티드 | Polymyxin derivatives and their use in combination therapy together with different antibiotics |
CN103396475B (en) * | 2013-08-06 | 2015-08-26 | 深圳翰宇药业股份有限公司 | A kind of method of pure solid-phase synthetic peptide class microbiotic Colistin |
TWI709575B (en) * | 2014-03-11 | 2020-11-11 | 美商斯佩羅治療學股份有限公司 | Polymyxin derivatives and their use in combination therapy together with different antibiotics |
CN103993059B (en) * | 2014-05-28 | 2016-08-17 | 河北圣雪大成制药有限责任公司 | Optimize the method for E1 and E2 component ratio in bacillus polymyxa zymotic fluid |
AU2015326947B2 (en) * | 2014-09-29 | 2020-04-16 | Sulfotools Gmbh | Method for peptide synthesis and apparatus for carrying out a method for solid phase synthesis of peptides |
GB2530776A (en) * | 2014-10-01 | 2016-04-06 | Randox Lab Ltd | Detection of polymyxins |
JO3669B1 (en) * | 2015-01-06 | 2020-08-27 | Ferring Bv | CGRP Antagonist Peptides |
CA3035473A1 (en) | 2016-09-13 | 2018-03-22 | Allergan, Inc. | Non-protein clostridial toxin compositions |
CN106868079B (en) * | 2017-04-26 | 2020-12-08 | 山东鲁抗医药股份有限公司 | Culture medium for fermenting polymyxin B sulfate and method for producing polymyxin B sulfate through fermentation |
JP2021501783A (en) * | 2017-11-02 | 2021-01-21 | ザ ユニバーシティー オブ クイーンズランド | Peptide antibiotics |
LT3810633T (en) | 2018-06-25 | 2024-01-10 | Spero Therapeutics, Inc. | Compounds |
CN111410682A (en) * | 2020-04-28 | 2020-07-14 | 梯尔希(南京)药物研发有限公司 | Cyclization preparation method of polymyxin B related compound |
CN113215185B (en) * | 2021-04-22 | 2022-04-29 | 浙江工业大学 | Recombinant gene sequence for recombinant expression of echinocandin B deacylase |
Family Cites Families (119)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3854480A (en) | 1969-04-01 | 1974-12-17 | Alza Corp | Drug-delivery system |
US3753970A (en) | 1970-08-04 | 1973-08-21 | Rhone Poulenc Sa | Cyclopeptides derived from polymyxins and their preparation |
FR2124060B1 (en) * | 1971-02-02 | 1974-04-12 | Rhone Poulenc Sa | |
US4091092A (en) | 1976-09-20 | 1978-05-23 | E. R. Squibb & Sons, Inc. | Polymyxin F and process of producing polymyxin F |
IE52535B1 (en) | 1981-02-16 | 1987-12-09 | Ici Plc | Continuous release pharmaceutical compositions |
US5366734A (en) | 1981-02-16 | 1994-11-22 | Zeneca Limited | Continuous release pharmaceutical compositions |
US4812470A (en) | 1982-02-27 | 1989-03-14 | Beecham Group P.L.C. | Antibacterial monic acid derivatives |
DE3378250D1 (en) | 1982-04-22 | 1988-11-24 | Ici Plc | Continuous release formulations |
USRE32311E (en) | 1982-05-21 | 1986-12-16 | Eli Lilly And Company | Derivatives of A-21978C cyclic peptides |
USRE32310E (en) | 1982-05-21 | 1986-12-16 | Eli Lilly And Company | Derivatives of A-21978C cyclic peptides |
US4482487A (en) | 1982-05-21 | 1984-11-13 | Eli Lilly And Company | A-21978C cyclic peptides |
US4524135A (en) | 1982-05-21 | 1985-06-18 | Eli Lilly And Company | A-21978C cyclic peptides |
US4399067A (en) | 1982-05-21 | 1983-08-16 | Eli Lilly And Company | Derivatives of A-21978C cyclic peptides |
US4537717A (en) | 1982-05-21 | 1985-08-27 | Eli Lilly And Company | Derivatives of A-21978C cyclic peptides |
GB2128617A (en) | 1982-10-06 | 1984-05-02 | Martti Vaara | Polypeptides for use in antibacterial therapy |
