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Peptide antibiotics and peptide intermediates for their prepartion

a technology of antibiotics and intermediates, applied in the field of peptide antibiotics and peptide intermediates for their preparation, can solve the problems of limited chromatographic purification methods, limited use of bacteria, and often susceptible to polymyxins, and achieve the effect of preparing for aqueous solution

Undetermined Publication Date: 2006-01-05
BIOSOURCE PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025] 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.
[0026] 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.
[0027] 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.
[0029] 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.

Problems solved by technology

These bacteria are often susceptible to the polymyxins (Refs.
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).
Chromatographic purification methods were limited and many of the products seemed to be relatively impure.
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.

Method used

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  • Peptide antibiotics and peptide intermediates for their prepartion
  • Peptide antibiotics and peptide intermediates for their prepartion
  • Peptide antibiotics and peptide intermediates for their prepartion

Examples

Experimental program
Comparison scheme
Effect test

example 1

N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)]5-polymyxin B

[0067] 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 ...

example 2

Preparation of the Deacylase

[0069] 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...

example 3

Deacylation of the Protected Polymyxin B by the Enzyme in Cells

[0071] 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.

[0072] Resin Method: Cells from 1 lite...

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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

FIELD OF THE INVENTION [0001] 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. BACKGROUND OF THE INVENTION [0002] 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): [0003] 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. [0004] 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. [0005] 3. Chihara, S., Ito, A., Yahata, M., Tobita, T., Koyama, Y. 1974. Chemical Synth...

Claims

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Application Information

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IPC IPC(8): C07K1/02
CPCA61K38/00C07K5/06086C07K7/64C07K7/62C07K5/0815Y02P20/55A61P31/00A61P31/04A61P43/00Y02A50/30
Inventor LEESE, RICHARD A.FRANCIS, NOREENCURRAN, WILLIAM V.BORDERS, DONALD B.JAROLMEN, HOWARD
Owner BIOSOURCE PHARM INC
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