US4452775A (en) | 1982-12-03 | 1984-06-05 | Syntex (U.S.A.) Inc. | Cholesterol matrix delivery system for sustained release of macromolecules |
US4601893A (en) | 1984-02-08 | 1986-07-22 | Pfizer Inc. | Laminate device for controlled and prolonged release of substances to an ambient environment and method of use |
US4895566A (en) | 1986-07-25 | 1990-01-23 | C. R. Bard, Inc. | Coating medical devices with cationic antibiotics |
AU607929B2 (en) | 1986-08-07 | 1991-03-21 | Medice Chem.-Pharm. Fabrik Putter Gmbh & Co. Kg | Pharmaceutical preparations |
US4830860A (en) | 1986-10-30 | 1989-05-16 | Pfizer Inc. | Stressed polymeric device for controlled release of a substance to an ambient environment |
US5755788A (en) | 1987-02-19 | 1998-05-26 | Rutgers, The State University | Prosthesis and implants having liposomes bound thereto and methods of preparation |
US20010021697A1 (en) | 1987-09-14 | 2001-09-13 | Henning Lowenstein | Methods and compositions for the treatment of mammalian infections employing medicaments comprising hymenoptera venom, proteinageous or polypeptide components thereof, or analogues of such proteinaceous or polypeptide components |
US5028590A (en) | 1988-04-11 | 1991-07-02 | Eli Lilly And Company | Derivatives of A54145 cyclic peptides |
US5039789A (en) | 1988-04-11 | 1991-08-13 | Eli Lilly And Company | A54145 cyclic peptides |
US5254535A (en) | 1989-04-17 | 1993-10-19 | The Children's Hospital Of Pennsylvania | Composition and treatment with biologically active peptides and antibiotic |
US5112614A (en) | 1989-09-14 | 1992-05-12 | Alza Corporation | Implantable delivery dispenser |
US5459237A (en) | 1990-02-08 | 1995-10-17 | Magainin Pharmaceuticals Inc. | Peptide compositions and uses therefor |
JPH04167172A (en) | 1990-10-31 | 1992-06-15 | Nec Corp | Vector processor |
US5082653A (en) | 1990-10-31 | 1992-01-21 | Warner-Lambert Company | Anti-plaque compositions comprising a combination of morpholinoamino alcohol and antibiotic |
US5686065A (en) | 1991-03-27 | 1997-11-11 | Special Advanced Biomaterials, Inc. | Topical siloxane sunscreen compositions having enhanced performance and safety |
CA2107674A1 (en) | 1991-04-08 | 1992-10-09 | W. Lee Maloy | Novel peptide compositions and uses therefor |
WO1993005802A1 (en) | 1991-09-13 | 1993-04-01 | Magainin Pharmaceuticals Inc. | Biologically active amphiphilic peptide compositions and uses therefor |
TW274552B (en) | 1992-05-26 | 1996-04-21 | Hoechst Ag | |
US6348445B1 (en) | 1992-06-01 | 2002-02-19 | Magainin Pharmaceuticals, Inc. | Biologically active peptides with reduced toxicity in animals and a method for preparing same |
US5283005A (en) | 1992-10-08 | 1994-02-01 | Olin Corporation | Synergistic biocide combination for industrial fluids |
US5424290A (en) | 1992-10-26 | 1995-06-13 | Magainin Pharmaceuticals Inc. | Biologically active peptides and uses therefor |
US5360788A (en) | 1992-11-10 | 1994-11-01 | Olin Corporation | Personal care composition containing pyrithione and a basic lipopeptide |
US5370876A (en) | 1993-01-08 | 1994-12-06 | Microbarriers | Antimicrobial protective skin composition and method for protecting skin from body fluids |
US5654451B1 (en) | 1993-01-14 | 2000-02-22 | Magainin Pharma | Amino acids and peptides having modified c-terminals and modified n-terminals |
CA2112776C (en) | 1993-01-21 | 2002-11-12 | Masakazu Tsuchiya | Process for inhibiting activity of endotoxin |
US5652332A (en) | 1993-03-12 | 1997-07-29 | Xoma | Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof |
US20040023884A1 (en) | 1993-03-12 | 2004-02-05 | Xoma Corporation | Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof |
US5733872A (en) | 1993-03-12 | 1998-03-31 | Xoma Corporation | Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof |
US6180134B1 (en) | 1993-03-23 | 2001-01-30 | Sequus Pharmaceuticals, Inc. | Enhanced ciruclation effector composition and method |
US5635216A (en) | 1993-12-16 | 1997-06-03 | Eli Lilly And Company | Microparticle compositions containing peptides, and methods for the preparation thereof |
EP0712405A1 (en) | 1993-08-13 | 1996-05-22 | Smithkline Beecham Plc | Derivatives of monic acids a and c having antibacterial, antimycoplasmatical, antifungal and herbicidal activity |
US5587358A (en) | 1994-05-09 | 1996-12-24 | Asahi Kasei Kogyo Kabushiki Kaisha | Potentiators of antimicrobial activity |
FR2735983B1 (en) | 1995-06-29 | 1997-12-05 | Centre Nat Rech Scient | PEPTIDE FOR MODIFYING THE ACTIVITY OF THE HUMAN OR ANIMAL IMMUNE SYSTEM |
US5849761A (en) | 1995-09-12 | 1998-12-15 | Regents Of The University Of California | Peripherally active anti-hyperalgesic opiates |
US20020150964A1 (en) | 1995-12-19 | 2002-10-17 | Centre National De La Recherche Scientifique | Peptides for the activation of the immune system in humans and animals |
US6011008A (en) | 1997-01-08 | 2000-01-04 | Yissum Research Developement Company Of The Hebrew University Of Jerusalem | Conjugates of biologically active substances |
US6004573A (en) | 1997-10-03 | 1999-12-21 | Macromed, Inc. | Biodegradable low molecular weight triblock poly(lactide-co-glycolide) polyethylene glycol copolymers having reverse thermal gelation properties |
US6846478B1 (en) | 1998-02-27 | 2005-01-25 | The Procter & Gamble Company | Promoting whole body health |
US6350438B1 (en) | 1998-02-27 | 2002-02-26 | The Procter & Gamble Company | Oral care compositions comprising chlorite and methods |
US6350738B1 (en) | 1998-03-06 | 2002-02-26 | Brigham Young University | Steroid derived antibiotics |
US6767904B2 (en) | 1998-03-06 | 2004-07-27 | Bringham Young University | Steroid derived antibiotics |
EP1115417B1 (en) | 1998-09-25 | 2006-04-05 | Cubist Pharmaceuticals, Inc. | Use of daptomycin |
IT1308180B1 (en) | 1999-02-12 | 2001-12-07 | Chiesi Farma Spa | SYNTHETIC PEPTIDES HAVING THE ABILITY TO DECREASE SURFACE TENSION AND THEIR USE IN THE PREPARATION OF A SURFACTANT |
US6515104B1 (en) | 1999-06-25 | 2003-02-04 | Xoma Technology Ltd. | Therapeutic peptide-based constructs derived from domain II of bactericidal/permeability-increasing protein |
US6964948B2 (en) | 1999-06-25 | 2005-11-15 | Xoma Technology Ltd. | Therapeutic peptide-based constructs derived from Domain II of bactericidal/permeability-increasing protein |
HUP0202315A2 (en) * | 1999-07-15 | 2002-10-28 | Lilly Co Eli | Process for deacylation of lipodepsipeptides |
US6380356B1 (en) | 1999-12-07 | 2002-04-30 | Advanced Medicine, Inc. | Multivalent polymyxin antibiotics |
KR20020063228A (en) | 1999-12-15 | 2002-08-01 | 큐비스트 파마슈티컬즈 인코포레이티드 | Lipopeptides as antibacterial agents |
KR20020063230A (en) | 1999-12-15 | 2002-08-01 | 큐비스트 파마슈티컬즈 인코포레이티드 | Novel lipopeptides as antibacterial agents |
EP2295444A3 (en) | 1999-12-15 | 2011-03-23 | Cubist Pharmaceutical Inc. | Lipopeptides as antibacterial agents |
US7074392B1 (en) | 2000-03-27 | 2006-07-11 | Taro Pharmaceutical Industries Limited | Controllled delivery system of antifungal and keratolytic agents for local treatment of fungal infections |
JP3292723B2 (en) * | 2000-05-26 | 2002-06-17 | アルス電子株式会社 | Semiconductor package and manufacturing method thereof |
CA2409188A1 (en) | 2000-05-30 | 2001-12-06 | Viridis Biotech Inc. | Polyubiquitin based hydrogel and uses thereof |
PT1294383E (en) | 2000-06-30 | 2008-10-08 | Procter & Gamble | Oral compositions comprising antimicrobial agents for the prevention of systemic diseases |
AU7893301A (en) * | 2000-07-17 | 2002-01-30 | Intrabiotics Pharmaceuticals | Antimicrobial sulfonamide derivatives of lipopeptide antibiotics |
US6511962B1 (en) | 2000-07-17 | 2003-01-28 | Micrologix Biotech Inc. | Derivatives of laspartomycin and preparation and use thereof |
IL156942A0 (en) * | 2001-01-16 | 2004-02-08 | Univ Ramot | Compounds, pharmaceutical compositions and methods for treatment of bacteremia and/or septicemia |
EA200300622A1 (en) | 2001-01-31 | 2003-12-25 | Пфайзер Продактс Инк. | DERIVATIVES OF AMIDES TIAZOLYL-, OXAZOLYL, PYRROLYL-AND IMIDAZOZYL ACID, USEFUL AS AN INHIBITORS OF PDE4 ISOFERMENT |
ATE477804T1 (en) | 2001-03-15 | 2010-09-15 | Soligenix Inc | METHOD FOR TREATING INFLAMMATORY DISEASES OF THE DIGESTIVE TRACT USING TOPICAL CORTICOSTEROIDS |
AU2002353775A1 (en) | 2001-08-06 | 2003-03-10 | Cubist Pharmaceuticals, Inc. | Lipopeptide stereoisomers, methods for preparing same, and useful intermediates |
US6681765B2 (en) | 2001-12-18 | 2004-01-27 | Sheree H. Wen | Antiviral and antibacterial respirator mask |
US7345018B2 (en) | 2002-04-25 | 2008-03-18 | Reception Aps | Method of treating side effects induced by therapeutic agents |
EP1360961A1 (en) | 2002-05-07 | 2003-11-12 | AM-Pharma B.V. | Use of antimicrobial peptides for potentiating the activity of antimicrobial agents |
US6767718B2 (en) * | 2002-05-10 | 2004-07-27 | Biosource Pharm, Inc. | Lipodepsipeptide antibiotics and methods of preparation |
JP2006505523A (en) | 2002-08-12 | 2006-02-16 | ダイナバックス テクノロジーズ コーポレイション | Immunomodulatory composition, method for making and using the same |
US20040037895A1 (en) | 2002-08-23 | 2004-02-26 | Alex Zhu | Methods of treating involuntary facial spasms and facial wrinkles |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
US7871607B2 (en) | 2003-03-05 | 2011-01-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
US20090123367A1 (en) | 2003-03-05 | 2009-05-14 | Delfmems | Soluble Glycosaminoglycanases and Methods of Preparing and Using Soluble Glycosaminoglycanases |
EP1982772A1 (en) | 2003-05-16 | 2008-10-22 | Cinvention Ag | Bio-compatible coated medical implants |
CA2540742A1 (en) | 2003-09-30 | 2005-04-14 | Terry Hicks | Compositions and methods for treating burns |
WO2005046746A2 (en) | 2003-11-10 | 2005-05-26 | Angiotech International Ag | Medical implants and fibrosis-inducing agents |
US7364747B1 (en) | 2004-02-02 | 2008-04-29 | S.S. Steiner, Inc. | Process and product for inhibiting or preventing bacterial infections |
US20060004185A1 (en) | 2004-07-01 | 2006-01-05 | Leese Richard A | Peptide antibiotics and peptide intermediates for their prepartion |
WO2006024741A2 (en) | 2004-07-30 | 2006-03-09 | Palumed S.A. | Hybrid qa molecules wherein q is an aminoquinoline and a is an antibiotic residue, their synthesis and their uses as antibacterial agent |
US7541046B1 (en) | 2005-01-04 | 2009-06-02 | Gp Medical, Inc. | Nanoparticles for protein drug delivery |
US20060195183A1 (en) | 2005-02-18 | 2006-08-31 | The Cleveland Clinic Foundation | Apparatus and methods for replacing a cardiac valve |
US20060259135A1 (en) | 2005-04-20 | 2006-11-16 | The Cleveland Clinic Foundation | Apparatus and method for replacing a cardiac valve |
CA2615467A1 (en) | 2005-07-15 | 2007-01-25 | The Cleveland Clinic Foundation | Apparatus and method for remodeling a cardiac valve annulus |
WO2007041677A2 (en) | 2005-10-03 | 2007-04-12 | Combinatorx, Incorporated | Soft tissue implants and drug combination compositions, and use thereof |
US20070299043A1 (en) | 2005-10-03 | 2007-12-27 | Hunter William L | Anti-scarring drug combinations and use thereof |
WO2007041584A2 (en) | 2005-10-03 | 2007-04-12 | Combinatorx, Incorporated | Implantable sensors, implantable pumps, and anti-scarring drug combinations |
US20070208134A1 (en) | 2005-10-03 | 2007-09-06 | Hunter William L | Anti-scarring drug combinations and use thereof |
KR100750658B1 (en) | 2005-12-09 | 2007-08-20 | 한국생명공학연구원 | Polymyxin synthetase and gene cluster thereof |
CA2632871A1 (en) | 2006-01-30 | 2007-08-09 | Angiotech Pharmaceuticals, Inc. | Sutures and fibrosing agents |
EP2028936A4 (en) | 2006-02-13 | 2014-02-26 | Univ Boston | Compositions and methods for antibiotic potentiation and drug discovers |
WO2007095631A2 (en) | 2006-02-15 | 2007-08-23 | The Regents Of The University Of California | New drug delivery system for crossing the blood brain barrier |
WO2007100663A2 (en) | 2006-02-22 | 2007-09-07 | University Of Kansas | Polyamines and their use as antibacterial and sensitizing agents |
US8585753B2 (en) | 2006-03-04 | 2013-11-19 | John James Scanlon | Fibrillated biodegradable prosthesis |
US20070243275A1 (en) | 2006-04-13 | 2007-10-18 | Gilbard Jeffrey P | Methods and compositions for the treatment of infection or infectious colonization of the eyelid, ocular surface, skin or ear |
US8652201B2 (en) | 2006-04-26 | 2014-02-18 | The Cleveland Clinic Foundation | Apparatus and method for treating cardiovascular diseases |
US8771712B2 (en) | 2006-05-09 | 2014-07-08 | Emisphere Technologies, Inc. | Topical administration of acyclovir |
JP2009545588A (en) | 2006-08-03 | 2009-12-24 | プロセンサ テクノロジーズ ビー.ブイ. | Antibiotic composition |
US7807637B2 (en) | 2006-08-11 | 2010-10-05 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
WO2008017734A1 (en) | 2006-08-11 | 2008-02-14 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US20100028334A1 (en) | 2006-12-15 | 2010-02-04 | Trustees Of Boston University | Compositions and methods to potentiate colistin activity |
ES2401045T3 (en) | 2007-04-02 | 2013-04-16 | Mount Sinai School Of Medicine | Methods to prevent or treat infectious diseases using gallium compounds |
US20090048155A1 (en) | 2007-08-15 | 2009-02-19 | Endacea, Inc. | Methods for preventing and treating tissue injury and sepsis associated with Yersinia pestis infection |
US20090214601A1 (en) | 2007-09-28 | 2009-08-27 | Chappa Ralph A | Porous Drug Delivery Devices and Related Methods |
US20090227018A1 (en) | 2007-10-25 | 2009-09-10 | Revalesio Corporation | Compositions and methods for modulating cellular membrane-mediated intracellular signal transduction |
US8329645B2 (en) | 2008-02-08 | 2012-12-11 | Northern Antibiotics Ltd. | Polymyxin derivatives and uses thereof |
US8193148B2 (en) | 2008-02-08 | 2012-06-05 | Northern Antibiotics Ltd. | Short fatty acid tail polymyxin derivatives and uses thereof |
WO2010075416A1 (en) | 2008-12-23 | 2010-07-01 | Biosource Pharm, Inc. | Antibiotic compositions for the treatment of gram negative infections |
US8415307B1 (en) | 2010-06-23 | 2013-04-09 | Biosource Pharm, Inc. | Antibiotic compositions for the treatment of gram negative infections |
-
2004
- 2004-07-01 US US10/881,160 patent/US20060004185A1/en active Pending
-
2005
- 2005-07-01 KR KR1020077002649A patent/KR20070047770A/en not_active Application Discontinuation
- 2005-07-01 MX MXPA06015239A patent/MXPA06015239A/en not_active Application Discontinuation
- 2005-07-01 EP EP05856867A patent/EP1761554A2/en not_active Withdrawn
- 2005-07-01 AU AU2005326770A patent/AU2005326770B2/en not_active Ceased
- 2005-07-01 JP JP2007519447A patent/JP2008505858A/en active Pending
- 2005-07-01 CN CNA2005800294199A patent/CN101010336A/en active Pending
- 2005-07-01 BR BRPI0512941-9A patent/BRPI0512941A/en not_active IP Right Cessation
- 2005-07-01 CA CA002571944A patent/CA2571944A1/en not_active Abandoned
- 2005-07-01 EP EP10184953A patent/EP2332965A1/en not_active Withdrawn
- 2005-07-01 NZ NZ552730A patent/NZ552730A/en not_active IP Right Cessation
- 2005-07-01 US US11/630,847 patent/US8889826B2/en not_active Expired - Fee Related
- 2005-07-01 RU RU2007103811/04A patent/RU2428429C2/en not_active IP Right Cessation
- 2005-07-01 NZ NZ579261A patent/NZ579261A/en not_active IP Right Cessation
- 2005-07-01 WO PCT/US2005/023343 patent/WO2006083317A2/en active Application Filing
-
2006
- 2006-12-21 ZA ZA200610818A patent/ZA200610818B/en unknown
- 2006-12-31 IL IL180458A patent/IL180458A0/en unknown
-
2007
- 2007-01-30 NO NO20070563A patent/NO20070563L/en not_active Application Discontinuation
-
2011
- 2011-04-15 RU RU2011115077/04A patent/RU2011115077A/en not_active Application Discontinuation
- 2011-08-01 JP JP2011168634A patent/JP2011256189A/en active Pending
Cited By (39)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007142611A1 (en) * | 2006-06-02 | 2007-12-13 | Biotika A.S. | Method of polymyxin b recovery from fermentation broth |
EP3078672A1 (en) | 2006-08-11 | 2016-10-12 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
WO2008017734A1 (en) | 2006-08-11 | 2008-02-14 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US20080287345A1 (en) * | 2006-08-11 | 2008-11-20 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US9067974B2 (en) | 2006-08-11 | 2015-06-30 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US8680234B2 (en) | 2006-08-11 | 2014-03-25 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US7807637B2 (en) | 2006-08-11 | 2010-10-05 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US20100292136A1 (en) * | 2006-08-11 | 2010-11-18 | Northern Antibiotics Oy | Polymyxin derivatives and uses thereof |
US8329645B2 (en) | 2008-02-08 | 2012-12-11 | Northern Antibiotics Ltd. | Polymyxin derivatives and uses thereof |
US8193148B2 (en) | 2008-02-08 | 2012-06-05 | Northern Antibiotics Ltd. | Short fatty acid tail polymyxin derivatives and uses thereof |
JP2011511045A (en) * | 2008-02-08 | 2011-04-07 | ノーザン アンチバイオティクス オイ | Polymyxin derivatives having short-chain fatty acid tails and uses thereof |
US8642535B2 (en) | 2008-02-08 | 2014-02-04 | Northern Antibiotics Ltd. | Short fatty acid tail polymyxin derivatives and uses thereof |
US20090239792A1 (en) * | 2008-02-08 | 2009-09-24 | Martti Sakari Vaara | Short fatty acid tail polymyxin derivatives and uses thereof |
US20090215677A1 (en) * | 2008-02-08 | 2009-08-27 | Martti Sakari Vaara | Polymyxin derivatives and uses thereof |
US9090669B2 (en) | 2008-02-08 | 2015-07-28 | Northern Antibiotics Ltd. | Short fatty acid tail polymyxin derivatives and uses thereof |
US9096649B2 (en) | 2008-02-08 | 2015-08-04 | Northern Antibiotics Ltd. | Polymyxin derivatives and uses thereof |
JP2015145399A (en) * | 2008-02-08 | 2015-08-13 | ノーザン アンチバイオティクス オイ | Polymyxin derivatives having short fatty acid tail and uses thereof |
EP3263586A1 (en) | 2008-02-08 | 2018-01-03 | Northern Antibiotics Oy | Short fatty acid tail polymyxin derivatives and uses thereof |
WO2009098357A1 (en) * | 2008-02-08 | 2009-08-13 | Northern Antibiotics Oy | Short fatty acid tail polymyxin derivatives and uses thereof |
US20130053305A1 (en) * | 2010-03-10 | 2013-02-28 | Universidad De Barcelona | Peptide compounds that can be used as antibacterial agents |
US10234460B2 (en) | 2013-08-26 | 2019-03-19 | The University Of Tokyo | Macrocyclic peptide, method for producing same, and screening method using macrocyclic peptide library |
EP3126376A4 (en) * | 2014-04-01 | 2017-10-25 | Monash University | Polymyxin derivatives as antimicrobial compounds |
US10047126B2 (en) | 2014-04-01 | 2018-08-14 | Monash University | Polymyxin derivatives as antimicrobial compounds |
USRE48335E1 (en) | 2014-04-01 | 2020-12-01 | Monash University | Polymyxin derivatives as antimicrobial compounds |
WO2016100578A2 (en) | 2014-12-16 | 2016-06-23 | Micurx Pharmaceuticals, Inc. | Antimicrobial polymyxins for treatment of bacterial infections |
WO2016100578A3 (en) * | 2014-12-16 | 2016-08-11 | Micurx Pharmaceuticals, Inc. | Antimicrobial polymyxins for treatment of bacterial infections |
CN107257803A (en) * | 2014-12-16 | 2017-10-17 | 盟科医药技术公司(开曼群岛) | Polymyxins antiseptic for treating bacterium infection |
KR102585108B1 (en) * | 2014-12-16 | 2023-10-05 | 상하이 미큐알엑스 파마슈티컬 컴퍼니 리미티드 | Antimicrobial polymyxins for treatment of bacterial infections |
US9771394B2 (en) | 2014-12-16 | 2017-09-26 | Micurx Pharmaceuticals, Inc. | Antimicrobial polymyxins for treatment of bacterial infections |
KR20170086671A (en) * | 2014-12-16 | 2017-07-26 | 미큐알엑스 파마슈티컬스, 인코포레이티드 | Antimicrobial polymyxins for treatment of bacterial infections |
EP3045469A1 (en) | 2015-01-15 | 2016-07-20 | Northern Antibiotics Oy | Polymyxin derivative and uses thereof |
WO2016113470A1 (en) | 2015-01-15 | 2016-07-21 | Northern Antibiotics Oy | Polymyxin derivative and uses thereof |
US9763996B2 (en) | 2015-01-16 | 2017-09-19 | Northern Antibiotics, Ltd. | Polymyxin derivative and uses thereof |
US11225505B2 (en) | 2015-09-29 | 2022-01-18 | Monash University | Antimicrobial polymyxin derivative compounds |
US12012466B2 (en) | 2015-10-27 | 2024-06-18 | Hoffmann-La Roche Inc. | Peptide macrocycles against Acinetobacter baumannii |
EP3556769A4 (en) * | 2016-12-16 | 2021-03-03 | Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences | Polymyxin derivative, preparation method and application thereof |
US11819532B2 (en) | 2018-04-23 | 2023-11-21 | Hoffmann-La Roche Inc. | Peptide macrocycles against Acinetobacter baumannii |
WO2021150792A1 (en) | 2020-01-21 | 2021-07-29 | Micurx Pharmaceuticals, Inc. | Novel compounds and composition for targeted therapy of kidney-associated cancers |
CN113174398A (en) * | 2021-04-22 | 2021-07-27 | 浙江工业大学 | Expression cassette for recombinant expression of echinocandin B deacylase and application |
Also Published As
Publication number | Publication date |
---|---|
JP2008505858A (en) | 2008-02-28 |
ZA200610818B (en) | 2008-07-30 |
NZ579261A (en) | 2011-02-25 |
RU2011115077A (en) | 2012-10-20 |
NZ552730A (en) | 2009-09-25 |
WO2006083317A2 (en) | 2006-08-10 |
US8889826B2 (en) | 2014-11-18 |
NO20070563L (en) | 2007-03-27 |
IL180458A0 (en) | 2007-06-03 |
KR20070047770A (en) | 2007-05-07 |
EP2332965A1 (en) | 2011-06-15 |
WO2006083317A9 (en) | 2007-03-01 |
AU2005326770A1 (en) | 2006-08-10 |
CA2571944A1 (en) | 2006-08-10 |
BRPI0512941A (en) | 2007-10-30 |
RU2428429C2 (en) | 2011-09-10 |
WO2006083317A3 (en) | 2007-01-18 |
JP2011256189A (en) | 2011-12-22 |
EP1761554A2 (en) | 2007-03-14 |
US20080207874A1 (en) | 2008-08-28 |
AU2005326770B2 (en) | 2011-11-03 |
MXPA06015239A (en) | 2007-12-10 |
CN101010336A (en) | 2007-08-01 |
RU2007103811A (en) | 2008-08-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060004185A1 (en) | Peptide antibiotics and peptide intermediates for their prepartion | |
CA2118757C (en) | Aza cyclohexapeptide compounds | |
BOECK et al. | Deacylation of echinocandin B by Actinoplanes utahensis | |
CA2979273C (en) | Short fatty acid tail polymyxin derivatives and uses thereof | |
US9090669B2 (en) | Short fatty acid tail polymyxin derivatives and uses thereof | |
US8415307B1 (en) | Antibiotic compositions for the treatment of gram negative infections | |
US6784283B2 (en) | Peptide antibiotics | |
US9096649B2 (en) | Polymyxin derivatives and uses thereof | |
US5516756A (en) | Aza cyclohexapeptide compounds | |
JPH10505100A (en) | New antifungal cyclohexapeptide | |
JPH075638B2 (en) | A-21978C cyclic peptide derivative | |
US4396543A (en) | Derivatives of A-21978C cyclic peptides | |
WO1994009033A1 (en) | An improved process for cyclohexapeptidyl bisamine compounds | |
US5962407A (en) | Loloatin derivatives and analogs | |
EP2493843A1 (en) | 2-amino-3-methyl-hex-5-enoic acid and its use in the production of peptides such as bacitracins | |
MXPA98008484A (en) | New lantibiotic related with actagardine, procedure for its preparation and employment of the mi |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: BIOSOURCE PHARM, INC., NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LEESE, RICHARD A.;FRANCIS, NORMAN D.;CURRAN, WILLIAM V.;AND OTHERS;REEL/FRAME:015558/0839 Effective date: 20040629 |